Applied Environmental Science and Engineering

for a Sustainable Future

Arindam Sinharoy
Piet N. L. Lens Editors

Renewable Energy
Technologies
for Energy Efficient
Sustainable
Development
Applied Environmental Science and Engineering
for a Sustainable Future

Series Editors
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Australia
Li Shu, LJS Environment, Melbourne, Australia
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Chart Chiemchaisri, Kasetsart University, Bangkok, Thailand
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Arindam Sinharoy • Piet N. L. Lens
Editors

Renewable Energy
Technologies for Energy
Efficient Sustainable
Development
Editors
Arindam Sinharoy Piet N. L. Lens
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Applied Environmental Science and Engineering for a Sustainable Future
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Contents

Part I Process Fundamentals

1 Fundamentals of Biofuel Production Using Anaerobic Digestion:
Metabolic Pathways and Factors Affecting the Process . . . . . . . . . . 3
Adriana Ferreira Maluf Braga and Marcelo Zaiat
2 Engineering Direct Interspecies Electron Transfer for Enhanced
Methanogenic Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Changsoo Lee

Part II Pretreatment
3 Adsorbents for the Detoxification of Lignocellulosic Wastes
Hydrolysates to Improve Fermentative Processes to Bioenergy
and Biochemicals Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Itzel Covarrubias-García and Sonia Arriaga
4 Pretreatment of Lignocellulosic Materials to Enhance their
Methane Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
A. Oliva, S. Papirio, G. Esposito, and P. N. L. Lens
5 Biogas Production from Dairy Cattle Residues: Definition
of the Pretreatment Approach Through a Bibliometric Analysis
of Publications and Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Ricardo Müller, Marcio Antonio Vilas Boas, Mônica Sarolli S. M.
Costa, Felipe Souza Marques, Douglas Alves Santos,
Günther Bochmann, Marcelo Bevilacqua Remor,
and Daiana Gotardo Martinez

Part III AD of Specific Waste-Streams

6 Anaerobic Digestion of Dairy Industry Wastewater . . . . . . . . . . . . 141
Sridhar Pilli, K. Bella, Y. Manojkumar, Vasam Vinila,
and Venkateswara Rao Polisetty
v
vi Contents

7 Solid State Anaerobic Digestion of Agricultural Waste

for Bioenergy Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Kunwar Paritosh, Himanshi Singh, Nupur Kesharwani, Nidhi Pareek,
and Vivekanand Vivekanand
8 Food Waste Biorefinery for Bioenergy and Value Added
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Miguel Ladero, Jesús Esteban, Juan Manuel Bolívar, Victoria E.
Santos, Víctor Martín-Domínguez, Alberto García-Martín,
Álvaro Lorente, and Itziar A. Escanciano

Part IV Downstream Processing for Resource Recovery

9 Valorisation of Anaerobic Digestate: Towards Value-Added
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
F. Guilayn, J. Jimenez, F. Monlau, and C. Vaneeckhaute
10 Biochar Produced from Organic Waste Digestate and Its Potential
Utilization for Soil Remediation: An Overview . . . . . . . . . . . . . . . . 263
Suchanya Wongrod, Gilles Guibaud, Stephane Simon,
Piet N. L. Lens, David Huguenot, Yoan Pechaud,
and Eric D. van Hullebusch

Part V Integration of AD in Biorefineries

11 Integration of Bio-electrochemical Systems with Anaerobic
Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
M. M. Ghangrekar, S. M. Sathe, and C. N. Khuman
12 Use of Biogas for Electricity-Driven Appliances . . . . . . . . . . . . . . . 319
Afshin Davarpanah
13 Syngas Fermentation for Bioenergy Production: Advances
in Bioreactor Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Arindam Sinharoy, Kannan Pakshirajan, and Piet N. L. Lens

Part VI Life Cycle Analysis

14 Up and Downstream Technologies of Anaerobic Digestion
from Life Cycle Assessment Perspective . . . . . . . . . . . . . . . . . . . . . 361
Seyedeh Nashmin Elyasi, Hadis Marami, Li He, Ali Kaab,
Junting Pan, Hongbin Liu, and Benyamin Khoshnevisan
15 Life Cycle Assessment of Anaerobic Digestion Systems:
An Approach Towards Sustainable Waste Management . . . . . . . . . 391
Marlia M. Hanafiah, Iqbal Ansari, and Kalppana Chelvam
 Part I
Process Fundamentals
Chapter 1
Fundamentals of Biofuel Production Using
Anaerobic Digestion: Metabolic Pathways
and Factors Affecting the Process

Adriana Ferreira Maluf Braga and Marcelo Zaiat

Abstract The need for a change in the global energy matrix from a fossil fuel based
to a renewable energy one is critical for sustainable development. In this context,
biofuels from anaerobic digestion of agro-industrial waste and wastewater, i.e.,
biohydrogen and biomethane, represent an attractive option. The dark fermentation
process for biohydrogen production involves several possible microbial pathways
that are dynamics and need to be understood to overcome limitations and process
optimization. Methanogenesis for biomethane production occurs close to thermody-
namic limitations. Thus, the system needs to be balanced to achieve stability and
satisfactory biofuel production. Several operational parameters interfere with the
process, and the knowledge about them allows to maximize the methane yield.
Besides problems with gas supersaturation, the role of micronutrients and adequate
removal of toxic compounds released during the biomass decay represent themes
that still must be solved to achieve the full potential of biofuels generation. This
chapter provides fundamental knowledge to overcome the technological limitation
and advance towards a wide production of biofuels from organic waste using
anaerobic digestion.

Keywords Biohydrogen · Biomethane · Dark fermentation · Methanogenesis ·

Renewable energy

1.1 Introduction

The growing concern on climate change has driven attention to alternative energy
supplies to convert the current energy matrix based on fossil fuels to renewable ones.
The world’s energy consumption from fossil fuels, i.e., oil, gas, and coal, represented

A. Ferreira Maluf Braga (*) · M. Zaiat

Biological Processes Laboratory, São Carlos School of Engineering (EESC), University of São
Paulo (USP), São Carlos, São Paulo, Brazil
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_1
4 A. Ferreira Maluf Braga and M. Zaiat

Fig. 1.1 Global consumption by source in the last 50 years (Data source: Ritchie and Roser 2020)

84.3% in 2019 (Ritchie and Roser 2020), compared with 4.3% from nuclear and
11.4% from renewable sources. A decrease of only 9.4% in the last 50 years
(Fig. 1.1) demonstrating the urge to improve the sustainable energy grid.
Among the renewable sources, i.e., wind, solar, and hydropower, biofuels have
received increasing attention in the last decades. Biofuels can be produced by
microbial fermentation of biomass and presented as solid, liquid, and gaseous
fuels, such as ethanol produced by yeast from sugarcane and corn, biodiesel from
microalgae, and biomethane upgraded biogas from anaerobic fermentation of waste
and wastewater by syntrophic interaction between bacteria and archaea.
CH4 produced from organic matter degradation through anaerobic digestion
(AD) can be upgraded to biomethane (BioCH4) by removing CO2, achieving
>96% purity of CH4 and an energy density of 50–55 kJ g
1 (Beil and Beyrich
2013). The AD process occurs in a closed reactor in the absence of oxygen. It is
divided into (1) hydrolysis of macromolecules, (2) fermentation of monomers into
organic acids, hydrogen (H2), CO2 and alcohols, (3) conversion of previous inter-
mediate products into acetate, and (4) final conversion of acetate and hydrogen to
biogas (mixture of CH4 and CO2).
Hydrogen thus produced, also called BioH2, can also be considered a biofuel, as it
is a clean energy carrier with an energy density of 122 kJ g
1, 2.75-fold higher than
that of fossil fuels and generating only water when converted to electricity in fuel
cells. The BioH2 production through anaerobic fermentation of organic matter is a
process called dark fermentation. BioH2 is generated to balance the cell redox
potential during fermentation by using the electrons eliminated in the process for
reducing protons and then excreted from the cells as dissolved gas (Cabrol et al.
2017; Schwartz and Friedrich 2006).
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . . 5

As the dark fermentation process corresponds to the acidogenic step of AD, it can
be followed by acetogenesis and methanogenesis. The latter results in the formation
of biogas, from which the CH4 can be enriched to BioCH4. Arreola-Vargas et al.
(2016) demonstrated that the overall energy recovery from bagasse is higher in a
system performing BioH2 followed by BioCH4 production (two-phase) than a
system producing only BioCH4 (single-phase). Therefore, coupling both processes
can improve the biofuel production, representing advances towards renewable and
sustainable energy production. Therefore, its fundamentals and process interferences
will be addressed in this chapter.

1.2 Metabolic Pathways

1.2.1 BioH2 Production Via Dark Fermentation

The organic fraction of waste and wastewater provided to microorganisms for

biofuel production through anaerobic digestion is composed of a combination of
carbohydrates, proteins, and lipids. Carbohydrates and amino acids are the most
common carbon source utilized by fermentative microorganisms, and glucose is
considered a model substrate (Cabrol et al. 2017). After hydrolysis, the monomers
entering the microbial cell follow a series of chemical reactions to provide energy
and components for the microorganisms’ metabolism.
The breakdown of 1 mol of glucose through the Embden-Meyerhof-Parnas
(EMP) glycolysis results in 2 moles of pyruvate, 2 moles of ATP, and 2 moles of
NADH (Fig. 1.2). The pyruvate can be metabolized through pyruvate-formate-lyase
(PFL), resulting in formate and Acetyl-CoA production. The former is secreted out
of the cell, and Acetyl-CoA can be used for ATP generation. This pathway is
associated with facultative microorganisms, such as Enterobacteria, and BioH2
generation is triggered by extracellular formate (pKa ¼ 3.75) excess and pH
dropping of the medium (Pinske and Sargent 2016). After a formate threshold
level is reached, it is transported back into the cell to be oxidized by formate
hydrogen lyase (FHL), catalysed by a molybdenum-dependent selenoenzyme
(McDowall et al. 2014). The electrons are used for a reduction of H+, with the
participation of a [NiFe] Ech (Escherichia coli hydrogenase 3) hydrogenase or a
formate-dependent (COO
) [FeFe] hydrogenase (Cabrol et al. 2017; McDowall
et al. 2014).
Strict anaerobes, such as Clostridia, break down pyruvate through the ferredoxin
oxidoreductase (PFOR) pathway. In these microorganisms, PFOR catalyzes the
oxidative pyruvate decarboxylation, and the electron acceptor is a ferredoxin,
which can be re-oxidized by a ferredoxin-dependent hydrogenase (Fd[FeFe]), releas-
ing BioH2 (Cabrol et al. 2017; Chabrière et al. 1999). The regeneration of NADH
produced during the glycolysis, coupled with reduced ferredoxin via ferredoxin
oxidoreductase (NFOR), can yield 4 moles of BioH2 per mol of glucose, whether
the acetyl CoA is driven to acetate production (Eq. 1.1) (Cai et al. 2011).
6 A. Ferreira Maluf Braga and M. Zaiat

Fig. 1.2 Main metabolic pathways and products associated with dark fermentation from glucose,
considering the Embden–Meyerhof–Parnas (EMP) glycolysis. Pyruvate can be converted into
Acetyl-CoA either through the pyruvate-formate-lyase (PFL) or pyruvate ferredoxin
oxydoreductase (PFOR) pathway depending if the microorganisms are facultative or strict anaer-
obes. In the PFL pathway, the BioH2 and CO2 production from formate might follow a reaction
catalyzed by formate hydrogen lyase (FHL) along with either a [NiFe] Ech (Escherichia coli
hydrogenase 3) hydrogenase or a formate-dependent (COO
) [FeFe] hydrogenase. In the PFOR
pathway, a ferredoxin-dependent (Fd-[FeFe]) hydrogenase is involved in the production of BioH2
via NADH: ferredoxin oxidoreductase (NFOR) or NADP+ reducing factor (NRF). The acetyl CoA
branch generates organic acids and alcohol. Pyruvate can directly generate L-lactate, which might
lead to propionate for reducing NADH. Propionate can also be formed through the dicarboxylic acid
(DCA) cycle. Numbers between parentheses represent stoichiometry for 1 mol of glucose. Adapted
from Cabrol et al. (2017) and Hoelzle et al. (2014)

∘
C6 H 12 O6 þ 2H 2 O ! 2 CH 3 COOH þ 4H 2 þ 2CO2 ΔG

206:0 kJ mol
1 ð1:1Þ

Acetate and butyrate are the primary pathways in the acetyl Co-A branch, as these
products result in the highest ATP production (Hoelzle et al. 2014). Butyrate is
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . . 7

favoured over acetate when an alternative electron sink is necessary. Ethanol,

butanol, and acetone are other alternatives to sink electrons onto and avoid the
increase of the hydrogen partial pressure and pH drop (Hoelzle et al. 2014).
Isopropanol might be produced by reducing acetone and sinking more electrons.
Although the alcohol production decreases the BioH2 production yield, the
solventogenic pathway described can be explored for liquid biofuel production.
Microorganisms without PFOR are unable to oxidize NADH with BioH2 pro-
duction via NFOR, and the NAD+ release occurs during the production of reduced
organic acids and alcohols. Additionally, dark fermentation is affected by BioH2
accumulation at different levels depending on the electron carriers. BioH2 continues
at a hydrogen partial pressure lower than 0.3 atm, while NADH activity can be
hindered at a hydrogen partial pressure higher than 0.6x10
4 atm (Angenent et al.
2004).
Lactate is produced directly via pyruvate and does not act as an electron sink.
Nevertheless, it is the preferred pathway for propionate producers resulting in the
re-oxidation of 2 moles of NADH (Eq. 1.2) (Saady 2013).

CH 3 COCOOH þ 2NADH þ 2H þ ! CH 3 CH 2 COOH þ 2NADþ þ H 2 O

∘ ð1:2Þ
ΔG
186:2 kJ mol
1

Propionate can also be generated by the dicarboxylic acid (DCA) cycle carried
out by Propionibacterium species. The cycle begins when the oxaloacetate is
reduced to L-malate, followed by fumarate and succinate production, which gener-
ates propionate when succinate receives the CoA from the propanoyl-CoA (Hoelzle
et al. 2014). The succinyl-CoA leads to the regeneration of oxaloacetate by trans-
ferring CoA to pyruvate to form propanoyl-CoA (Hoelzle et al. 2014).

1.2.2 BioCH4 Via Methanogenesis

Methanogenesis is a series of biochemical reactions composing the last step in AD

and promoting organic matter degradation into CH4 and CO2. Methanogens can
metabolize three classes of substrates: CO2-type substrate, methyl substrates, and
acetotrophic substrate (Madigan et al. 2010).
Methanogenesis from acetate cleavage requires ATP consumption and results in
acetyl-CoA formation. Acetyl-CoA decarbonylase/synthase (ACS) catalyses the
breaking down of the acetyl-CoA into CoA, CO, and a methyl group, generating a
methylated corrinoid (Can et al. 2014; Mulrooney and Hausinger 2003). The
oxidation of CO catalysed by CO-dehydrogenase (CODH) forms CO2, whereas
the CH4 is produced from the reduction of the corrinoid-bound methyl group with
the participation of methyl-coenzyme M reductase, coenzyme B, and the cofactor
F430 (Mulrooney and Hausinger 2003). The autotrophic pathway to produce acetate,
the Wood-Ljungdahl pathway, is energy conservative, and methanogens use it in the
8 A. Ferreira Maluf Braga and M. Zaiat

reductive direction for CO2 fixation (Ragsdale and Pierce 2008). This pathway is
known as the homoacetogenic pathway and responsible for depletion in BioH2
production, and it is also spread among Clostridium genus (Castelló et al. 2020).
The CH4 production can occur in a single-phase reactor using raw waste or
wastewater or using a two-phase system that receives the effluent of the dark
fermentation reactor. In two-phase systems, the methanogenic reactor is fed with a
substrate formed by a mixture of organic acids and alcohols. Therefore, it is
necessary to convert these compounds into the substrates required by methanogenic
archaea, mainly acetate, CO2 and H2.
Generally, the acetogenic reactions are thermodynamically not favourable, as no
energy is released (Table 1.1). Nevertheless, the consumption of the dissolved
hydrogen and acetate by methanogenic archaea allows a syntrophic relationship
that alters the energetic balance, allowing sufficient energy production to allow the
growth of the acetogenic microorganisms (Madigan et al. 2010).
The global reaction (Eq. 1.13) demonstrates that the propionate yields 56.6 kJ per
mol converted, divided among the groups involved (acetoclastic bacteria,
hydrogenotrophic and acetoclastic methanogens), hence explaining their low growth
rate and biomass yield.

CH3 CH2 COO
þ 3H2 O ! CH3 COO
þ HCO
þ

3 þ H þ 3H2 +76.1 kJ (1.3)
propionate acetate
CH3 COO
þ H2 O ! CH4 þ HCO
3
31 kJ (1.9)
acetate methane
3H2 þ 0:75 HCO
3 þ 0:75 H
þ
! 0:75 CH4 þ 2:25 H2 O
101.7 kJ (1.10)
hydrogen methane
CH3 CH2 COO
þ 1:75 H2 O ! 1:75 CH4 þ 1:25 HCO
3 þ 0:25 H
þ
56.6 kJ (1.13)
propionate methane

Harper and Pohland (1986) used the results from thermodynamic calculations of
main anaerobic reactions versus hydrogen partial pressure (Fig. 1.3) to establish a
methanogenic niche, in which the hydrogen partial pressure ranges from 10
6 to
10
4 atm. This range is thermodynamically favourable for the syntrophism between
organic acid producers and consumers, consequently, for CH4 production.
Operational failures, such as shock loads, biomass wash-out, changes in temper-
ature and pH, might lead to BioH2 and acetate accumulation and inhibiting the
degradation of reduced organic acids, as acetogenic reactions occur close to the
thermodynamic equilibrium. Proprionate concentrations exceeding 900 mg L
1
decrease the acetogenic bacterial activity, hampering other organic acids degradation
and leading to more accumulation in the system (Wang et al. 2009). The organic
acids accumulation causes the drop of the system pH, as the alkalinity of the medium
is consumed, hindering methanogenic archaea growth and activity, which causes
acetate accumulation until the CH4 is completely ceased (Wang et al. 2009).
Therefore, methanogenic systems are more sensitive to variations and susceptible
to failures.
Table 1.1 Main reactions involved in acetate and methane production from organic waste and wastewater (Harper and Pohland 1986; Thauer et al. 1977)
Stage Reaction ΔG (kJ) Eq.
Acetogenesis

þ +76.1 (1.3)
CH3 CH2 COO þ 3H2 O ! CH3 COO þ HCO

þ H þ 3H2
3
propionate acetate
CH3 CH2 COO
þ 2HCO
3 ! CH3 COO
þ Hþ þ 3HCOO
+72.2 (1.4)
propionate acetate
CH3 CH2 CH2 COO
þ 2H2 O ! 2CH3 COO
þ Hþ þ 2H2 +48.1 (1.5)
butyrate acetate
CH3 CH2 OH þ H2 O ! CH3 COO
þ Hþ þ 2H2 +9.6 (1.6)
ethanol acetate
þ
4.2 (1.7)
CH3 CHOHCOO
þ 2H2 O ! CH3 COO
þ HCO
3 þ H þ 2H2
lactate acetate
þ
(1.8)
2HCO
3 þ 4H 2 þ H ! CH 3 COO þ 4H 2 O
104.6
bicarbonate acetate
Methanogenesis CH3 COO
þ H2 O ! CH4 þ HCO
3
31 (1.9)
acetate methane
4H2 þ CO2 ! CH4 þ 2 H2 O
131 (1.10)
hydrogen methane
HCOO
þ ¼ H2 O þ ¼ Hþ ! ¼ CH4 þ ¾ HCO
3
32.6 (1.11)
formate methane
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . .

þ (1.12)
HCO
3 þ 4H2 þ H ! CH4 þ 3H2 O
135.6
bicarbonate methane
9
10 A. Ferreira Maluf Braga and M. Zaiat

Fig. 1.3 Diagram indicating the favourable niche for BioCH4 production from anaerobic degra-
dation of organic waste and wastewater, based on the hydrogen-dependent thermodynamics of the
main reactions: (1) Propionic acid oxidation to acetic acid. (2) Butyric acid oxidation to acetic acid.
(3) Ethanol to acetic acid. (4) Lactic acid to acetic acid. (5) Acetogenic respiration of bicarbonate.
(6) Methanogenic respiration of bicarbonate. (7) Methanogenic cleavage of acetic acid. Acetic acid,
25 mM; propionic, butyric, lactic acids, and ethanol, 10 mM; bicarbonate, 20 mM; methane 0.7 atm.
(Source: Harper and Pohland 1986)

1.3 Factors Affecting the Process

1.3.1 Inoculum Source

The microorganisms established in the system are the most crucial factor affecting
the metabolic pathway, and consequently, the biofuel production and yield of the
process. Therefore, the inoculum source plays the primary role.
A pure culture can be selected and used to ensure higher control of a specific step
of the biofuel production through the desired pathway. Microorganisms have an
optimal condition for growth and metabolic activity. Hence, a pure culture allows
product yield maximization during the industrial process. The enhancement of the
BioH2 production rate from monomers, i.e., glucose, by Thermotoga neapolitana
cf. Capnolactica was demonstrate via heterofermentation pathway (acetate and
lactate) varying the inoculum concentration by Dreschke et al. (2018). A
co-culture of isolated strains can collaborate to overcome technical aspects of the
conversion of substrates in biofuel. The combination of pure cultures strictly anaer-
obic and facultative can eliminate O2 in the medium; strains able to hydrolyse
cellulose can provided carbohydrate monomers for high BioH2 producers in dark
fermentation; strains able to produce BioH2 at different pH can maintain the pro-
duction without require buffering (Elsharnouby et al. 2013). A co-culture of E. coli
CECT432 strain and Enterobacter spH1 showed a three-fold higher H2 productivity
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . . 11

from pure glycerol compared with pure of E. coli CECT432 strain (Maru et al. 2016).
However, isolated cultures require sterilization before using equipment and feeding
the system, turning this option infeasible for real substrates, such as wastewater
containing indigenous microorganisms.
Diverse mixed consortia obtained from indigenous/autochthonous substrate and
inoculum provide the system with notable robustness regarding operational changes,
such as variation in substrate composition and characteristics due the higher micro-
bial diversity without the cost for sterilizing the system and substrate (Cabrol et al.
2017). However, mixed non-isolated consortia for BioH2 production must avoid the
presence of consuming H2 populations, such as methanogenic archaea and sulfate-
reducing bacteria. Therefore, inoculum pre-treatment is an option to select a group of
microorganisms. Thermal (temperatures from 90 to 100  C), acidic (pH lower than
3), and alkaline (pH higher than 11) pre-treatment or a combination of them are
widely adopted to select acidogenic bacteria for dark fermentation and to avoid the
presence of methanogenic microorganisms in the dark fermentation reactor. Some
acidogenic bacteria can form spores under extreme conditions, while archaea typi-
cally present in anaerobic sludge are sensitive to the pre-treatment conditions.
Mockaitis et al. (2020) applied acidic, thermal, and acidic-thermal and thermal-
acidic pre-treatments to a mixed-culture for BioH2 production from xylose. The
authors obtained better results with acidic pre-treatment, achieving a BioH2 yield
equal to 1.57 mol H2 mol xylose
1. Rafieenia et al. (2018) evaluated inoculum
pre-treatment for BioH2, followed by BioCH4. Heat shock, aeration, alkaline and an
innovative pre-treatment with saponified frying oil (WFO) were evaluated using
synthetic food waste as the substrate. The authors found better results using the WFO
for BioH2 and BioCH4, respectively, 76.1 mL gVS
1 and 598.2 mL gVS
1.
Penteado et al. (2013) compared BioH2 production in packet-bed reactor using
pre-treated inoculum from an UASB reactor treating poultry slaughterhouse waste-
water, an UASB fed with swine wastewater and natural fermentation of the substrate.
These authors found higher BioH2 yield using the inoculum from natural fermenta-
tion (2.1  1.8 molH2 molsucrose
1) and higher stability using inoculum pre-treated
with an acid shock (2.0  1.1 molH2 molsucrose
1).
Natural fermentation was also applied using sugarcane vinasse from ethanol and
sugar mill using packed bed (Ferraz et al. 2014) and structured bed reactor (Fuess
et al. 2019) continuous reactors. This inoculation strategy exploits the development
of an adapted consortium in the substrate exposed to the ambient conditions, which
will be further recirculated in the reactor for microorganism attachment (Leite et al.
2008). Autochthonous mixed consortia developed naturally in the substrate such as
mushroom farm waste (Lin et al. 2017) and banana waste (Mazareli et al. 2020) were
also used for BioH2 production in batch experiments and represent a suitable
inoculation approach for BioH2 production from real waste and wastewater.
12 A. Ferreira Maluf Braga and M. Zaiat

1.3.2 pH

Microorganisms have an optimal pH growth and are classified into neutrophile

(5.5 < pH < 8), acidophile (pH < 5.5) and alkaliphile (8  8), which is measured
externally, as the intracellular pH remains close to neutrality (Madigan et al. 2010).
Changes in pH values from the optimal can cause cellular lyase or denaturation of
enzymes necessary for microbial activities and affect the speciation of compounds
present in the medium. Acetic and butyric acids (HA) equilibrium with their ionic
counterparts, acetate and butyrate (A
), depends on the pH according to Eq. 1.14.
The undissociated forms of these acids can cross the cell membrane and release
protons, altering the internal pH, reducing or inhibiting the cellular activities
(Castelló et al. 2020).
 
A

pH ¼ pK a þ log ð1:14Þ
HA

Dark fermentation is generally performed under acidic conditions. Horiuchi et al.

(2002) observed a shift from butyrate and acetate to propionate production when
increasing the pH value from 5.0–7.0 to 8.0 in a completely mixed fermenter fed
with glucose. Fuess et al. (2019) concluded that an increase in pH value from <5.0 to
5.0–5.5 leads to a shift from lactate production to BioH2 production via butyrate,
with a further increase (pH >6.0) associated with propionate and sulfate-reducing
activity using sugarcane vinasse as the substrate. Similar results had already been
reported by Hwang et al. (2009), who found a decrease in BioH2 production at pH
values equal 5.8 and 6.2 compared to a pH value of 5.5, due an increase in the
activity of sulfate-reducing bacteria using glucose as the substrate and adding iron
sulfate.
Methanogenic archaea are able to live in extreme environments. However, the
adequate pH range for the anaerobic process for methane production was found to be
around 7.0–7.5, narrower than the range for dark fermentation (Liu and Sung 2002;
Sprott et al. 1984). Additionally, a more robust methodology was proposed by
Ripley et al. (1986) to monitor the methanogenic system and avoid process failure.
According to the authors, the ratio of the intermediate alkalinity (IA): partial
alkalinity (PA) at the range 0.1 to 0.35 allowed to ensure adequate bicarbonate
buffering and avoid excess of organic acids. Other authors have adopted the IA:total
alkalinity (TA) ratio as a stability parameter, although still considering values lower
than 0.3 (Garcia et al. 2007).

1.3.3 Temperature

Microorganisms possess an optimal temperature for developing their metabolic

functions at the maximum rate, although their activities are still developed within
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . . 13

Fig. 1.4 Solubility of H2, CH4, and CO2 in water. The measurements are normalized for standard
conditions (0  C and 1 atm). (Data source: Dean 1999)

at a certain temperature range. At the lower range value, the semifluid cytoplasmic
membrane begins a solidification process, hindering the transport in the microbial
cell (Madigan et al. 2010). The upper range value indicates the temperature triggers
the collapse of the cytoplasmic membrane, with enzyme denaturation, and thermal
lysis (Madigan et al. 2010). Psychrophilic microorganisms are adapted to tempera-
tures ranging from 0 to 10  C; mesophiles possess optimal temperature at 40  C, but
their metabolism is functioning from 10 to 45  C; thermophiles have an optimal
temperature around 60  C, with activity in the range from 45 to 65  C; and
hyperthermophiles are able to live in environments with temperatures from 90 to
115  C, with an optimum at 105  C (Madigan et al. 2010).
The temperature also affects the solubility of the main gases participating in
AD. The solubility values decrease with the increase in temperature (Fig. 1.4),
indicating that higher operational temperatures might help overcome mass transport
and thermodynamic limitations.
Regarding dark fermentation, the lower solubility of H2 increases the BioH2 yield
by enhancing the mass transfer from the liquid to the gaseous phase and limiting the
homoacetogenesis due to the lower CO2 solubility. In methanogenesis, the lower
solubility of CH4 allows the higher mass transfer of CH4 to the gaseous phase.
However, the lower CO2 solubility might impact the activity of hydrogenotrophic
methanogens. Labatut et al. (2014) found that mesophilic continuously-stirred
anaerobic digesters (CSADs) producing CH4 from cow manure co-digested with
dog fod waste was more robust and stable than a thermophilic CSAD, despite a
slightly lower CH4 yield.
Supersaturation of CH4 in the liquid phase has already been reported by Yeo et al.
(2015). Despite the low solubility of H2 (<CH4 << CO2), BioH2 supersaturation
was observed in several studies under mesophilic and thermophilic conditions
(Beckers et al. 2015; Dreschke et al. 2019a, b; Obazu et al. 2012; Zhang et al.
14 A. Ferreira Maluf Braga and M. Zaiat

2012). Values up to seven-fold the values calculated for equilibrium conditions were
found by Beckers et al. (2015) during BioH2 production using the pure Clostridium
butyricum strain, demonstrating that other strategies besides increasing temperature
must be applied to promote a higher BioH2 yield.

1.3.4 Nutrients and Potentially Toxic Compounds

An interesting alternative substrate of biofuel production from AD is organic waste

and wastewater generated from municipal solid waste, food and beverage industries,
and agro-industrial activities. The use of residues as a resource for biofuel produc-
tion through AD relies on the biorefinery concept and can reduce production costs
while enhancing environmental gains. However, the composition of a substrate from
agro-industrial activities differs depending on the industrial process, soil and climate
conditions and varies seasonally. Consequently, these substrates might contain
inhibitory compounds and provide unbalanced macro and micronutrient supply for
the microorganisms.
Macro and micronutrients are frequently found in poor concentrations in the
substrate, as pointed out by Thanh et al. (2016). Nutritional deficiency in anaerobic
reactors might limit microbial growth and metabolic activities, as trace elements
often composes the reactive centres of metalloenzymes and cofactors (Vignais and
Colbeau 2004). Zhang et al. (2019) observed complete inhibition of methanogenic
activity in a semi-continuous reactor fed with food waste and demonstrated that
supplementation of Fe, Co, Mo and Ni at concentrations, respectively, 100, 1, 5 and
5 mg L
1 eliminated all factors causing failure in the system. Molaey et al. (2019)
observed an increase in methane production from chicken manure with a total
ammonia nitrogen concentration of 3000 mg L
1 by supplementing the system
with concentration up to 5 mg L
1 Ni, 5 mg L
1 Co, 1 mg L
1 Mo, 1 mg L
1 Se,
1 mg L
1 W, and 25 mg L
1 Fe. Conversely, concentrations over two-fold caused a
decrease in CH4 production and rate (Molaey et al. 2019).
The presence of sulfate in the substrate might impact biofuels production, as the
activity of sulfate-reducing bacteria and the hydrogen sulfide generated might
hamper the BioH2 (Fuess et al. 2019) and the CH4 (Kiyuna et al. 2017) production
from sugarcane vinasse. Ammonia has also demonstrated to have an inhibitory effect
on CH4 production at concentrations exceeding 400 mg L
1 in a CSTRs fed with
soluble non-fat dry milk as organic substrate and operated under thermophilic
(55  1  C) conditions (Liu and Sung 2002).
For the biofuel production from lignocellulosic materials, a physicochemical
pre-treatment is necessary to turn the monosaccharides available for AD. The
pre-treatments include mechanical (high-pressure homogenizer and bead mills),
physical (microwave and ultrasonic), thermal (autoclave and steam explosion) and
chemical (catalysis and enzymes) techniques (Onumaegbu et al. 2018). These pro-
cedures also release phenols and furans, their concentration depends on the
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . . 15

lignocellulosic material type and the pre-treatment technique applied (Monlau et al.
2014).
The BioH2 production can be inhibited by furanic and phenolic compounds with
a concentration range of 250–1000 mg L
1 (Elbeshbishy et al. 2017). The inhibition
by phenolic compounds is related to the damage in the microbial cell membrane,
while the furans interfere with enzyme activity (Elbeshbishy et al. 2017). Fang et al.
(2006) demonstrated 70% phenol degradation at a concentration of 630 mg L
1 in a
thermophilic UASB at HRT of 28 h. A specific methanogenic activity (SMA) test
demonstrated that the sludge was able to degrade the phenols at a range concentra-
tion of 600–1000 mg L
1 and produce CH4; however, no phenol degradation was
achieved at 2000 mg L
1 (Fang et al. 2006).

1.3.5 Reactor Configuration

Ideally, the liquid flow pattern in continuous reactors can be classified in plug-flow
and mixed flow, determined by models such as tank-in-series and dispersion
(Levenspiel 1999). It directly impacts the kinetics of the process. Some reactor
configurations applied to biofuels production are presented in Table 1.2. The
mixed flow present in the continuous stirred tank reactor (CSTR) allows a homog-
enous composition throughout the reactor and favours the mass transfer of the
products excreted by the microorganisms (Bailey and Ollis 1986). The plug-flow
pattern allows a longitudinal microorganism separation, and consequently, the
composition of the medium changes throughout the reactor, and it is suitable for
substrates with potentially toxic compounds (Parkin and Speece 1983). Plug-flow
reactors can be used in large-scale for CH4 production from cattle manure in long
term operation (Dong et al. 2019). However, most of the reactors possess a non-ideal
flow pattern, standing in between mixed and plug-flow.
Important reactors characteristics are the presence or absence of a support mate-
rial for microbial attachment, recirculation of liquid or gas, and up or downflow
velocity. Reactor modifications that promote sludge settling and aggregation of
biomass, e.g. three-phase separator in UASB reactors, disconnect the hydraulic
retention time (HRT) from the sludge retention time (SRT), allowing a high-rate
reactor (Lettinga et al. 1980). This also occurs when adding support material such as
in packed and structured bed reactors. Liquid recirculation can be used to increase
the upflow velocities as in expanded granular sludge blanket (EGSB) reactors,
increasing the contact between substrate and microorganisms (Verstraete et al.
1996). It can also improve the alkalinity in methanogenic reactors and dilute toxic
compounds present in the system. Gas recirculation can be applied to overcome H2
supersaturation (Beckers et al. 2015; Dreschke et al. 2019a).
Table 1.2 Reactors used for biofuels production through anaerobic digestion
16

ORL
(gCOD Temperature
Stage Reactor Substrate Inoculum L
1 d
1) ( C) pH initial Biofuel yield Biofuel production Reference
Dark Continuous Glucose Thermotoga – 80 7 3.5  0.2 molH2 850  71 mLH2 Dreschke
fermentation stirred tank neapolitana mol
1 L
1 h
1 et al.
reactor cf. capnolactica (2019b)
(CSTR)
Dark Internal cir- Glucose Mixed culture – 35  1 6.5 1.4  0.21 molH2 10.78  0.84 L
1 Su et al.
fermentation culation (thermal mol
1 d
1 (2020)
(IC) reactor pre-treatment)
Dark CSTR Tequila Vinasse Mixed culture 32.8 37 7.5 1.68 molH2 mol
1 0.42  0.02 NL H2 García-
fermentation (thermal L
1 d
1 Becerra
pre-treatment) et al.
(2019)
Dark Up-flow Glucose Mixed culture 64 48  2 6.0  0.5 0.89 molH2 mol
1 4.73 LH2 L
1 d
1 Karapinar
fermentation packed bed (thermal et al.
reactor pre-treatment) (2020)
(UPBR)
Dark Anaerobic Sugarcane vinasse Natural 56 70 6.5 2.6  0.5 molH2 630  47 mLH2 Niz et al.
fermentation structured fermentation mol
1 L
1 d
1 (2019)
bed reactor
(AnSTBR)
Methanogenesis Plug-flow Cattle manure Mixed culture 3.6 37–40 7.85  0.05 – 0.43–0.75m3 m
3 Dong
reactor d
1 et al.
(2019)
Methanogenesis Fixed-bed Municipal organic Mixed culture 0.7 37–40 7–8 – 335 N-LCH4 Knoop
digester waste percolate kgVS-1 a et al.
(two-phase (2018)
system)
Methanogenesis Horizontal- Cassava Mixed culture 2.3–8.5 28.5  1.5 7-8b 0.3–0.15 LCH4 0.5–4.5 LCH4 d
1 Palma
flow reac- processing waste- gCODremoved
1 et al.
tors with water CPWW (2016)
A. Ferreira Maluf Braga and M. Zaiat
 dolomite
rocks
Methanogenesis CSTR Sugarcane trash Mixed culture 2.5 (gVS 37  1 7.3–7.8b 121 (ST), 303 (ST), Paulose
untreated (ST) and L
1 d
1)c 226 (TT), 565 (TT), and
treated 148 (SB), and 370 (SB) and Kaparaju
(TT) sugarcane 236 (TB) (mLCH4 590 (TB) (mLCH4 (2021)
bagasse untreated gVSfed
1) L
1 d
1)
(SB) and treated
(TB)
Methanogenesis CSTR and Swine manure Mixed culture 0.165 lab 23–24 BLC 7.4 BLC 0.9 lab and 0.56 0.2 lab and 0.18 Tápparo
Covered solid (CSTR) and and 0.356 34–37 CSTR and 7.8 full CLB, 0.46 lab full CLB, 0.61 lab et al.
lagoon liquid (CLB) full CLB; CSTR and 0.38 full CSTR and 0.65 full CSTR (2021)
biodigester phase 1.340 lab (NLbiogas gVSadd
1 (NLbiogas L
1 d
1)
(CLB) lab and 1.618
and full full CSTR
scale
a
Value from biochemical potential assay
b
Effluent pH
c
Best OLR
1 Fundamentals of Biofuel Production Using Anaerobic Digestion: Metabolic. . .
17
18 A. Ferreira Maluf Braga and M. Zaiat

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Chapter 2
Engineering Direct Interspecies Electron
Transfer for Enhanced Methanogenic
Performance

Changsoo Lee

Abstract Producing biogas from organic waste streams through anaerobic diges-
tion (AD) is a well-established bioenergy technology. Efficient electron transfer
between syntrophic bacteria and methanogens is critical for balancing acidogenesis
and methanogenesis, which is necessary for stable digester operation. The recently
discovered direct interspecies electron transfer (DIET) links syntrophic partners via
cell-to-cell electrical connections without using diffusive electron carriers such as
H2. Promoting DIET by adding conductive materials has been suggested as a
possible method to accelerate syntrophic degradation of organic compounds, and
many studies have demonstrated the enhancement of methanogenesis by the addition
of conductive materials. Although further research is needed for practical applica-
tions, accumulated evidence indicates that engineering DIET is a promising strategy
to enhance the performance and stability of AD processes. A few recent studies have
also demonstrated the scale-up potential of DIET-aided AD.

Keywords Anaerobic digestion · Conductive material · Direct interspecies electron

transfer · Electric syntrophy · Electrotrophic methanogens · Exoelectrogenic
bacteria · Methanogenesis

2.1 Introduction

Anaerobic digestion (AD) is a multi-step process involving biochemical reactions

mediated by taxonomically and metabolically very diverse microorganisms, and
their concerted activity is necessary for methanogenic degradation of organic matter
under anaerobic conditions. Various microorganisms with different ecophysiologi-
cal characteristics coexist in AD processes even if a single substrate is utilized

C. Lee (*)
Department of Urban and Environmental Engineering, Ulsan National Institute of Science and
Technology (UNIST), Ulsan, Republic of Korea
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 23

A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_2
24 C. Lee

(Fernandez et al. 2000). The microbial community structure and interspecies inter-
actions will be even more complicated in anaerobic digesters treating complex
organic wastes. Therefore, facilitating and harmonizing the activities of individual
community members is the key to stable and robust AD.
Methanogenesis, the last step of AD, is performed by a unique group of strictly
anaerobic microorganisms belonging to the domain Archaea, known as
methanogens. All methanogens identified so far are classified into one phylum,
Euryarchaeota, while the existence of putative methanogens in uncultured phyla
Bathyarchaeota (Evans et al. 2015) and Verstraetearchaeota (Vanwonterghem et al.
2016) was suggested in recent metagenomic studies. Methanogens use a narrow
range of substrates such as H2/CO2, formate, acetate, methylated compounds and
CO, and acetoclastic and hydrogenotrophic pathways are often the main routes of
methanogenesis in anaerobic digesters (Enzmann et al. 2018). Therefore,
methanogens at the end of the trophic chain of AD need other microorganisms
that hydrolyze and ferment organic macromolecules into substrates for
methanogenesis. Methanogenesis is usually considered the rate-limiting step of the
overall AD process because of the slow growth rate and high sensitivity to environ-
mental conditions of methanogens (Lee et al. 2018). However, hydrolysis can be the
rate-limiting step when digesting organic substances with low bioavailability, such
as waste activated sludge and lignocellulosic biomass. Furthermore, the degradation
of C3 or higher volatile fatty acids (VFAs) to acetate and H2/CO2 (i.e., acetogenesis)
also becomes rate-limiting if the syntrophic association between VFA oxidizers and
methanogens is not well developed (Baek et al. 2018). In this case, an imbalance
between the production and consumption of VFAs (and H2) can occur and result in a
buildup of VFAs (and H2 partial pressure) and even in a digester failure. Therefore,
efficient electron transfer between syntrophic microorganisms involved in anaerobic
VFA degradation is critical in maintaining the balance between acidogenesis and
methanogenesis for stable AD.
The interspecies electron transfer (IET) between VFA oxidizers and methanogens
has been thought to be mediated exclusively by microbially produced H2 or formate
as electron carriers. It was recently revealed that there exists an alternative IET route
where electrons are transferred directly from exoelectrogenic VFA oxidizers to
electrotrophic methanogens through a cell-to-cell electrical connection. This direct
IET (DIET) is energetically and kinetically advantageous over the H2/formate-
mediated indirect IET (IIET), because complex reactions for synthesizing and
consuming H2 or formate are not required (Lovley 2011). This suggests the possi-
bility that thermodynamically more favorable conditions for rapid methanogenic
degradation of organic matter can be achieved by promoting DIET (Barua and Dhar
2017). In support of such a possibility, a mathematical modeling study estimated that
the cell-to-cell electron transfer rate can be more than eight-fold higher for DIET
than for IIET via H2 (Storck et al. 2016).
The potential for DIET in methanogenic systems was experimentally observed
for the first time only a decade ago by Morita et al. (2011) in a study of electron
transfer mechanisms within methanogenic granular sludge treating brewery waste.
In the next year, Kato et al. (2012) reported a significant acceleration of
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 25

methanogenesis by the addition of (semi)conductive materials (i.e., hematite (Fe2O3)

and magnetite (Fe3O4)) in methanogenic soil enrichment cultures, and suggested that
conductive materials promote DIET between electro-syntrophic partners by serving
as conduits for electron flow. Since this work, increasing attention has been paid to
whether the addition of conductive materials effectively promotes DIET, thereby
enhancing methanogenesis. Many studies have been made to explore this interesting
possibility in recent years, using different conductive materials, mostly carbon- or
iron-based (Liu et al. 2012; Chen et al. 2014a, b; Baek et al. 2015, 2016; Zhao et al.
2015; Dang et al. 2016; Lee et al. 2016), and it is now apparent that DIET and
methanogenic activities are enhanced in the presence of conductive materials.
Engineering DIET with conductive additives has recently emerged as a promising
strategy for enhancing the methanogenic performance and stability of AD systems,
and an increasing number of studies are looking into its potential for practical
applications. This chapter reviews the fundamentals of DIET-based electric
syntrophy and recent advances in engineering DIET in AD, and discusses challenges
and directions for future research.

2.2 Interspecies Electron Transfer in Anaerobic Digestion

The anaerobic degradation of C3 or higher VFAs is endergonic under standard

conditions and can only be achieved by syntrophic associations between VFA
oxidizers and methanogens via IET (Table 2.1). Therefore, the IET-based syntrophy
is critical for complete mineralization of organic matter in the AD process. The slow
rate of syntrophic VFA oxidation, particularly of propionate, the accumulation of
which is toxic to methanogens, often causes an imbalance between acidogenesis and
methanogenesis and limits the overall reaction rate in AD (Stams and Plugge 2009).
IET in AD can be divided into IIET and DIET, according to whether or not
extracellular electron carriers (i.e., H2 and formate) are required to mediate the
redox reactions between the syntrophic partners. Electrons released from the
syntrophic oxidation of VFAs are transferred via H2 or formate (electron donor for
CO2 reduction to CH4) to hydrogenotrophic methanogens in IIET, and via direct
electrical connections between cells to electrotrophic methanogens in DIET (Baek
et al. 2018). In this section, the roles, mechanisms, and biochemical characteristics of
IIET and DIET in AD are described.

2.2.1 Indirect Interspecies Electron Transfer

IIET between VFA oxidizers and methanogens via H2 or formate has been consid-
ered the major route for syntrophic electron exchange necessary for methanogenic
degradation of C3 or higher VFAs (Fig. 2.1). This syntrophic relationship is very
sensitive to the accumulation of oxidation products of VFAs (i.e., H2 and formate)
26 C. Lee

Table 2.1 Reactions involved in syntropic oxidation of propionate and butyrate by interspecies
electron transfer via hydrogen or formate (adapted from Baek et al. 2018)
Reaction ΔG0 a (kJ/mol)
Methanogenic degradation of propionate via H2 as electron carrier
4CH3CH2COO
+ 12H2O ! 4CH3COO
+ 4HCO3
+ 4H+ + 12H2 +76.5
3HCO3
+ 3H+ + 12H2 ! 3CH4 + 9H2O
101.7
(overall) 4CH3CH2COO
+ 3H2O ! 4CH3COO
+ HCO3
+ H+ + 3CH4
25.2
Methanogenic degradation of propionate via formate as electron carrier
4CH3CH2COO
+ 8H2O + 8CO2 ! 4CH3COO
+ 12HCOO
+ 4H+ +65.3
12HCOO
+ 12H+ ! 3CH4 + 9CO2 + 6H2O
144.5
(overall) 4CH3CH2COO
+ 8H+ + 2H2O ! 4CH3COO
+ CO2 + 3CH4
79.2
Methanogenic degradation of butyrate via H2 as electron carrier
2CH3CH2CH2COO
+ 4H2O ! 4CH3COO
+ 2H+ + 4H2 +48.3
HCO3
+ H+ + 4H2 ! CH4 + 3H2O
67.8
(overall) 2CH3CH2CH2COO
+ H2O + HCO3
! 4CH3COO
+ H+ + CH4
19.5
Methanogenic degradation of butyrate via formate as electron carrier
2CH3CH2CH2COO
+ 4H2O + 4CO2 ! 4CH3COO
+ 4HCOO
+ 4H+ +38.5
4HCOO
+ 4H+ ! CH4 + 3CO2 + 2H2O
96.3
(overall) 2CH3CH2CH2COO
+ 2H2O + CO2 ! 4CH3COO
+ CH4
57.8
a
ΔG0 , Standard Gibbs free energy change of reaction at pH 7

Fig. 2.1 Indirect interspecies electron transfer (IIET) between syntrophic VFA-oxidizing bacteria
and hydrogenotrophic methanogens via H2 and formate

because of the unfavorable thermodynamics of anaerobic VFA degradation

(Table 2.1). High H2 partial pressure inhibits the regeneration of the cytoplasmic
pool of oxidized coenzymes in acetogenic bacteria (Thiele and Zeikus 1988), and
needs to be kept below approximately 10
4 and 10
3 atm for degradation of
propionate and butyrate, respectively, through IIET in methanogenic environments
(Schmidt and Ahring 1993). Therefore, H2 (and formate) should be rapidly con-
sumed by hydrogenotrophic methanogens for stable AD. When the balanced pro-
duction and degradation of VFAs is disturbed, acidogenic products, such as H2,
formate, and acetate, rapidly accumulate, leading to further inhibition of syntrophic
VFA oxidation through IIET. The vicious cycle of process imbalance and acidifica-
tion can result in significant deterioration or even total failure of the AD process.
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 27

Methanogenesis is a major terminal electron-accepting process in anaerobic

environments, and H2 serves as a primary electron donor for different microbial
reduction reactions occurring in AD processes, including the methanogenic reduc-
tion of CO2 (Stams and Plugge 2009). The key enzyme in the synthesis and
utilization of H2 is hydrogenase, a family of iron-containing metalloenzymes (Fe,
Fe-Fe, and Fe-Ni) that catalyze the reversible oxidation of H2 (Lubitz et al. 2014).
Bacteria containing H2-producing hydrogenase reduce protons through the oxidation
of reduced intracellular redox mediators, such as ferredoxin, NADH, and FADH2
(Stams et al. 2006). The oxidation of reduced ferredoxin or NADH is thermody-
namically feasible when the H2 partial pressure is kept low by H2-consuming
microorganisms, preferably hydrogenotrophic methanogens, while ATP is required
for the oxidation of FADH2 (Schink 1997). Metabolic H2 produced by the oxidation
of VFAs (and other organic matter) is taken up by hydrogenotrophic methanogens
and oxidized by H2-uptake hydrogenase for the reduction of oxidized ferredoxin and
coenzyme F420 that provide electrons to convert CO2 into CH4 (Kim and Geoffrey
2008).
Another important diffusive electron carrier mediating IIET between VFA oxi-
dizers and hydrogenotrophic methanogens is formate. Many syntrophic VFA oxi-
dizers produce both H2 and formate, and most hydrogenotrophic methanogens can
use both of them as electron donors to reduce CO2 to CH4 (de Bok et al. 2004;
Schink et al. 2017). In addition, many H2/formate-metabolizing microorganisms,
such as hydrogenotrophic methanogens and acetogenic bacteria, are able to inter-
convert H2/CO2 and formate (Lemaire et al. 2020; Nielsen et al. 2019). This
complexity makes it difficult to determine whether H2 or formate is a more important
electron carrier for IIET. H2 has a much lower solubility in water (0.8 mM at 1 atm,
20  C) than formate, and therefore the concentration gradient between the producers
and consumers (>1000-fold) can be much larger with formate than with H2 (de Bok
et al. 2004; Felchner-Zwirello et al. 2013). Meanwhile, the diffusion coefficient in
water (25  C) is more than four times higher for H2 (5.11  10
3 mm2/s) than for
formate (0.96  10
3 mm2/s) (Lide 1995; Prüsse et al. 2000). Given that the
diffusive flux of interspecies electron carriers is proportional to their diffusion
coefficients and concentration gradients between the syntrophic partners (Fig. 2.2),
it is likely that H2 and formate are primarily involved in short- and long-distance
IIET, respectively (de Bok et al. 2004).
Thiele and Zeikus (1988) found that formate-mediated IIET was the main IET
mechanism during the syntrophic conversion of ethanol to methane in cultures of
methanogenic flocs from a lab-scale digester treating whey. Boone et al. (1989)
calculated that H2 diffusion alone could not support the syntrophic degradation of
propionate and butyrate in suspended cultures (i.e., IIET between dispersed cells)
and that formate diffusion contributed 98 times more to IET than did H2 diffusion. In
contrast, Goodwin et al. (1991) reported that the H2 diffusion rate was fast enough to
account for the methanogenesis rate in anaerobic microbial aggregates degrading
lactate. Schmidt and Ahring (1995) also suggested that H2 was the dominant electron
carrier in methanogenic granular sludge degrading propionate and butyrate. Since
the diffusion distance between syntrophic partners is mainly determined by cell
28 C. Lee

Fig. 2.2 Diffusive flux of interspecies electron carriers between a producing and a consuming
microorganism (adapted from de Bok et al. 2004). A, the surface area of the electron carrier
producer; D, the diffusion coefficient of the electron carrier; Cp, the concentration of the electron
carrier at the surface of the producer; Cc, the concentration of the electron carrier at the surface of
the consumer; and d, the distance between the producer and consumer

density, it is understandable that IIET via formate may be preferred in planktonic

cultures, whereas H2 diffusion may dominate IIET in methanogenic aggregates or
biofilms. Recent measurements in different anaerobic digesters suggest that the pool
sizes of H2 and formate are energetically nearly equivalent, and both H2- and
formate-mediated IIET likely occur at the same time in a methanogenic system
(Schink et al. 2017). Therefore, the electron transfer efficiency can be expected to
increase with increasing cell density (i.e., reducing intercellular distance). Accord-
ingly, previous studies have reported that the syntrophic degradation rate of VFAs
could be enhanced by forming compact microbial aggregates/granules, reducing the
mixing level, or adding H2/formate-utilizing microorganisms (Schmidt and Ahring
1995; Stroot et al. 2001; Kim et al. 2002; Kaparaju et al. 2008).

2.2.2 Direct Interspecies Electron Transfer

2.2.2.1 Introduction

Recent discoveries revealed that DIET is an important alternative IET route for the
use of electrons derived from the oxidation of acidogenic metabolites, such as VFAs
and ethanol, for the methanogenic reduction of CO2 in AD environments (Morita
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 29

et al. 2011; Kato et al. 2012; Rotaru et al. 2014a, b). In contrast to IIET, DIET
excludes the involvement of electron carriers (i.e., H2 and formate) because electrons
released by exoelectrogenic bacteria flow directly via intercellular electrical connec-
tions to electrotrophic methanogens. Therefore, electrically coupled syntrophic
partners by DIET can avoid complex reactions for the synthesis, diffusion, and
utilization of interspecies electron carriers, which makes DIET energetically and
kinetically advantageous over IIET (Lovley 2011). An imbalance between the
production and consumption of VFAs often occurs in AD processes due to the
large difference in growth rate between acidogens and methanogens, especially
under high organic loading conditions. In such cases, VFAs (and H2 partial pressure)
build up to levels that cannot be effectively stabilized by IIET-driven
hydrogenotrophic methanogenesis, resulting in severe inhibition of methanogenic
activity. DIET-driven electrotrophic methanogenesis can accelerate the stabilization
of VFA accumulation and help to maintain a stable methanogenic system because of
the higher electron transfer efficiency and smaller intermediate loss in DIET com-
pared to IIET (Kato et al. 2012; Leng et al. 2018). For example, the Gibbs free
energy change at pH 7 (ΔG0 ) of the propionate oxidation through DIET (Eq. 2.1) is
significantly lower than that of the propionate oxidation through IIET via H2
(Eq. 2.2), with the former being negative (i.e., spontaneous reaction) and the latter
being positive (i.e., non-spontaneous reaction) (Jing et al. 2017).
The concentration gradient of electron carriers between syntrophic partners is the
main limiting factor for IIET, while that for DIET is considered to be the activation
loss (or overpotential) during the flow of electrons through cellular components
comprising cell-to-cell electrical connections (Storck et al. 2016). Activation loss is
the energy lost to overcome the energy barrier and initiate electron transfer, for
example, between terminal membrane-bound redox proteins and e-pili in DIET. It
takes place as a result of slow charge transfer in an electrochemical redox reaction,
and a certain portion of the electron energy is consumed to drive the reaction at a
certain rate (Petrovic 2021).

C2 H5 COO
þ 0:75H2 O ! CH3 COO
þ 0:25HCO
þ

3 þ 0:25H þ 0:75CH4

0 

ΔG ¼
26:4 kJ=mol 37 C, pH 7
ð2:1Þ
C2 H5 COO
þ 3H2 O ! CH3 COO
þ HCO
þ
3 þ H þ 3H2
 
ð2:2Þ
ΔG 0 ¼ 72:7 kJ=mol 37 C, pH 7

The possibility of DIET between exoelectrogenic bacteria and electrotrophic

methanogens was first raised by Stams et al. (2006) based on the observation that
acetogens and methanogens form direct physical contact in some methanogenic
environments. Experimental evidence for the electro-syntrophic methanogenesis
through DIET was first presented in the early 2010s by Morita et al. (2011) with
anaerobic granules treating brewery waste, and subsequently by many others in
different methanogenic environments (Kato et al. 2012; Liu et al. 2012; Chen et al.
30 C. Lee

2014b; Rotaru et al. 2014a, b; Shrestha et al. 2014). Given the compact and dense
structure of granular sludge, DIET can explain the efficient degradation of propio-
nate by anaerobic granules even when the Gibbs free energy change calculated from
the dissolved H2 available for the granules was positive (Dubé and Guiot 2015).
DIET mechanisms can be classified into two types: biological DIET (bDIET) and
mineral DIET (mDIET). The former involves direct cell-to-cell contact via biotic
components, whereas abiotic conductive materials support electrical connections
between cells in the latter (Shrestha and Rotaru 2014). Electrons are transferred via
electron transport proteins, such as outer surface c-type cytochromes, or electrically
conductive pili (e-pili), also known as nanowires, in bDIET (Fig. 2.3). Electric
syntrophy through bDIET was first described by Summers et al. (2010) in defined
co-cultures of Geobacter metallireducens and Geobacter sulfurreducens with etha-
nol and fumarate as the sole electron donor and acceptor, respectively. G.
metallireducens, which can oxidize ethanol to obtain energy for growth but cannot
use fumarate as an electron acceptor, and G. sulfurreducens, which cannot use
ethanol as an electron donor but can reduce fumarate as an electron acceptor,
developed dense electrically conductive aggregates and syntrophically metabolize
ethanol with the reduction of fumarate in the co-cultures, even when an hyb-deleted
mutant strain of G. sulfurreducens, unable to utilize H2 or formate, was used.
However, deletion of the genes encoding OmcS (a multi-heme c-type cytochrome)
or PilA (the structural protein for e-pili) prevented the syntrophic metabolism
between the Geobacter strains and their growth. Studies with different mutant strains
and transcriptome assays proved the development of DIET via e-pili networks
associated with c-type cytochromes in the Geobacter co-cultures (Summers et al.
2010; Rotaru et al. 2012; Shrestha et al. 2013; Vargas et al. 2013).

2.2.2.2 Biological Direct Interspecies Electron Transfer (bDIET)

The first example of bDIET involving methanogens was demonstrated in co-cultures

of G. metallireducens and Methanothrix (formerly Methanoseata) harundinacea,
which were isolated from an anaerobic digester treating brewery waste with ethanol
as the sole substrate for growth (Rotaru et al. 2014b). The co-cultures converted one
mole of ethanol to 1.5 moles of methane, indicating that M. harundinacea produced
methane not only by splitting acetate derived from the oxidation of ethanol by G.
metallireducens, but also by using other electron donors produced during the ethanol
oxidation. M. harundinacea, an acetoclastic methanogen that cannot use H2 or
formate as electron donor for methanogenesis, expressed genes for the reduction
of CO2 to CH4 at high levels. Radiotracer experiments using 14C-bicarbonate
showed that one-third of the methane production was derived from CO2, according
to the stoichiometric reactions involving electrotrophic methanogenesis (Eqs. 2.3–
2.6). Additionally, the PilA-deficient mutant strain of G. metallireducens failed to
develop syntrophic IET with M. harundinacea and to metabolize ethanol to methane
in the co-culture. The multiple lines of evidence proved that the two species
were capable of exchanging electrons through DIET for the electrotrophic
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 31

Fig. 2.3 Direct interspecies electron transfer between syntrophic VFA-oxidizing bacteria and
hydrogenotrophic methanogens via biotic compounds (A) and abiotic conductive material (B)

methanogenesis of CO2 and that e-pili played an important role in building their
electrical connections. Although the genes for CO2 reduction were previously found
in Methanothrix species (Zhu et al. 2012), the electrotrophic conversion of CO2 to
CH4 by Methanothrix was first demonstrated by Rotaru et al. (2014b). This was a
32 C. Lee

surprising finding because Methanothrix species were by then known to be strictly

acetoclastic and unable to reduce CO2 (Smith and Ingram-Smith 2007).

Ethanol oxidation : CH3 CH2 OH þ H2 O ! CH3 COOH þ 4Hþ þ 4e
ð2:3Þ

Acetoclastic methanogenesis : CH3 COOH ! CH4 þ CO2 ð2:4Þ
Electrotrophic methanogenesis : 0:5CO2 þ 4e
þ 4Hþ ! 0:5CH4 þ H2 O ð2:5Þ
Overall reaction : CH3 CH2 OH ! 1:5CH4 þ 0:5CO2 ð2:6Þ

A further study by Rotaru et al. (2014a) demonstrated that another acetoclastic

methanogen, Methanosarcina barkeri, which is metabolically much more diverse
than Methanothrix species (Boone and Castenholz 2001), could exchange electrons
with G. metallireducens through DIET via e-pili networks in a similar way, and
syntrophically convert ethanol to methane in defined co-cultures. Several other
Methanosarcina and Methanothrix species/strains were additionally shown to
be able to accept electrons directly from exoelectrogenic syntrophic partners, and
it has been assumed until very recently that only the members of the order
Methanosarcinales, which have multiheme c-type cytochromes, are capable of
bDIET among all methanogens (Yee et al. 2019; Yee and Rotaru 2020). However,
although the direct electron uptake mechanisms of these methanogens are yet to be
fully understood, it was revealed that multiheme c-type cytochromes are not neces-
sary in extracellular electron uptake via DIET by Methanosarcinales (Yee
and Rotaru 2020). Furthermore, the first discovery of DIET involving
Methanobacterium was very recently reported in defined co-cultures of
Methanobacterium sp. strain YSL and G. metallireducens fed with ethanol
(Zheng et al. 2020). This finding contrasts with previous studies that showed the
inability of strictly hydrogenotrophic methanogens, Methanospirillum hungatei,
Methanoculleus marisnigri (both belonging to the order Methanomicrobiales), and
Methanobacterium formicicum (belonging to the order Methanobacteriales) to grow
via DIET in syntrophic associations with exoelectrogenic Geobacter species (Rotaru
et al. 2014b; Yee et al. 2019; Yee and Rotaru 2020). The results of the iterative
defined co-culture studies raise the possibility that much more diverse methanogens
are capable of participating in bDIET, which certainly deserves further investigation
to better understand (electro-)syntrophic interactions in methanogenic communities.
A recent study thermodynamically analyzed the effects of carbon sources and
conductive components electrically connecting cells (biotic or abiotic) on DIET in
methanogenesis and reported that ethanol may be the most favorable organic carbon
source for DIET (Gu et al. 2019).
The bDIET capacity of methanogens has so far only been confirmed in defined
co-cultures with exoelectrogenic Geobacter species using ethanol as the electron
donor. However, Yee and Rotaru (2020) have recently reported that Rhodoferax
ferrireducens showed significantly higher electron recovery as methane from glu-
cose in co-cultures with electrotrophic methanogens than with strictly
hydrogenotrophic methanogens. The use of ethanol that favors the enrichment of
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 33

Geobacter species to develop e-pili networks was likely an important factor that
facilitated the electro-syntrophic interactions in the DIET co-cultures (Martins et al.
2018). This corresponds to the fact that the existence of bDIET involved in
methanogenic systems was first observed in studies of conductive methanogenic
aggregates derived from brewery waste digesters (Morita et al. 2011; Rotaru et al.
2014b; Shrestha et al. 2014). Many (putative) exoelectrogenic bacteria other than
Geobacter species have been suggested (not yet proven) in recent studies to partic-
ipate in DIET with (putative) electrotrophic methanogens (Barua and Dhar 2017;
Park et al. 2018a). Therefore, in order to gain a comprehensive understanding of
DIET mechanisms and the involved microorganisms in diverse methanogenic envi-
ronments, more studies are needed, for example, in defined co-cultures or complex
mixed cultures with different electron donors other than ethanol.

2.2.2.3 Mineral Direct Interspecies Electron Transfer (mDIET)

The potential of mDIET in methanogenic environments was first documented by

Kato et al. (2012) in enrichment cultures from rice paddy soil. The methane
production rates from both acetate and ethanol increased significantly with the
addition of (semi)conductive iron oxides, magnetite and hematite (20 mM Fe), in
the enrichment cultures. Dissolution of the iron oxides and the use of iron ions as a
nutrient or electron shuttle were unlikely to have contributed to the enhancement of
methanogenesis, because the ferrous ion concentration was negligible in the cultures.
Adding ferrihydrite, an insulative iron oxide, did not accelerate, and even
suppressed, methanogenesis. The accelerated methanogenesis was attributed to the
stimulation of DIET between Geobacter and Methanosarcina, based on their pre-
dominance in the cultures to which (semi)conductive iron oxides had been added,
where the growth of Geobacter was suppressed by the addition of 2-bromoethane
sulfonate (BES), a methanogenic inhibitor.
Soon after, Liu et al. (2012) demonstrated in defined co-cultures with ethanol as
the sole electron donor that granular activated carbon (GAC) could promote DIET
between G. metallireducens and M. barkeri and accelerate the syntrophic conversion
of ethanol to methane. The microorganisms were tightly attached to the surface of
GAC but did not form direct physical contact as required for bDIET. The DIET-
promoting effect of GAC was even more pronounced in the cultures of
methanogenic aggregates from a digester treating brewery waste, which was previ-
ously reported to be DIET-active (Morita et al. 2011), with the methane production
rate from ethanol being 2.5-fold higher with GAC than without. Later, carbon cloth
(Chen et al. 2014a) and biochar (Chen et al. 2014b) were also demonstrated in
co-cultures of G. metallireducens and M. barkeri to stimulate the syntrophic metab-
olism of ethanol through DIET. Conductive materials serve as an electrical conduit
for DIET by connecting syntrophic partners, which aggregate to form cell-
conductive material complexes (Lovley 2017; Baek et al. 2019). The physical
structure of such complexes varies with the particle size of the abiotic conductive
materials involved. Microorganisms attach and form biofilms on the surface of
34 C. Lee

Table 2.2 Electrical conductivity of abiotic conductive materials used for promoting DIET
Conductive materials Conductivity (mS/cm) Reference
Granular activated carbon 3–1200 Liu et al. (2012), Cheng et al. (2018)
Carbon cloth 4350 Lei et al. (2016)
Biochar 0.002–220 Chen et al. (2014b), Cheng et al. (2018)
Magnetite 105–106 Cornell and Schwertmann (2003)
Graphene 8.5 3 105 Lin et al. (2018)
Carbon nanotube >105 Yan et al. (2019)
Stainless steel 0.667 Li et al. (2017)
Polyaniline nanorod ~740 Hu et al. (2017)
G. Sulfurreducens e-pilia 51 Adhikari et al. (2016)
a
Pili from wild-type cells were measured in a hydrated state at pH 7

conductive materials that are large enough to serve as supporting media for attached
growth, while submicron/nanoparticles agglomerate together with microorganisms
to form aggregates such as flocs or granules (Baek et al. 2019).
Conductivity is the key characteristic of abiotic additives for promoting mDIET,
bypassing the need for electrical contacts via biological components necessary for
bDIET (Shrestha and Rotaru 2014). In support, magnetite nanoparticles were able to
compensate for the absence of OmcS, which, together with e-pili, forms electrical
conduits for DIET, in co-cultures of a mutant G. sulfurreducens strain deficient in
OmcS and G. metallireducens with ethanol as the electron donor (Liu et al. 2015). In
contrast to magnetite, which could only marginally compensate for the lack of PilA
(Liu et al. 2015), GAC was able to promote mDIET between the two Geobacter
species in co-cultures with ethanol, and to effectively compensate for the lack of both
PilA and OmcS (Liu et al. 2012). The findings of these studies, although not
performed with methanogenic cultures, confirm that abiotic conductive materials
can electrically connect DIET partners and stimulate their electric syntrophy, inde-
pendent of bDIET, and that the DIET mechanisms may differ among different
conductive additives with different characteristics, such as conductivity and size
(Baek et al. 2019). Biochar (Chen et al. 2014b) was shown to be comparably
effective to GAC (Liu et al. 2012) or carbon cloth (Chen et al. 2014a) in promoting
DIET, although the conductivity of biochar is approximately 1000-fold lower than
that of the others (Table 2.2). Furthermore, semiconductive hematite and conductive
magnetite showed similar methanogenesis-accelerating effects in DIET-stimulated
soil enrichment cultures with acetate or ethanol (Kato et al. 2012). By contrast, Baek
et al. (2015) and Zhuang et al. (2015) reported that the addition of iron oxides with
different conductivities (i.e., goethite, hematite, and magnetite) promoted DIET to
different extents in methanogenic cultures degrading whey and benzoate, respec-
tively. These results indicate that the DIET-promoting effect of a conductive additive
is not determined solely by its conductivity and will thus not continuously increase
in proportion to its electrical conductivity (Barua and Dhar 2017).
Following the studies in defined co-cultures, mostly pairing methanogens with
Geobacter species, which confirmed the existence of DIET in anaerobic
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 35

environments, many studies have investigated DIET in more environmentally rele-

vant, mixed cultures. In those studies, assessing whether the addition of conductive
materials could promote DIET and enhance methanogenesis was of primary interest.
Different conductive materials (mostly carbon- or iron-based), such as GAC (Lee
et al. 2016; Zhao et al. 2016b; Dang et al. 2017; Yan et al. 2017; Yang et al. 2017;
Capson-Tojo et al. 2018; Park et al. 2018b; Chowdhury et al. 2019; Xu et al. 2020;
Zhang et al. 2020a), biochar (Luo et al. 2015; Li et al. 2018; Wang et al. 2019b,
2020b; Lü et al. 2020), carbon cloth (Dang et al. 2017; Zhao et al. 2017c), carbon
nanotubes (Li et al. 2015b; Zhang and Lu 2016; Yan et al. 2017), graphene (Lin et al.
2017; Tian et al. 2017), magnetite (Cruz Viggi et al. 2014; Baek et al. 2015; Li et al.
2015a; Yamada et al. 2015; Zhuang et al. 2015; Yang et al. 2016; Zhang and Lu
2016; Jing et al. 2017; Yin et al. 2017b; Wang et al. 2018c, 2020a; Zhang et al.
2019a), hematite (Zhuang et al. 2015), and stainless steel (Li et al. 2017) have been
tested for their DIET-promoting potential in batch methanogenic cultures with
different inocula and substrates. The key observations and culture conditions, such
as conductive material dose, inoculum source, and carbon/electron source, of the
previous studies are described in detail in recent reviews (Barua and Dhar 2017;
Baek et al. 2018; Park et al. 2018a; Gahlot et al. 2020; Zhao et al. 2020a).
Enhancement of methanogenesis by promoting mDIET in mixed-culture studies
has been demonstrated on various substrates (or electron donors) besides ethanol, for
example, VFAs, glucose, sucrose, benzoate, phenol, whey, waste activated sludge,
livestock manure, food waste, and waste cooking oil. Many studies have examined
the effect of promoting mDIET on the methanogenesis of VFAs, the main interme-
diates of AD, and the methanogenic conversion of C3 or higher VFAs was found to
be significantly higher in the presence of conductive materials (Cruz Viggi et al.
2014; Li et al. 2015a, 2017; Yamada et al. 2015; Lee et al. 2016; Yang et al. 2016;
Zhang and Lu 2016; Zhao et al. 2016b; Jing et al. 2017). Cruz Viggi et al. (2014) first
demonstrated the acceleration of methanogenesis from propionate (up to a 33%
increase in the degradation rate) by the addition of submicron magnetite particles in
methanogenic cultures derived from a sewage sludge digester. Li et al. (2015a)
reported the enhanced methanogenesis from butyrate by supplementation with
magnetite nanoparticles in paddy soil enrichments. Yang et al. (2016) showed in a
similar study using an equimolar mixture of C2–C6 VFAs that the methanogenic
degradation rates of the VFAs, particularly higher VFAs, were significantly
increased by the addition of magnetite nanoparticles. Furthermore, Lee et al.
(2019) demonstrated that the methanogenic conversion rates of C2–C4 VFAs could
be significantly increased by the addition of magnetite even under high ammonia
stress (6.5 g total ammonia N/L). The results of these and other studies suggest that
both methanogenic performance and process stability of anaerobic digesters can be
improved by promoting DIET with conductive additives. In support, changes in
microbial community structure and metabolic properties, accompanied by the DIET-
facilitated degradation of VFAs, have been identified by different meta-omic
approaches (Li et al. 2015a; Jing et al. 2017; Lee et al. 2019). Besides the accelerated
degradation of VFAs, metabolic shifts towards more acetic-type fermentation, which
is favorable for methanogenesis in the presence of (semi)conductive iron oxides,
36 C. Lee

were noted in methanogenic reactors treating whey (Baek et al. 2015). The accu-
mulated evidence suggests that promoting mDIET by adding conductive materials
can provide a simple and effective means for enhancing methanogenic performance
and maintaining stable operation of AD processes.

2.3 Engineering Direct Interspecies Electron Transfer

with Conductive Additives

Since the observation by Kato et al. (2012) that methanogenesis was accelerated by
adding (semi)conductive iron oxides in anaerobic mixed cultures with acetate or
ethanol, the potential of promoting mDIET as a means to improve the performance
of anaerobic digesters has gained much attention. As described in the preceding
section, many subsequent studies confirmed the widespread occurrence of DIET-
active methanogenic consortia in anaerobic environments and demonstrated the
DIET-promoting and methanogenesis-boosting effects of various conductive mate-
rials in defined co-cultures and mixed cultures. Adding inexpensive conductive
materials is currently considered a promising approach to enhance methanogenic
performance at a more fundamental level, and significant research efforts have
recently been made to investigate this possibility under more realistic conditions
for practical applications. This section describes the recent advances in engineering
DIET with conductive additives for enhanced methanogenesis in (semi-)continuous
anaerobic digesters treating complex organic wastes, including the latest studies at
pilot scale. Particular attention is given to the use of different (mostly carbon- or
iron-based) conductive additives and their effects on the digester performance.

2.3.1 Carbon-Based Materials

2.3.1.1 Granular Activated Carbon (GAC)

Carbon-based conductive additives, such as activated carbon (both granular and

powdered), anthracite, biochar, carbon cloth/felt/fiber, carbon nanotube, graphene,
and graphite, have been extensively used to promote mDIET in different types of
(semi-)continuous anaerobic digesters (Table 2.3). GAC, which is commonly used
as a support medium for biofilm development in attached-growth bioreactors, is
among the most used additives to promote mDIET because of its high surface area
and electrical conductivity (Table 2.2). Lee et al. (2016) reported in a study using
continuously stirred tank reactors (CSTRs) fed with synthetic wastewater containing
acetate that the methane production rate was 1.8-fold higher in the digester
supplemented with GAC than in the control digester without GAC. Zhao et al.
(2017b) operated four two-phase AD systems (i.e., combinations of acidogenic
reactors with or without magnetite, and methanogenic reactors with or without
Table 2.3 Studies on engineering DIET in (semi-)continuous anaerobic digesters with conductive additives
Additivea Doseb Inoculumc Substrated Reactor typee Tempf ( C) Reference
GAC 10 g/L ADS SWW (Hac) CSTR 35 Lee et al. (2016)
40 g/L ADS SWW (glucose + milk powder) Two-phase SBR 37 Zhao et al. (2017b)
25 g/L ADS SWW (glucose) UASB 20.0  0.5 Zhang et al. (2020c)
15 g/L ADS SWW (glucose) UASB 37 Guo et al. (2020a)
5 g/L SSS SWW (ethanol + glucose) UASB 35  2 Xu et al. (2015)
100% (v/v) ADS SWW (fructose + polyethylene glycol) UPBR 35 Mei et al. (2018)
100 g/L ADS Commercial dog food SBR 37 Dang et al. (2016)
75 g/L ADS MSW incineration plant leachate UASB 33  1 Lei et al. (2019)
10 g/L ADS HTLWW from sewage sludge SBR 37 Usman et al. (2019)
80 g/L ADS HTLWW from swine manure UPBR 37 Yang et al. (2020b)
25 g/L ADS Municipal primary effluent UASB 16.5  2.0 Zhang et al. (2020b)
PAC 5 g/L SSS SWW (EtOH + glucose) UASB 35  2 Xu et al. (2015)
5–35 g/L ADS Food waste CSTR 35 Zhang et al. (2017)
7.5–30 g/L ADS Food waste CSTR 37 Zhang et al. (2018)
Biochar 2.5 g/L ADS SWW (EtOH) UASB 37 Zhao et al. (2015)
5 g/L ADS SWW (EtOH + Hpr) UASB 37 Zhao et al. (2016a)
5 g/L ADS SWW (EtOH + Hbu) UASB 37 Zhao et al. (2016a)
4 g/L – SWW (sucrose) UASB 35  2 Wang et al. (2018a)
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . .

4 g/L – SWW (sucrose) UASB 35  2 Wang et al. (2021a)

5–10 g/L ADS Food waste Two-phase CSTR 35/55g Lim et al. (2020)
Carbon cloth 200 cm2/L ADS SWW (EtOH) UASB 37 Zhao et al. (2015)
500 cm2/L ADS SWW (butanol) SBR 37 Zhao et al. (2017c)
667 cm2/L ADS SWW (glucose + glycine) Two-phase CSTR 35  1 Feng et al. (2020)
977 cm2/L ADS Commercial dog food SBR 37 Dang et al. (2016)
(continued)
37
Table 2.3 (continued)
38

Additivea Doseb Inoculumc Substrated Reactor typee Tempf ( C) Reference

833 cm2/L ADS MSW incineration plant leachate UASB 33  1 Lei et al. (2016)
Graphite 200 cm2/L ADS SWW (EtOH) UASB 37 Zhao et al. (2015)
106 cm2/L ADS SWW (EtOH + Hpr) SBR 37 Zhang et al. (2019b)
106 cm2/L ADS SWW (EtOH + Hbu) SBR 37 Zhang et al. (2019b)
60% (v/v) MECE SWW (glucose) UPBR 25  3 Guo et al. (2018)
1000 cm2/L ADS Commercial dog food SBR 37 Dang et al. (2016)
Carbon felt 30 g/L ADS SWW (glucose) Upflow SBR 35  3 Xu et al. (2016)
977 cm2/L ADS Commercial dog food SBR 37 Dang et al. (2016)
Anthracite 100% (v/v) ADS SWW (fructose + polyethylene glycol) UPBR 35 Mei et al. (2018)
Carbon fiber 1.583 m2/L ADS SWW (Hpr + Hbu) Fed-batch 37  1 Barua et al. (2018)
CNT 1.5 g/L ADS Beet sugar wastewater EGSB 36  1 Ambuchi et al. (2017)
Graphene 30–120 mg/L ADS SWW (glucose) UASB 10–20 Tian et al. (2017)
Magnetite 100 g/L ADS High-salinity SWW (Hac) UASB 37 Chen et al. (2020)
10 g Fe/L – SWW (Hac) UASB 35  2 Wang et al. (2020a)
10 g Fe/L – SWW (Hpr + Hbu) UASB 35  2 Wang et al. (2020a)
10 g Fe/L – SWW (sucrose) UASB 35  2 Wang et al. (2020a)
20 mM Fe ADS Sulfate-rich SWW (glucose) UASB 37  1 Jin et al. (2019)
10 g/L ADS Sulfate-rich SWW (EtOH + Hac + Hpr) SBR 35  1 Liu et al. (2019)
10 g Fe/L – SWW (sucrose) UASB 35  2 Wang et al. (2019a)
10 g/L ADS SWW (Hac + starch + tryptone) SBR 35 Yin et al. (2017b)
10 g/L ADS SWW (tryptone) SBR 35  1 Yin et al. (2017a)
10 g/L SSS SWW (tryptone) SBR 35  1 Yin et al. (2018)
20 mM Fe ADS Whey CSTR 35  2 Baek et al. (2016)
20 mM Fe ADS Whey CSTR 35 Baek et al. (2017)
10 g/L ADS MSW incineration plant leachate UASB 35  1 Lei et al. (2018)
C. Lee
 0.1–0.3 g/L ADS Fischer-Tropsch wastewater SBR 37 Wang et al. (2018b)
2–20 mM Fe ADS Ulva/whey mixture CSTR 35  2 Jung et al. (2020)
IONP 0.75 g/L ADS Beet sugar wastewater EGSB 36  1 Ambuchi et al. (2017)
a
GAC, granular activated carbon; PAC, powdered activated carbon; CNT, carbon nanotube; IONP, iron oxide (Fe2O3) nanoparticle
b
Concentration (mass, molar, or volumetric) or geometric surface aera per reactor working volume
c
ADS, anaerobic digester sludge; SSS, settled secondary sludge; MECE, microbial electrolysis cell effluent
d
SWW, synthetic wastewater (main carbon sources); MSW, municipal solid waste; HTLWW, hydrothermal liquefaction wastewater; Hac, acetate; Hpr, propionate;
Hbu, butyrate; EtOH, ethanol
e
CSTR, continuously stirred tank reactor; SBR, sequencing batch reactor; UASB, upflow anaerobic sludge blanket; UPBR, upflow packed bed reactor; EGSB,
expanded granular sludge bed
f
Temp, reactor operation temperature
g
Temperature-phased (acidogenic reactor/methanogenic reactor)
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . .
39
40 C. Lee

GAC) with synthetic dairy wastewater in sequencing batch mode. They found that
GAC could accelerate the syntrophic methanogenesis of simple organics, such as
ethanol and VFAs, when efficient acidogenesis was achieved in the presence of
magnetite.
Zhang et al. (2020c) and Guo et al. (2020a) reported significant increases in
chemical oxygen demand (COD) removal and methane production in upflow anaer-
obic sludge blanket (UASB) reactors treating low-strength synthetic wastewater
containing glucose (500 mg COD/L) at psychrophilic and mesophilic temperatures,
respectively. Xu et al. (2015) compared the effects of GAC (0.84–2.00 mm) and
powdered activated carbon (PAC; 75–177 μm) on the anaerobic treatment of
synthetic brewery wastewater in UASB reactors. Both GAC and PAC enhanced
the methanogenic degradation of ethanol and VFAs and facilitated the enrichment of
methanogens, while PAC with more abundant micro-mesoporous structures for the
colonization of DIET partners induced a greater increase in methane production,
particularly at higher organic loading rates (OLRs). Mei et al. (2018) reported, in a
study of the DIET-stimulated methanogenic degradation of synthetic soft drink
processing wastewater using upflow packed bed reactors (UPBRs), that the daily
methane production relative to the biomass concentration was 43.3% and 31.5%
greater in the digesters filled with GAC and anthracite, respectively, than in the
control digester with non-conductive ceramic media. They suggested a syntrophic
association between a novel Geobacter population and Methanothrix in the DIET-
stimulated digesters based on the metagenomic analysis results.
Additionally, several studies have reported the promotion of mDIET with acti-
vated carbon in (semi-)continuous digesters treating real waste/wastewater of dif-
ferent characteristics. Dang et al. (2016) compared four carbon-based conductive
materials (GAC, carbon cloth, carbon felt, and graphite rod; with similar geometric
surface areas of ~1000 cm2/L) for their effects on the performance of anaerobic
sequencing batch reactors (SBRs) treating commercial dog food (as a proxy for food
waste). The digesters supplemented with GAC, carbon cloth, and carbon felt showed
better and more stable AD performance than the control digesters with polyester
cloth or no material added, particularly at higher OLRs, whereas adding graphite
rods was ineffective in promoting or stabilizing methanogenesis.
Usman et al. (2019) and Yang et al. (2020b) studied the effect of adding GAC in
anaerobic digesters treating hydrothermal liquefaction wastewaters from sewage
sludge (SBR) and swine manure (UPBR), respectively. Both studies claimed that
GAC enhanced the methanogenic performance of the digesters by promoting DIET,
although the adsorption of inhibitors by GAC also must have contributed to the
improved methane yield. Lei et al. (2019) reported that GAC dosing promoted DIET
involving electroactive Geobacter and Methanosarcina and enhanced the
methanogenic performance in UASB reactors fed with municipal solid waste incin-
eration leachate. The leachate was effectively treated without a lag period in the
GAC-added digester at increasing OLRs from 5.9 to 36.7 g COD/Ld, while the
control digester without GAC failed within the first 17 days of operation at the
lowest OLR of 5.9 g COD/Ld. Zhang et al. (2020b) compared low-temperature
(16.5  2.0  C) UASB reactors with and without GAC addition for the
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 41

methanogenic treatment of municipal sewage (primary-treated effluent). Potential

electro-syntrophic partners including Geobacter were enriched by the addition of
GAC, and accordingly the GAC-amended digester showed significantly higher COD
removal and methane production.
Zhang et al. (2017) investigated the effects of different doses of PAC (5–35 g/L)
in anaerobic CSTRs treating food waste. PAC addition facilitated the colonization of
microorganisms, particularly methanogens, and accelerated the degradation of
lipids, thereby enhancing the conversion of food waste to methane, with the optimal
PAC dose to promote methanogenesis being 15 g/L. Zhang et al. (2018) conducted a
similar study of food waste AD in lab-scale CSTRs, with a similar PAC dose range
(7.5–30 g/L), and then operated a pilot-scale CSTR (working volume, 700 L)
supplemented with the optimal amount of PAC as determined from lab-scale
experiments (15 g/L). Enhancements of methane production and operation stability
were achieved by the addition of PAC in the lab- and pilot-scale experiments, with
the methane yield of the pilot-scale digester being 41% higher with PAC supple-
mentation than without.

2.3.1.2 Biochar

Biochar, particularly when produced from waste biomass, is a low-cost alternative to

GAC. Although much less conductive than other carbon-based materials (Table 2.2),
biochar is highly porous and rich in electroactive surface functional groups such as
quinones (Sun et al. 2017), which is beneficial for enriching electroactive microor-
ganisms and promoting DIET (Chen et al. 2014b; Zhao et al. 2016a). Several studies
have demonstrated the enhancement of methane production, mostly from synthetic
wastewater, by the addition of biochar in (semi-)continuous experiments (Table 2.3).
Zhao et al. (2016a) reported, in a study using UASB reactors fed with ethanol/
propionate or ethanol/butyrate mixtures, that biochar effectively enriched
electroactive Geobacter and Methanothrix species and promoted the DIET-based
syntrophic degradation of propionate or butyrate. Similar DIET-promoting effects,
along with the enrichment of potential electro-syntrophic partners, were also
observed with the addition of biochar in UASB reactors treating synthetic wastewa-
ter containing sucrose (Wang et al. 2018a, 2021a).
Zhao et al. (2015) compared biochar, carbon cloth, and graphite rod for their
effectiveness in enhancing the AD of synthetic wastewater containing ethanol in
UASB reactors. The three conductive additives significantly enhanced the
syntrophic conversion of ethanol to methane, resulting in greater organic removal
and methane production compared to those of the control digester without conduc-
tive material. The differences became more pronounced with increasing OLR
(4.11–12.33 g COD/Ld), and the enhancement of methanogenic performance
disappeared immediately when the conductive additives were removed from the
digesters. Lim et al. (2020) examined the effect of biochar at different doses (5 and
10 g/L) on the food waste AD in temperature-phased two-phase CSTRs (i.e.,
mesophilic acidogenesis and thermophilic methanogenesis). The biochar-
42 C. Lee

supplemented digesters showed comparable methane yields, which were signifi-

cantly higher than those of the control digester without biochar, and the 5 g/L dose
of biochar (representing an 18% increase in methane yield) was found to be optimal
for the two-phase AD system. Biochar addition enriched methanogens (i.e., increase
in the archaea-to-bacteria ratio) and electroactive bacteria, thereby enhancing the
degradation of VFAs by promoting both DIET and acetoclastic methanogenesis.

2.3.1.3 Carbon Cloth, Felt and Fiber

Carbon cloth, felt, and fiber with high electrical conductivity have also been dem-
onstrated to promote syntrophic metabolism and accelerate methanogenesis in
several (semi-)continuous digesters fed with synthetic wastewaters with different
organic compounds as the main electron donor, such as ethanol (Zhao et al. 2015),
butanol (Zhao et al. 2017c), glucose (Xu et al. 2016), a propionate/butyrate mixture
(Barua et al. 2018), and a glucose/glycine mixture (Feng et al. 2020). As mentioned
above, carbon cloth and felt were tested, together with other conductive additives, in
a study using SBRs treating commercial dog food, and their effectiveness in
promoting DIET and improving digester performance and stability was demon-
strated (Dang et al. 2016). Lei et al. (2016) reported in a study using UASB reactors
treating municipal solid waste incineration leachate, that the carbon cloth-added
digester maintained stable performance with high organic removal at OLRs up to
49.4 g COD/Ld, where the control digester without carbon cloth failed with an
accumulation of VFAs. They found that the addition of carbon cloth enriched
potential exoelectrogenic bacteria and electrotrophic methanogens and increased
sludge conductivity by approximately two-fold, indicating the development of a
DIET-active methanogenic community.

2.3.1.4 Carbo Nanotubes

Ambuchi et al. (2017) assessed the effects of adding multi-well carbon nanotubes or
iron oxide (Fe2O3) nanoparticles in expanded granular sludge bed reactors treating
beet sugar wastewater. The digester supplemented with Fe2O3 nanoparticles (0.75-g/
L dose) produced more methane than the one with carbon nanotubes (1.5-g/L dose),
while both digesters showed significantly higher methane production than the
control without conductive additive, although the cumulative methane content in
biogas was as low as 32–33% in all three digesters. Both carbon nanotubes and
Fe2O3 nanoparticles were suggested to provide electrical connections between DIET
partners and accelerate methane production. However, the former promoted the
enrichment of bacteria rather than methanogens, whereas the latter showed the
opposite tendency. Notably, carbon nanotubes have been reported to enhance
methanogenesis in pure cultures of methanogens, independently of DIET (Salvador
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 43

et al. 2017). This effect was attributable to the reduction of the oxidation-reduction
potential (only in the presence of reducing agents) and provision of large
electroactive surface area for microbial adhesion, both of which are beneficial for
the growth of methanogens, by the addition of carbon nanotubes (Martins et al.
2018). The market price of carbon nanotubes is US$100–150/kg (Fan et al. 2020),
which is 10–100 times more expensive than activated carbon (Yunus et al. 2020).
Hence, its continuous addition to digesters, despite its demonstrated effectiveness, is
not economically feasible.

2.3.1.5 Graphite

Graphite is a crystalline form of carbon that is highly conductive and commonly

used as electrode material. Promotion of DIET and enhancement of methanogenesis
by the addition of graphite in the form of felt, granules, and rods have been
demonstrated in SBRs, UPBRs, and UASB reactors fed with synthetic wastewaters
containing ethanol, VFAs, and/or glucose as the main electron donors (Zhao et al.
2015; Guo et al. 2018; Zhang et al. 2019b). In contrast, Dang et al. (2016) reported
that graphite rods did not have any promoting effect, while the other carbon-based
materials tested in parallel (i.e., GAC, carbon cloth, and carbon felt) significantly
enhanced methanogenic performance, in anaerobic SBRs treating commercial dog
food. This inconsistency was not specifically addressed in the study and requires
further investigation for clarification.
Different doses of graphene, or single-layer graphite, were applied to UASBs fed
with synthetic wastewater containing glucose in a study on the DIET-promoting
effect of graphene during AD (Tian et al. 2017). The low-dose digester (30 mg
graphene/L) showed a significantly higher methane production rate compared to the
control digester without graphene, whereas the high-dose digester (120 mg
graphene/L) showed a slight inhibitory effect on methanogenesis, during the exper-
iments at room temperature (10–20  C). Although the reasons for this inhibition
were not explored, microbial community structure analysis indicated that the long-
term exposure to graphene in the high-dose digester may have suppressed the growth
of dominant microorganisms, such as Methanothrix, Lactococcus, and Anaerolinea.
The addition of graphene helped to maintain a high relative abundance of
Methanothrix and enriched Geobacter in the digesters, reflecting the development
of DIET-based electric syntrophy. Although graphene was effective in promoting
DIET at a much lower concentration compared to other carbon-based conductive
materials described above (Table 2.3), its price is still prohibitive for use in (semi-)
continuous applications. The market price of graphene nanoplatelets ranges from
several hundred to well over one hundred thousand US dollars per kilogram,
depending on the grade (La et al. 2020).
44 C. Lee

2.3.2 Iron-Based Materials: Magnetite

2.3.2.1 Synthetic Wastewater

In contrast to batch studies, use of iron-based conductive additives other than

magnetite to promote mDIET during AD has rarely been reported in (semi-)
continuous experiments. However, it provides many advantages owing to its low
cost (US$0.35–2.7/kg 100-mesh powder at Alibaba.com [accessed 5 Jan 2021]),
high abundance in nature, and non-toxicity (He et al. 2017). Yin et al. (2017b)
demonstrated in SBRs treating synthetic wastewater containing acetate, soluble
starch, and tryptone that the addition of magnetite accelerated methane production.
They suggested Methanosarcina species as the major methanogens involved in
DIET in the digesters, based on high-throughput sequencing data. However,
Methanosarcina remained as the most abundant methanogen genus throughout the
experiment in both the magnetite-added and control digesters. This interpretation
may need to be reconsidered and supported by further evidence, particularly given
that the operation time of 43 days (cf. sludge retention time of 33 days) would not be
sufficient for reshaping microbial community structure. The same group reported
similar results in other studies using SBRs fed with synthetic wastewater containing
tryptone (Yin et al. 2017a, 2018). Methane production rate and electron transport
system activity were significantly enhanced by the addition of magnetite in both
studies. The latter study showed that genes involved in the CO2-reducing
methanogenic pathway were assigned mainly to Methanosarcina, and the conduc-
tivity of sludge increased in the magnetite-added digester, suggesting that magnetite
stimulated DIET-driven electrotrophic methanogenesis by Methanosarcina.
Wang et al. (2019a) studied the effect of magnetite addition on the start-up and
starvation recovery of UASB reactors fed with synthetic wastewater containing
sucrose. Both biogas production and organic removal efficiency were enhanced by
the addition of magnetite, and the recovery time after a 60-day starvation was
shortened by half in the magnetite-supplemented digester compared to that in the
control digester without magnetite. Potential DIET partners, such as Syntrophaceae
and Methanothrix, were enriched during the recovery from starvation in the presence
of magnetite.
Wang et al. (2020a) compared the AD performance of two UASB reactors added
with the same mass of magnetite and silica, respectively, at increasing OLRs from
1 to 10 g COD/Ld while changing the substrate (acetate, propionate/butyrate
mixture, and sucrose in order) in synthetic wastewater fed to the digesters. The
magnetite supplemented digester showed better COD removal and methane produc-
tivity than did the one with silica, for all substrates tested, with the enhancement of
biogas production being greatest for sucrose-containing wastewater (23% increase).
Magnetite addition also improved the intensity, hydrophobicity, and electroactivity
of granular sludge and enriched different potential DIET partners on different sub-
strates. Chen et al. (2020) demonstrated that magnetite addition enhanced the
methanogenic treatment of synthetic wastewater containing acetate with high
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 45

salinity (0.4 M NaCl) in UASB reactors. Significant acceleration of VFA degrada-

tion and methanogenesis was observed in the magnetite-supplemented digester
compared to the control digester without conductive additive. Microbial community
analysis revealed that the addition of magnetite enriched biofilm-forming
exoelectrogenic bacteria, especially Pseudomonas, and electrotrophic methanogens,
especially Methanosarcina, which potentially interacted via DIET under the high-
salinity experimental conditions.

2.3.2.2 Sulfate Rich Wastewaters

Jin et al. (2019) tested the effect of magnetite addition in UASB reactors treating
sulfate-rich synthetic wastewater containing glucose while varying the COD/sulfate
ratio from 5.0 to 2.5. They observed simultaneous enhancement of methanogenesis
and sulfate removal in the magnetite-supplemented digester and suggested the
establishment of electric syntrophy via DIET between Fe(III)/sulfate-reducing bac-
teria and methanogens. By contrast, Liu et al. (2019) reported that the sulfate-
reducing activity was reduced by the addition of magnetite, while VFA degradation
and methanogenesis were promoted, in anaerobic SBRs fed with sulfate-rich syn-
thetic wastewater (COD/sulfate ratio ¼ 6.0) containing ethanol, acetate, and
propionate.
Furthermore, a more recent study that examined the effect of magnetite in
anaerobic CSTRs treating a sulfur-rich organic waste mixture (Ulva biomass and
cheese whey; 0.8% w/w dry weight) observed no apparent enhancement of
methanogenic performance by the addition of magnetite (Jung et al. 2020). It was
found instead that the H2S content in biogas decreased remarkably with magnetite
addition through anaerobic oxidation of sulfide to elemental sulfur, which was
suggested to be coupled via DIET with electrotrophic methanogenesis. These
different observations may be attributed in part to the differences in experimental
conditions, such as inoculum source, magnetite dose, substrate/electron donor, and
reactor type/operation mode, and at the same time underscore the need for more
studies in sulfate/sulfur-rich environments, where sulfate-reducing bacteria can
compete strongly with methanogens for common electron donors.

2.3.2.3 Industrial Wastewaters

The DIET-promoting effect of magnetite was also demonstrated with different real
waste streams in (semi-)continuous anerobic digesters. Baek et al. (2016) demon-
strated the beneficial effect of magnetite on the methanogenic performance and
stability of anaerobic CSTRs treating whey in long-term experiments over a
one-year period. As an extension study, they investigated the potential of magnetic
separation and recycling of magnetite from the digester effluent as a means to
maintain enhanced stable digester performance without continuous supplementation
with magnetite (Baek et al. 2017). Magnetite recycling effectively maintained
46 C. Lee

promoted DIET and methanogenic activities over a long period of time (>250 days)
and helped to maintain high biomass retention by returning active microorganisms
associated with magnetite to the digester. In both of the above studies, magnetite
addition significantly affected the microbial community structure, and Methanothrix
was suggested to be responsible for electrotrophic methanogenesis via DIET.
Lei et al. (2018) reported the enhancement of methane production and COD
removal with the addition of magnetite in UASB reactors treating municipal solid
waste incineration leachate. The microbial community structure was significantly
different between the digesters with and without magnetite addition, and
exoelectrogenic bacteria and electrotrophic methanogens (Methanosarcina and
Methanothrix), which were potentially involved in DIET, were enriched in the
magnetite-supplemented digester. Wang et al. (2018b) compared anaerobic SBRs
added with different doses of magnetite (0–0.3 g/L) for the treatment of Fischer-
Tropsch wastewater from a coal-to-liquids plant. The digesters supplemented with
0.1 or 0.2 g/L magnetite showed a significantly higher organic removal and methane
production compared to the control digester, with 0.2 g/L being the optimal magne-
tite dose, while the digester dosed with 0.3 g/L magnetite showed the worst
performance. Magnetite addition promoted the enrichment of Geobacter and
Methanothrix and their electric syntrophy via DIET, particularly at the optimal
magnetite dose.

2.3.3 Other Conductive Additives

New and other conductive materials besides the above-mentioned carbon- and iron-
based materials have been evaluated for effectiveness in promoting DIET during AD
in recent studies. However, most of the studies have been performed in batch mode
with synthetic wastewater, and limited information is available on the practicability
of the materials in (semi-)continuous processes. Polyaniline, a conductive polymer,
was applied in the form of a hydrogel (Zhou et al. 2021) and nanorod (Hu et al. 2017)
to promote DIET in the methanogenic treatment of synthetic wastewater containing
sucrose in batch cultures, and proved effective in accelerating methane production.
Wang et al. (2021b) prepared magnetite-contained biochar using iron-rich Fenton
sludge and demonstrated its promoting effect on DIET and methanogenesis in batch
cultures anaerobically treating synthetic dairy wastewater.
Yang et al. (2020a) reported that composite GAC-MnO2 was significantly more
effective than GAC in enhancing electron transfer between syntrophic microorgan-
isms and thus methanogenesis during the batch AD of synthetic wastewater
containing starch. Different non-iron transition metal compounds (i.e., WO2, WC,
W2N, W18O49, TaOx, Nb2O5, and HfO2) were shown to be effective in stimulating
DIET and enhancing methane production in anaerobic batch cultures fed with dairy
manure (Wang et al. 2020c; Yun et al. 2021). Guo et al. (2020b) examined the effect
of nickel foam addition on the batch methanation of synthetic wastewater containing
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 47

ethanol and reported that nickel foam effectively promoted DIET and accelerated
methane production.
Liu et al. (2020) compared the performance of three anaerobic integrated floating
fixed-film activated sludge (An-IFFAS) reactors filled with high-density polyethyl-
ene (HDPE)-based carriers with 3%, 5%, and 7% (w/w) graphite contents with two
control reactors, one anaerobic floc reactor and one An-IFFAS reactor filled with
plain HDPE carriers, for the continuous anaerobic treatment of synthetic wastewater
containing ethanol. All graphite-modified carrier-filled reactors performed signifi-
cantly better than the controls, and the reactors filled with the carriers with higher
graphite contents showed greater methanogenic performance. The addition of
graphite-modified carriers enriched potential DIET partners, Geobacter and
Methanothrix, which could accelerate syntrophic methanogenesis.

2.4 Future Perspectives

VFA oxidizers and methanogens have been conventionally thought to exchange

electrons exclusively through IIET via microbially produced H2 or formate as
electron carriers. However, the existence of DIET which transfer electrons through
cell-to-cell electrical connections was recently revealed, and it is now clear that
different exoelectrogenic bacteria and electrotrophic methanogens involved in AD
can exchange electrons through DIET. IIET and DIET coexist in methanogenic
environments, and together play an important role in balancing acidogenesis and
methanogenesis during the conversion of organic matter into methane. Over the last
decade, considerable efforts have been made to enhance the performance and
stability of AD processes by promoting DIET, mostly by adding conductive mate-
rials (i.e., promoting mDIET), because DIET is energetically and kinetically advan-
tageous over IIET.
The effectiveness of this simple strategy in enriching DIET-active methanogenic
consortia and accelerating methanogenesis has been well demonstrated in many
recent studies using different conductive additives (mostly carbon- or iron-based) in
batch and (semi-)continuous cultures. Our understanding of the role of DIET in
methanogenic environments has evolved significantly in recent years, and promoting
mDIET using inexpensive conductive additives is considered a promising strategy to
accelerate methanogenesis in anaerobic digesters. However, biochemical mecha-
nisms and microbial interactions underlying DIET during AD, particularly in
engineered systems treating complex organic wastes, are still poorly understood,
and much more work needs to be done to engineer DIET-based electric syntrophy in
anaerobic digesters in the field for enhanced methanogenic performance. Outlined
below are some points to deliberate in future studies of engineering DIET for
practical applications.
48 C. Lee

2.4.1 Retention of Additives in the Reactor

For field application, preventing or minimizing the loss of conductive additives by

washout from (semi-)continuous digesters is important. Continuous addition of
conductive materials is not an economical and scalable solution. For example, a
pilot-scale food waste digester (1 m3) added with PAC (15 g/L) lost 0.345 kg PAC
daily in the discharged digestate, and the cost of replenishing the loss accounted for
up to 81.8% of the income from biogas production (Zhang et al. 2018; Zhao et al.
2020a). Discharge of conductive additives in digestate increases the volume of
residual solids that needs further treatment, resulting in an increase in operating
costs. Baek et al. (2017) demonstrated that the enhanced methanogenic performance
of a CSTR was stably maintained over a long period of time (>250 days) by
magnetically separating and recycling magnetite from the digestate. However, this
method is not applicable to non-magnetic materials, and the efficiency of magnetic
separation (and physical screening) would be low for small-size particles.
Many studies applied upflow reactors (i.e., UASB and EGSB) or SBRs to retain
conductive additives, which are heavy enough to settle down and/or aggregate well
with biomass, in the digesters with minimum washout loss. Although effective in
delaying the loss, this approach cannot prevent the discharge of conductive materials
with the excess sludge. Furthermore, UASB/EGSB-type digesters are not suitable
for treating suspended solids-rich waste streams, such as food waste, sewage sludge,
and manure, which are major feedstocks for biogas production. A promising
approach is fixing conductive media in, for example, anaerobic filter or packed
bed reactors, although the specific surface area for the attached growth of DIET
syntrophic partners will be reduced compared to using small particles. Fabricating
and applying non-toxic, non-reactive support media with high conductivity and large
specific surface area is a key in this respect. The moving-bed biofilm reactor, which
is advantageous in mass transport over fixed-bed reactors, is also a reactor design of
choice to apply conductive media for promoting DIET (Liu et al. 2020). However,
these biofilm reactors are also not applicable to high-suspended solids waste streams,
which can cause biofilm damage and clogging problems. Further research is needed
to minimize the consumption of conductive additives while maintaining a desired
level of digester performance enhancement for long-term operation.

2.4.2 External Voltage Supply

Several studies applied the addition of conductive materials combined with the
application of external voltage as a strategy to further promote DIET and boost
syntrophic methanogenesis (Baek et al. 2020, 2021; Vu et al. 2020). Combining AD
with a microbial electrolysis cell (AD-MEC) has been suggested as a way to improve
AD performance, because the cathodic CO2 conversion to CH4 by electrotrophic or
hydrogenotrophic methanogens increases methane production. However, it is yet to
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 49

be clarified whether the main role of the electrodes in an AD-MEC system is

providing surfaces for the growth of biofilms (i.e, enhanced biomass retention) or
stimulating bioelectrochemical reactions involved in AD (Baek et al. 2021).
External voltage application enhances the activity of microorganisms capable of
extracellular electron exchange, and therefore it would be worth investigating
whether or not combining AD-MEC and mDIET promotion using conductive
additives has a synergistic effect on accelerating methanogenesis. Recent studies
using magnetite (Baek et al. 2020) and carbon brush (Baek et al. 2021) reported that
adding conductive materials (or providing conductive surface area) was significantly
more effective in enhancing AD performance than applying external voltage, which
contributed little to methane production. So far, only a few studies have addressed
this question, and more results are needed to draw valid conclusions.

2.4.3 Two Stage AD (Ethanol AD)

Another interesting approach which could be applied in combination with adding

conductive materials (or applied alone) to promote DIET is ethanol-type fermenta-
tion pretreatment. A two-stage process consisting of ethanol-type fermentation and
methanogenesis stages has been demonstrated to effectively promote DIET and
enhance energy recovery in several recent studies from a research group using
synthetic dairy wastewater (Zhao et al. 2017a), waste activated sludge (Zhao et al.
2018), corn straw (Zhu et al. 2019), bagasse wastes (Zhao and Zhang 2019), and
food waste (Zhao et al. 2020b). The two-stage approach is reasonable given that
ethanol is possibly the most favorable substrate for DIET (Gu et al. 2019) and that
DIET syntrophy prevailed in UASB granules treating ethanol-rich brewery waste-
water (Morita et al. 2011; Rotaru et al. 2014b; Shrestha et al. 2014). This bDIET-
promoting strategy may be combined with the addition of conductive materials for
possible synergistic effects on the methanogenic performance and biomass retention
(Zhao and Zhang 2019).

2.4.4 Improved Analytical Tools

Although numerous studies have empirically demonstrated the enhancement of

syntrophic degradation of fermentation intermediates and methanogenesis by adding
conductive materials in anaerobic digesters, to date, the evidence for promoted DIET
activity has been indirect and circumstantial. This is due to the lack of techniques to
quantitatively trace the flow of electrons released from the degradation of organic
matter in complex methanogenic consortia. Although still indirect, a combination of
meta-omic, electrochemical (e.g., cyclic voltammetry and sludge conductivity), and
metabolic (e.g., stable isotope probing) analyses is currently the best strategy to
evaluate the occurrence of DIET. A recent review article discusses different methods
50 C. Lee

that have been used to confirm the occurrence of DIET in methanogenic systems and
the need for the development of suitable DIET characterization methods in detail
(Van Steendam et al. 2019). Technologies for engineering DIET for enhanced AD
are still in the embryonic stage. Quantitative methods to monitor DIET activity will
not only help to advance our fundamental understanding of DIET but also to enable
better engineering and operation of DIET-aided AD processes. Furthermore, such
methods will provide the ability to unravel the conflicting results in the literature.
Although conductive materials have generally been reported to promote DIET
and enhance the methanogenic conversion of organic matter, a few studies have
demonstrated no or rather inhibitory effects for some carbon- and metal-based
conductive materials, such as ferrihydrite, carbon black, magnesium oxide, and
silver nanoparticles (Martins et al. 2018; Baek et al. 2019). It is understandable
that conductive materials may exert different effects on DIET and thus
methanogenesis according to their different physicochemical properties (e.g., elec-
tric conductivity, crystallinity, solubility, surface area, porosity, morphology, and
point of zero charge) and the digester environmental conditions (e.g., substrate,
inoculum, oxidation-reduction potential, pH, temperature, inhibitory substances,
and other operational parameters). However, the underlying reasons for the
conflicting results, especially for the inhibitory effects, are not well understood,
although their understanding is vital for effectively engineering DIET for enhanced
methanogenesis.

2.4.5 Other Electro-Syntrophic Interactions

Another point that needs to be addressed is the effect of the addition of conductive
materials on electro-syntrophic interactions other than electrotrophic
methanogenesis. Many studies have reported enhancement of microbial and enzy-
matic activities related to hydrolysis, fermentation, and acetoclastic methanogenesis,
besides those associated with CO2-reducing methanogenesis, in methanogenic cul-
tures supplemented with conductive additives. These observations are not surprising
given that highly diverse microorganisms, including many (potential) electroactive
microorganisms, coexist and interact in mixed-culture methanogenic systems. It is
not unlikely that mDIET-promoting approaches may affect other microbial redox
processes, either directly or indirectly.
For example, anoxygenic photosynthesis (green sulfur bacteria Prosthecochloris)
can be coupled to anaerobic respiration (Geobacter) via DIET under anoxic condi-
tions (Ha et al. 2017). This syntrophic anaerobic photosynthesis may occur in
methanogenic environments, although its effect on the overall AD process would
be limited, particularly in field-scale AD plants run under completely dark conditions
with no light for photosynthesis. Furthermore, a recent study reported that magnetite
addition did not enhance methane production but greatly reduced the H2S content in
biogas during the semi-continuous AD of sulfur-rich organic waste (Jung et al.
2020). The authors found that H2S was removed by microbial oxidation to S0 and
2 Engineering Direct Interspecies Electron Transfer for Enhanced. . . 51

suggested a novel DIET-based electric-syntrophy coupling the oxidation of sulfide

to S0 to electrotrophic methanogenesis of CO2. Dissimilatory sulfate reduction is a
major electron sink that competes with methanogenesis for electron donors, and the
effect of adding conductive materials on the flow of electrons could become even
more complex when other electron sinks (i.e., biologically reducible substances in
methanogenic environments) are abundant. Various electro-syntrophic associations
not directly involved in methane production likely occur in methanogenic systems,
and understanding how they function and affect methanogenesis in the presence of
conductive materials is important for engineering DIET in practical applications.
However, this aspect has been little studied and poorly understood.

Acknowledgement This work was supported by a grant from the National Research Foundation of
Korea (NRF-2020R1A2C2004368).

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 Part II
Pretreatment
Chapter 3
Adsorbents for the Detoxification
of Lignocellulosic Wastes Hydrolysates
to Improve Fermentative Processes
to Bioenergy and Biochemicals Production

Itzel Covarrubias-García and Sonia Arriaga

Abstract The depletion of fossil fuels and their environmental impact has moti-
vated the research on alternative sources of energy. Lignocellulosic wastes are
potential substrates for bioenergy and biochemicals production due to the carbohy-
drate content in the cell wall, which is composed of cellulose (40–80%), hemicel-
lulose (10–40%), and lignin (5–25%). The sugar content of lignocellulosic biomass
can be converted into fermentable monomeric sugars by physicochemical or enzy-
matic process. The hydrolysates produced during those processes have a high
content of inhibitory substances such as phenolic and furanic compounds, i.e.
syringaldehyde, vanillin, furfural and 5 hydroxymethylfurfural. Thus, the need of a
detoxification pretreatment of hydrolysates before the fermentation process should
be addressed. There are several detoxification processes reported such as overliming,
ion exchange, membranes, use of enzymes or microorganisms and adsorption.
Adsorption processes with nanomaterials have emerged as a promising technique
for the removal of such inhibitors. This chapter shows the potential methods to
detoxify hydrolysates, specifically using the adsorption process with potential adsor-
bents such as nanomaterials. Furthermore, inhibitors should not be just considered as
obstacles for fermentation and hydrolysis, but could also be viewed as valuable
chemicals for other industries which is also highlighted in this chapter.

I. Covarrubias-García
División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica,
San Luis Potosí, Mexico
e-mail: [email protected]
S. Arriaga (*)
División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica,
San Luis Potosí, Mexico
Microbiology Department, School of Natural Sciences, National University of Ireland, Galway,
Galway, Ireland
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 63

A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_3
64 I. Covarrubias-García and S. Arriaga

Keywords Adsorbents · Nanomaterials · Hydrolysates · Inhibitors · Bioenergy

production

3.1 Introduction

The price rise, environmental issues and depletion of fossil fuels along with the
tremendous increase in the world’s energy demand have pushed countries to search
for alternative renewable energy options such as biofuel production from lignocel-
lulosic biomass (Onaran et al. 2020). Lignocellulosic biomass is one of the most
abundant biopolymers found on earth and includes agricultural residues such as
sugarcane bagasse, rice straw, wheat, soy, oats, corn, stump, sawdust, plant and tree
branches as well as urban solid residues. The secondary cell wall of plants contains
cellulose (40–80%), hemicellulose (10–40%), and lignin (5–25%). Lignocellulosic
biomass contain fermentable sugars that have been extensively evaluated for biofuel
production. The carbohydrate fraction of the plant cell wall is converted into
fermentable monomeric sugars through physical (e.g. milling, ultrasound irradiation
and extrusion), chemical (e.g. acid, alkaline, ammonia explosion, organo-solvents
and ionic liquids), physicochemical (e.g. steam explosion, microwave and CO2
explosion) and enzymatic hydrolysis (e.g. cellulose and xylanase) pretreatment or
a combination of these methods.
During the pretreatment of lignocellulosic biomass, formation of many inhibitory
compounds can take place. These compounds are divided in three main groups based
on their origin: weak acids, furan derivatives and phenolic compounds (Palmqvist
and Hahn-Hägerdal 2000a). Formation of these inhibitors during pretreatment is a
limiting factor in the production of valuable chemicals and bioenergy from ligno-
cellulosic biomass and the selection of an efficient pretreatment method can signif-
icantly reduce the production of inhibitory compounds and may lead to enhance in
production and yield of the desired product. Presence of inhibitory compounds have
adverse effect on the substrate utilization and product yield by fermentative
microbes. The unfavourable environment created by the presence of inhibitors
increases the length of the lag phase, reduces cell density and lower the growth
rates of fermenting microbes (Zabed et al. 2019; Wang et al. 2018; Kumar et al.
2020). Moreover, inhibitors can decrease the activity of several enzymes, break
down the DNA, inhibit protein and RNA synthesis in microorganisms (Jung et al.
2014, 2013; Liu et al. 2004; Palmqvist and Hahn-Hägerdal 2000a, 2000b;
Taherzadeh et al. 2000).
In order to decrease the amount of toxic compounds, several detoxification
methods have emerged such as alkaline detoxification, biological detoxification by
microorganisms or enzymes, and adsorption using polymeric sorbents or activated
carbon (Grzenia et al. 2010; Ludwig et al. 2013; Myoung et al. 2010; Zhang et al.
2011). Adsorption processes are advantageous to detoxify hydrolysates as they do
not modify the chemical composition of the inhibitor which can be desorbed to
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 65

recover and further utilized as valuable chemical in other industries (Sjulander and
Kikas 2020).
This book chapter is focused on examining the fundamentals that govern the
function of adsorbents for the detoxification of lignocellulosic wastes hydrolysates to
improve fermentative processes to bioenergy and biochemicals production. It also
reviews the commonly used adsorbents and discusses the advantages and disadvan-
tages of using each of these methods. Likewise, it will also present the emerging
methods used for detoxification and the challenges involved in their application.

3.2 Pretreatment of Lignocellulosic Residues

The main objective of pretreatment of lignocellulosic biomass is to break down the

lignin structure and to make the cellulose and hemicellulose more available for
bioconversion (Hernández-Beltrán et al. 2019). During the pretreatment, the disrup-
tion of the physical barriers such as lignin and hemicellulose take place to depoly-
merize and to reduce the cellulose crystallinity. The effectiveness of pretreatment
depends on the structural and compositional properties of lignocellulosic biomass,
such as crystallinity, degree of polymerization, degree of hemicellulose acylation,
surface area, pore volume and lignin content (Basak et al. 2020). Pretreatment has
been identified as the key step in the bioconversion of lignocellulosic biomass for its
use in biorefinery. A large number of pretreatment methods for lignocellulosic
biomass have been studied which can be classified into 4 groups: physical, chemical,
physicochemical and biological processes. Table 3.1 shows the most common
pretreatment methods reported along with their advantages and disadvantages.

3.2.1 Physical Pretreatment

Physical pretreatment includes biomass particle size reduction using grinding, cut-
ting, ball milling among other processes (Table 3.1). The advantage of this process is
that it does not produce inhibitory substances. Particle size reduction is necessary
prior to most physicochemical pretreatments to improve material handling and
enhances the efficiency of physicochemical pretreatments. However, the excessive
reduction of size makes the processes economically unfeasible for a potential
application, its inability to remove lignin is the main drawback (Taylor et al.
2019). Other physical methods include ultrasonic, extrusion, microwave irradiation,
pulsed electric field, freeze pretreatment and pyrolysis.
66 I. Covarrubias-García and S. Arriaga

Table 3.1 Physical, chemical and biological methods to pretreat lignocellulosic biomass (Bajpai
2016; Bhatia et al. 2020; Bhutto et al. 2017; Zabed et al. 2019)
Type of pretreatment Advantages Disadvantages
Physical • Milling • Increases surface area • Inability to remove
• Ultrasound • Decrystallizes cellulose lignin
• Gamma irradi- • Removes hemicellulose • High energy
ation • No formation of inhibitors requirement
• Extrusion • Improves hydrolysis when used
• Microwave in combination with other
radiation processes
• Hydrothermal
pyrolysis
Chemical • Acid • Solubilize hemicellulose • Inhibitors formation at
hydrolysis • Removes lignin high acid concentration
• Improves sugar yield • Causes corrosion of
equipment
• The acid recycling
process is expensive
• Requires neutralization
• Alkaline • Solubilize lignin and hemicel- • Reaction time takes
hydrolysis lulose days-weeks at room
• Increase crystallinity of cellu- temperature
lose • Harsh chemical
• Improves glucose yield from conditions
biomass
• Organosolv • Efficient lignin removal • Recycling of solvent
process • Pure lignin obtention • Risk in operating at
• High sugar yield from biomass high pressure
• Recovery of lignocellulosic
components
• Ionic liquids • Effective in lignin removal • Extremely viscous
(IL) • Decrystallize cellulose solution is formed which
• As they are non-volatile, bio- limit industrial applica-
mass is easily treated at high tion
temperature without solvent • Complex synthesis
losses by evaporation process
• ILs can be recovered and • High costs of ILs
recycled easily
• Ozonolysis • Selective lignin degradation • High cost of generating
• Produce biomass with great ozone
surface areas
• Minimal losses of hemicellu-
lose and cellulose
• Peroxide • Depolymerize lignin • Requires to maintain
• Mild conditions of temperature the pH constant to avoid
and pressure hemicellulose
• Reduce inhibitors formation elimination
Combined • Steam • Solubilize hemicellulose • Process cost is high
physical and explosion • High removal of lignin • High pressure required
chemical • Increase porosity and surface • High rate of degrada-
methods area by disruption of biomass tion of sugars
(continued)
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 67

Table 3.1 (continued)

Type of pretreatment Advantages Disadvantages
and reduce the crystallinity of
cellulose
• • Decrystallize cellulose • Less removal lignin
Thermochemical • Removes hemicellulose • Yield of sugars is low
• Removes lignin • Significant consump-
• Alters lignin structure tion of energy
• Ammonia fiber • Decrease crystallinity of cellu- • Generation of
explosion- lose by-products
AFEX • Increase surface area and • High initial overhead
porosity cost for ammonia
• Break down lignin structure reagent
• Liquid hot • Hydrolysis of hemicellulose • Formation of inhibitors
water and lignin content reduction like furfural and
• Cellulose hydration hydroxymethylfurfural
• Low capital costs (HMF)
• Operates under lower pressure
• No requires chemicals and cor-
rosion resistant materials
• Wet oxidation • Increase the solubilisation of • Yield of sugar is low
hemicellulose and breakdown • Not yet established at
lignin pilot scale
• High cellulose yields
• Microwave • Decrease crystallinity of cellu- • Not economically
chemical lose viable
pretreatment • Depolymerize lignin compo-
nents
• Increase available surface area
• CO2 explosion • Increase the surface area • High pressures of
• Significant hydrolysis of sugars operation
• Green technology as the CO2 is • High initial costs and
recycled maintenance costs
Biological • Microbial • Removes hemicellulose • Long process time
• Fungi • Removes lignin • Large space require-
• Alters lignin structure ment
• Environmentally friendly • Loss of carbohydrates
• Low inputs such as energy and
chemicals
• Low outputs like inhibitors and
wastes
• Enzymes (Cel- • Short residence time • High enzyme cost
lulases and • Low nutrition requirements
Xylanases) • Cheap process equipment

3.2.2 Chemical Pretreatment

Chemical pretreatment enhances the biodegradability of cellulose by removing

lignin and hemicellulose, thus the polymerization and crystallinity of the cellulose
68 I. Covarrubias-García and S. Arriaga

is changed. This pretreatment is highly selective for the biomass constituents and
requires harsh operational conditions (Peral 2016). These methods include acid
hydrolysis, alkaline hydrolysis, organosolv, peroxide and ozonolysis (Table 3.1).
The most known acids used are H2SO4, HNO4, HCl, H3PO4, HNO3 and organic
acids.
Acid hydrolysis is considered one of the most common lignocellulosic biomass
pretreatments and can be performed by using dilute or concentrated acids (4% to
40%). They are flexible in feedstock choice, high monomeric sugar yield, and mild
temperature operation conditions. The formation of toxic compounds such as
5 hydroxymethylfurfural (5-HMF), furfural, levulinic and formic acid occurs under
acid hydrolysis. A neutralization step is required which adds cost to the overall
process.
During alkaline treatment, little degradation of sugars to furfural, HMF and
organic acids occurs (Peral 2016). Organic solvent pretreatment involves the use
of an organic or aqueous organic solvent mixed with inorganic acid catalysts (HCl or
H2SO4). Oxalic, acetylsalicylic and salicylic acid can also be used as catalysts.
Solvents commonly used are methanol, ethanol, acetone and ethylene glycol (Peral
2016). Organosolv pretreatment hydrolyses lignin bonds as well as lignin-
carbohydrate bonds. Lignin is extensively removed and hemicellulose is almost
completely solubilized, while cellulose remains in solid form (Peral 2016). This
pretreatment is very selective to lignin which can be recovered and utilized for a
range of applications such as precursors for various chemicals or fuels in a
biorefinery. The challenge with this pretreatment is the recovery of the organic
solvents. The high cost involved in the organosolv pretreatment method due to
involvement of expensive chemicals and catalyst is the only drawback of this
method which make its application less desirable than the other commonly used
technologies (Peral 2016). Moreover, in many cases, high temperature (up to
200  C) is also applied in organosolv pretreatment which can increase the cost of
operation.

3.2.3 Physicochemical Pretreatment

Physicochemical treatment technology is nothing but combined methods involving

both physical and chemical pretreatment techniques (Table 3.1). The lignocellulosic
biomass is subjected to chemical modification and the cell wall structure is physi-
cally broken (Aslanzadeh et al. 2014). The high pressure and temperatures used in
these types of processes increase the costs of applying them in a biorefinery type
system. Steam pretreatment is the most common method applied to biomass due to
their wide ranging application on various types of biomass, however in case of
recalcitrant biomass sources, it may necessitates additional treatment using acid or
SO2 (Aslanzadeh et al. 2014). During steam explosion, fermentation inhibitors are
generated such as HMF, weak acids and phenolic compounds. This method requires
a high amount of energy and involves elevated operation costs (Peral 2016). Ionic
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 69

liquids are effective in decrystallizing the cellulose, fractionating lignin and poly-
saccharide contents into separate streams, making the following hydrolysis step
much easier (Aslanzadeh et al. 2014).

3.2.4 Biological Pretreatment

Biological pretreatment is performed by using wood-degrading microorganisms,

which includes white-rot fungi, brown-rot fungi, soft-rot fungi, and bacteria
(Table 3.1). These microorganism changes the chemical composition and/or struc-
ture of the lignocellulosic biomass so as to make the biomass less resistant during the
subsequent enzymatic hydrolysis step (Aslanzadeh et al. 2014). They operate under
standard conditions of pressure and temperature but long periods of operation are
needed (10–14 days).
Enzymatic hydrolysis form sugar monomers by depolymerisation reaction of
cellulose and hemicelluloses prior to fermentation (Aslanzadeh et al. 2014). In
contrast to the physicochemical treatment, enzymatic hydrolysis is generally
performed under milder conditions and does not generate any corrosive compounds.
This method can attain nearly complete cellulose conversion without forming any
inhibitory by-products (Mussatto et al. 2008; Aslanzadeh et al. 2014). On the other
hand, the main drawback is that this method may take longer time (1–4 d) for the
completion of the reaction. The commonly used enzymes for this method are
cellulases and hemicellulases with very high selectivity to the substrates
(Aslanzadeh et al. 2014).

3.3 Fermentation Inhibitors Formed during Pretreatment

of Lignocellulosic Biomass

As mentioned in Sect. 3.1, during the pretreatment of lignocellulosic biomass

inhibiting compounds are formed. Inhibitors can be divided into five major groups
(i) organic acids (acetic, formic and levulinic acids), (ii) furan derivatives (furfural
and 5-HMF), (iii) phenolic compounds, (iv) raw materials extractives (acidic resins,
tannic and terpene acids) and (v) heavy metal ions (iron, chromium, nickel and
copper) (Llano et al. 2017). Figure 3.1 shows the inhibiting compounds profile
derived from lignocellulosic materials after pretreatment.
The amount and nature of the formed degradation products depends directly on
the type of lignocellulosic biomass, pretreatment method and conditions used
(Jönsson and Martín 2016). The degradation products include acetic acid
(34–180 mg/L), formic acid (43–250 mg/L), levulinic acid (0.48–41 mg/L), vanillin
(1.7–6.7 mg/L), hydroxybenzaldehyde (1.5–4.4 mg/L), furfural (0.40–220 mg/L),
and 5-hydroxymethyl-furfural (0.89–44 mg/L) (Bonturi et al. 2017; Poontawee
70 I. Covarrubias-García and S. Arriaga

Fig. 3.1 Inhibitory compounds profile derived from lignocellulosic materials during pretreatment

et al. 2017; Yu et al. 2011). Table 3.2 shows the common inhibitors that can be
formed from pretreatment of lignocellulosic biomass and their principal effects on
fermentative microorganisms.
Table 3.2 and Fig. 3.1 show that inhibitors come from the degradation of each
component of the biomass. Cellulose can degrade to hexoses to different extent.
Such hexoses could further be dehydrated to 5-HMF, and 5-HMF can be further
dehydrated to form levulinic acid and formic acid (Kumar et al. 2018). On the other
hand, hemicellulose after pretreatment can degrade into sugar acids, aliphatic acids,
and furan aldehydes, where furfural is an abundant and potent inhibitor (Osorio-
González et al. 2019; Kumar et al. 2020). Formic acid, acrylic acid and levulinic acid
are other carboxylic acids found in hemicellulose hydrolysates (Zabed et al. 2019;
Kumar et al. 2020).
Regarding the degradation products from lignin, 4-hydroxybenzoic acid,
4-hydroxybenzaldehyde, vanillin, dihydroconiferyl alcohol, coniferyl aldehyde,
syringaldehyde and syringic acid are the most common phenols formed from lignin
degradation (Hodge et al. 2009; Kumar et al. 2020). There are different effects
associated with each inhibitor. For instance, weak acids in undissociated form can
permeate through and inside the cells to release the anion and proton disrupting the
intracellular pH (Wang et al. 2018). On the other hand, phenolics are more toxic than
aliphatic and furans with the same functional groups and have shown antibiofouling
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 71

Table 3.2 Common inhibitors formed during pretreatment of lignocellulosic biomass

Origin Inhibitors (mg/L) Effects References
Cellulose • 5-HMF • 5-HMF toxic to the growth of Basak et al. (2020);
(0.89–44) ethanolegens. Kumar et al. (2018)
• Levulinic acid • Furans enter the cytosol and
(0.48–41) inhibit NADH-dependent
• Formic acid enzymes.
(43–250) • Furfural and 5-HMF bind to
nitrogenous bases of DNA and
induce strand break, damaging
the structure.
• Furfural inhibits the synthe-
sis of TTP which lead to the
cessation of DNA replication.
Hemicellulose • Furfural • Furfural is the most abundant Kumar et al.
(0.40–220) and potent inhibitor. (2020); Zabed et al.
• Acetic acid (2019)
(34–180)
• Formic acid
(43–250)
• Levulinic acid
(0.48–41)
Lignin • Hydroxybenzaldehyde • Phenols are able to penetrate Basak et al. (2020);
(1.5–4.4) the cell membrane easily Kumar et al. (2018,
• Vanillin altering cell membrane struc- 2020); Lin et al.
(1.7–6.7) ture, permeability and integ- (2015)
• Syringaldehyde rity.
(0.6–1.2) • Phenolic compounds and
• Syringic acid furans generate reactive oxy-
(0.026–1.4) gen species (ROS) which
inactivate enzymes, damaging
DNA and cellular proteins.

effects on Gram negative bacteria (Pattrick et al. 2019). Likewise, furans present
negative effects on glycolytic and fermentative enzymes and can inhibit the synthe-
sis of ATP, which lead to the cessation of DNA replication (Basak et al. 2020).
Furfural and 5-HMF bind to nitrogenous bases of DNA and induce strand break,
damaging the structure (Liu et al. 2019). Both phenolic compounds and furans
generate Reactive Oxygen Species (ROS), which inactivate enzymes, damaging
DNA and cellular proteins (Basak et al. 2020; Lin et al. 2015).
In general, the effect of each inhibitor and the degree of inhibition will depend on
the fermenting microorganisms and its tolerance to the inhibitor. Additionally, the
toxicity of each inhibitor depends on its concentration, chemical structure and
reactivity, molecular weight and chemical polarity, which determines their inherent
capacity to penetrate the cell membrane and cause cellular damage (Jung et al. 2013;
Taherzadeh et al. 2000).
In general if there is more than one inhibitory compounds are present in the
lignocellulosic hydrolysates that can have a more severe recalcitrant effect on the
microorganisms and enzymatic activity (Almeida et al. 2007; Jayakody et al. 2017;
72 I. Covarrubias-García and S. Arriaga

Wikandari et al. 2019; Sjulander and Kikas 2020). For instance, the effects of
inhibitor combinations (i.e. acetate, furfural, 5-HMF, vanillin,
p-hydroxybenzaldehyde and syringaldehyde) on R. toruloides-Y4 at different con-
centrations have been analysed. The combinations of the different inhibitors gave a
much more complex inhibition effect on the fermentation process (Hu et al. 2009;
Zhao et al. 2012).
Currently, different strategies have been employed to tackle problems with
inhibition after pretreatment of lignocellulose. Some of the different approaches
used are: (i) feedstock selection, that is using less recalcitrant feedstocks in order to
generate less inhibitors during pretreatment (Chiaramonti et al. 2012; Larsen et al.
2012); (ii) selection of microorganisms, i.e. the selection of microorganisms that
produce less amount of inhibitors, should be done mainly based on specific produc-
tivity and product yields (Wimalasena et al. 2014); (iii) adaptive evolution, where
microorganism adapt to specific inhibitors (Almario et al. 2013; Koppram et al.
2012); (iv) genetic or metabolic engineering, based on creating tolerance to fermen-
tation inhibitors (Sanda et al. 2011; Wang et al. 2013); (v) culturing schemes such as
the use of a large inoculum size (Hoyer et al. 2010; Pienkos and Zhang 2009);
(vi) conditioning which consists of addition of chemicals that can improve the
fermentability of the substrates (e.g. NH4OH and NaOH), these agents can be
added during fermentation (Alriksson et al. 2011, 2006); and (vii) detoxification of
hydrolysate (e.g. distillation, solvent extraction and adsorption) (Mussatto and
Roberto 2001; Myoung et al. 2010; Palmqvist and Hahn-Hägerdal 2000a).

3.4 Strategies for the Detoxification of Lignocellulosic

Wastes Hydrolysates

So far, there is no single pretreatment method that can perform delignification

without sugar degradation. Therefore, the tendency is to apply successive treatment
to remove the generated by-products other than sugars (i.e. detoxification). The main
concern of detoxification processes is the simultaneous removal of fermentable
sugars which results in a reduction of final products (Almeida et al. 2009; Deng
and Aita 2018; Li et al. 2013; Myoung et al. 2010). All the different methods using
biological, chemical and physical means are employed for detoxification of ligno-
cellulosic hydrolysate (Kumar et al. 2020; Singh et al. 2017).
Physical methods mainly include distillation, solvent extraction and adsorption
(Cantarella et al. 2004). The main disadvantage of distillation is that only inhibitors
with high boiling point can be removed. On the other hand, solvent extraction
presents a low efficiency with a large consumption of solvent. Regarding chemical
methods, alkaline detoxification, adsorption and ion exchange have been reported.
The used alkali solutions control the pH (9–12) (Alriksson et al. 2006; Nilvebrant
et al. 2003) and remove the inhibitors by precipitation, these include Ca(OH)2,
NaOH, and NH4OH solutions. Likewise, sodium borohydride has been reported to
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 73

be able to effectively remove coniferyl aldehyde, p-benzoquinone,

2,6-dimethoxybenzoquinone, and furfural under mild reaction conditions (pH 6
and 20  C) (Cavka and Jönsson 2013).
Concerning biological detoxification, inhibitors are removed by microorganisms
and enzymes. Inhibitors such as furfural (100%), HMF (94%) and acetic acid (82%)
have been removed with Bordetella spp. without affecting the sugar concentration
(Singh et al. 2017). Otherwise, the laccase enzyme of Trametes maxima IIPLC-32
has been used to detoxify phenolics from sugarcane bagasse removing 66% of
lignin-derived phenolics inhibitors in 55 h (Suman et al. 2018). The main difficulty
of the enzymatic and microbial method is their production cost and the fact that the
process need to be performed under sterile conditions.
Apart from the conventional methods, there are also few emerging technologies
used. The main objective for these processes are the use of simultaneous detoxifi-
cation and product production which may further improve the process economics
and avoid an additional treatment step (Ahmaruzzaman 2008; Bhatia et al. 2020).
For example, the addition of chemicals such as ozone is investigated on a Norway
spruce hydrolysate and has been found that it effectively reduced the aromatic
compounds, furfural and HMF in the hydrolysate. However, the reactive ozone
treatment caused increase in the formic acid concentration in the hydrolysate,
which made the detoxified hydrolysate unsuited for subsequent fermentation
(Cavka et al. 2015).
Other approaches consists of the combination of nanomaterials to immobilize
enzymes for detoxification. Yin et al. (2020) developed a novel detoxification
strategy of rice straw hydrolysate using immobilized laccase on magnetic Fe3O4
nanoparticles for improving lipid production by Rhodotorula glutinis. Notably, the
immobilized laccase exhibited good reusability in repeated batch detoxification.
78.2% phenols, 43.8% furfural, 30.4% HMF and 16.5% formic acid in the hydro-
lysate were removed after the fourth batch (Yin et al. 2020). However, it is important
to take into account the toxicological issues associated with application of
nanomaterials and their fate in the environment.
In comparison to other methods, adsorption has advantages in terms of low
energy consumption, environmental friendliness and low cost, hence creating its
wider application in removal of fermentation inhibitors from hydrolysates (Chen
et al. 2020). Furthermore, use of adsorption as detoxification method compared to
other processes can provide the recovery of such inhibitory compounds by desorp-
tion, which further can be utilized as valuable chemical in other industries, as during
adsorption the inhibitor is not chemically altered (Sjulander and Kikas 2020). Thus,
the recovered adsorbed inhibitor has an added economic value. Worth mentioning
that the different detoxification methods cannot be strictly compared as the removal
efficiency of every method depends on the properties of the inhibitor, such as size,
polarity, chemical reactivity, and concentration (Cavka and Jönsson 2013; Qi et al.
2011; Sjulander and Kikas 2020). The adsorption process for detoxification of
lignocellulosic biomass is discussed in the following section.
74 I. Covarrubias-García and S. Arriaga

3.5 Detoxification of Hydrolysate Using Adsorption

3.5.1 Adsorbent Materials

Section 3.4 described that removal of inhibitors from hydrolysate is an essential step
before performing microbial fermentation. Table 3.3 overviews different adsorption
materials that have been tested for their ability to remove inhibitors from lignocel-
lulosic hydrolysates. The types of adsorbents include activated carbons, minerals,
resins, industrial and agricultural wastes. The adsorption efficiency of the inhibitors
depends on the adsorbent surface area, pore size and chemical properties, and is also
influenced by environmental conditions (i.e. temperature, pH, and contact time
between inhibitor and adsorbent) (Deng et al. 2018; Sjulander and Kikas 2020).
Activated carbon (AC) either stand-alone or in combination with other chemicals
(i.e. alkali and ion exchange resins) is the most common adsorbent used for detox-
ification. Activated carbons are usually produced from low-cost materials which is
one of its main advantage. Apart from that, reliability and consistency of the resource
supply, ease of activation, adsorption capacity, and selectivity are notable qualities
of AC. Literature has reported several studies where different inhibitors have been
removed with activated carbons, such as furfural, 5-HMF, acetic acid and phenolic
compounds (Chen et al. 2019; Myoung et al. 2010; Santana et al. 2018; Zhang et al.
2011). It has been suggested that removal of inhibitors is related to the strong
hydrophobic nature of the activated carbons (Lee and Park 2016). The main draw-
backs in the use of AC are its regenerability and cost, where there is 10% loss during
each regeneration cycle. In addition, it can retain up 30% of the fermentable sugars
which is not good from the economic point of view (Carvalho et al. 2006; Ranjan
et al. 2009). Therefore, the exploration and study of other adsorbents is of interest.
Alternatively, mineral adsorbents including siliceous materials, clay and natural
zeolites have been used (Wang and Peng 2010). Ranjan et al. (2009) demonstrated
the potential recovery of HMF, furfural and vanillin using hydrophobic zeolites.
Removal of model compounds using mineral adsorbents has been also reported
(Ahmaruzzaman 2008). On the other hand, different types of resins have been
reported to remove inhibitors such as fumaric acid, acetic acid, formic acid,
5-HMF and soluble lignin (Chen et al. 2019; Choi et al. 2017; Zheng et al. 2018).
The main advantages in the use of polymeric resins is that they are durable,
chemically inert and stable, and possess a high adsorption capacity, efficiency,
selectivity and ease of regeneration, with relatively low cost and limited toxicity
(Soto et al. 2011).
In addition, the implementation of nanoscale materials has emerged as an attrac-
tive alternative in several applications. Their size and their relatively large surface
area to volume ratio when compared to larger particles can result in a high adsorption
capacity (Alsaba et al. 2020). Few studies have reported the use of nanomaterials for
detoxification of hydrolysates. Covarrubias-García et al. (2021) used reduced
graphene oxide adorned with magnetite nanoparticles for the removal of fermenta-
tion inhibitors such as furfural, 5-HMF, levulinic acid, vanillic acid and vanillin from
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 75

Table 3.3 Summary of adsorbents used for detoxification of hydrolysates. “x” means the studies
where fermentation step is not performed to produce any product
Removal of
inhibitors
Type Adsorbent (%) Products Reference
Activated Activated carbon HMF (96) x Myoung et al.
carbons Furfural (2010)
(93)
Formic acid
(42)
Acetic acid
(14)
Activated carbon Furfural Ethanol Zhang et al.
(97) (2011)
Activated charcoal Phenolic Xylitol Santana et al.
compounds (2018)
(78)
Furfural
(99)
5-HMF (99)
Activated carbon HMF Acetone, Wang et al.
(50–60) butanol and (2019)
Furfural ethanol
(50–60)
Phenolic
compounds
(50)
Membranes Adsorptive membrane Acetic acid x Wickramasinghe
(Sartobind Q) (99) and Grzenia
(2008)
Hollow-structured porous 5-HMF (94) x Zhang et al.
aromatic polymer (2019)
Resins Amberchrom-CG71C resin Fumaric x Choi et al. (2017)
acid (99)
Acetic acid
(99)
Microporous polymeric Levulinic x Lin et al. (2017)
SY-01 resin acid (99)
Microporous resin (SY-01) Formic acid x Zheng et al.
(80) (2018)
Levulinic
acid (16)
5-HMF (63)
Resin CS-6 Soluble lig- Lipid Chen et al.
nin (70) (2019)
Nanostructures Fe0 nanoparticles on acti- Furfural x Sajab et al.
vated carbon (99) (2019)
Acid solu-
ble lignin
(81)
(continued)
76 I. Covarrubias-García and S. Arriaga

Table 3.3 (continued)

Removal of
inhibitors
Type Adsorbent (%) Products Reference
Laccase on magnetic Phenols Lipids Yin et al. (2020)
nanoparticles (78)
Furfural
(44)
HMF (30)
Formic acid
(16)
Reduced graphene oxide Furfural x Covarrubias-
adorned with magnetite 5-HMF García et al.
nanoparticles (72–76) (2021)
Levulinic
acid
(60–63)
Vanillic
acid
(54–57)
Vanillin
(39–45)
Others Zeolite 5-HMF Ethanol Ranjan et al.
(100) (2009)
Furfural
(100)
Vanillin
(100)
Sugarcane bagasse fly ash Phenolic x Freitas and
compounds Farinas (2017)
(80)

wood hydrolysates with a minimal sugar loss of 5.9, 6.2 and 7.6%, for 10, 20 and
50 mg of the adsorbent, respectively. There are other studies where nanomaterials
are immobilized on the AC surface (Sajab et al. 2019) and nanomaterials as a support
for enzyme immobilization (Yin et al. 2020) have been reported for removal of
toxicants from hydrolysate.
Other types of adsorbents such as sugarcane bagasse derived fly ash has been
successfully used to adsorb aromatic compounds, such as tannic acid and vanillin
from a lignocellulosic hydrolysate (Freitas and Farinas 2017).

3.5.2 Challenges of Adsorption

The main challenge related to the adsorbent based technology for detoxification of
lignocellulosic hydrolysates is related to the desorption and reusability of the
adsorbents. Desorbing eluents may not be effective for all the adsorbents and need
3 Adsorbents for the Detoxification of Lignocellulosic Wastes Hydrolysates. . . 77

to be optimized on a case to case basis. Requirement of large amounts of chemicals

as desorbing agent, cost associated with them and their disposal may again put a
question mark on the sustainability of adsorption. Another issue is the loss of
adsorption capacity due to repeated adsorption-desorption cycles: compounds that
can be used multiple times without significant loss in adsorption property are more
suited economically. It is also somewhat difficult to design a continuous system as
even the simplest setup will require at least two continuous columns, one in
operation and another in regeneration to avoid any time delay in operation. There
are also issues with the effectiveness of adsorption as in some cases an additional
step may be required for complete removal of toxic substances from hydrolysate.
This type of issues are more prevalent in hydrolysate where different inhibitory
compounds are present.
Regarding the emerging adsorbents such as the use of nanoadsorbents, additional
challenges are the release and fate of the nanoadsorbent in the environment as well as
the toxicity that could be caused. From the process point of view, the challenges are
related with the loss of the material in the downstream process. The strategy to avoid
its loss would be the immobilization of the nanomaterial in a granular support.

3.6 Recuperation of Commodities from Detoxification

The production of biofuels and value-added biochemicals from evenly-distributed

non-food lignocellulosic biomass would decrease net greenhouse gas emissions by
replacing the use of fossil fuels and would bring benefits of trade (Lynd et al. 2002;
Zhang 2011). Recovery of these compounds is an interesting option for biorefineries
because in addition to increasing the suitability of hydrolysates as substrates, it can
result in economic benefits. Therefore, it is of great interest to study the recovery of
such compounds. The promising products like ethanol, xylitol, organic acids and
2,3-butanediol may play a pivotal role in this direction (Kumar et al. 2020).
The alleviation of the inhibition of fermentation in hydrolysates decreases pro-
duction costs, however, it is very difficult to separate them from the biomass
hydrolysate when they are present in low concentrations (<5% by weight) (Travália
et al. 2019). Thus, it is of interest to understand the separation of fermentation
inhibitors from the hydrolysate. Fermentation inhibitors such as acetic acid, furfural,
HFM and formic acid can be valuable commodities in the industrial manufacture of
pharmaceuticals, dyes, solvents, adhesives, polymers, plastics, resins, lubricants and
fuels (Travália et al. 2019). Alternately, bio-oil can be obtained from lignin
depolymerisation and lignins can be transformed into other value-added products
such as carbon fibers, polyurethanes, biocomposites and nanomaterials for many
applications ranging from biomedicine to automobiles (Zevallos Torres et al. 2020).
Among the different approaches to extract the inhibitors, liquid–liquid extraction
has been extensively investigated as a recovery method. The major drawback of
liquid-liquid extraction is the biological toxicity of the extraction agents, usually
organic solvents (Tomek et al. 2015). Likewise, the use of surfactants-based cloud
78 I. Covarrubias-García and S. Arriaga

point extraction (CPE) has also been explored. Surfactants, which are soluble in
water at low temperatures but insoluble at high temperatures, are used in this
approach, allowing for a two-phase extraction. The main advantage of the method
is that the surfactants are non-toxic to microorganisms and no sugar loss has been
observed (Dhamole et al. 2013). Other studies have reported the extraction of
inhibitors with common solvents like deionized water and ethanol. Ranjan et al.
(2009) demonstrated the potential recovery of HMF, furfural and vanillin using
hydrophobic zeolites. They desorbed with deionized water and indicated that desorp-
tion isotherms almost overlapped with the adsorption isotherms. Lin et al. (2017)
found that the use of an ethanol solution as an eluent was easy and effective to
recover levulinic acid from a SY-01 resin with a 99.4% efficiency and the SY-01
resin was successfully regenerated.

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Chapter 4
Pretreatment of Lignocellulosic Materials
to Enhance their Methane Potential

A. Oliva, S. Papirio, G. Esposito, and P. N. L. Lens

Abstract Lignocellulosic materials (LMs) are the most abundant residues on the
planet and have a huge potential for methane production. Several strategies have
been tested to enhance the methane potential of LMs, with a particular emphasis on
environmentally friendly and economically convenient pretreatments. This chapter
revisits the potential of two chemical, i.e. organosolv and N-methylmorpholine
N-oxide (NMMO)-driven, and one physical, i.e. ultrasounds, pretreatments.
Organosolv pretreatment enables to obtain a pure lignin fraction from LMs, leaving
most of the fermentable sugars in the solid matrix. The result is a lignin-poor material
with an increased porosity and a higher bioavailability of the sugar fraction. Another
advantage is the cost-effectiveness and the easy recovery of the chemicals involved.
NMMO pretreatment focuses on the cellulosic component of the biomass, aiming to
reduce its crystallinity and to increase the porosity of the substrate. The main
advantage of NMMO lies in its high recovery percentage, which reaches up to
99%. Ultrasound pretreatment involves ultrasonic waves that allow fractionating
LMs, breaking the linkages between lignin, cellulose and hemicellulose, generally
leaving cellulose and most of the hemicellulose in the solid fraction and dissolving
the lignin in the liquor. Ultrasound pretreatment does not require chemicals and can
be easily combined with other pretreatment methods to enhance its effectiveness.

Keywords Lignocellulosic materials · Pretreatment · Organosolv · NMMO ·

Ultrasound · Anaerobic digestion · Methane

A. Oliva (*) · P. N. L. Lens

National University of Ireland Galway, Galway, Ireland
e-mail: [email protected]
S. Papirio · G. Esposito
Department of Civil, Architectural and Environmental Engineering, University of Naples
Federico II, Naples, Italy

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 85

A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_4
86 A. Oliva et al.

4.1 Introduction

The interest in renewable energy started when the world experienced an energy crisis
in the ‘70s and realised that fossil fuels are not an unlimitedly available resource.
More recently, the interest in renewable energy sources is related to global environ-
mental quality (Sen and Ganguly 2017) and geographic independence of the fossil
fuels reservoirs (Martins et al. 2019). The first emerging problem regarded the
emission of toxic compounds and oxides of nitrogen and sulfur upon combustion
of fossil fuels, which contributes to acid rain formation (Singh and Agrawal 2008).
At the moment, the main concern relates to global warming caused by the increase in
carbon dioxide concentration and other upper atmospheric pollutants deriving from
anthropogenic activities (Kweku et al. 2018; Qazi et al. 2019).
The preservation of the environment and the necessity of renewable energy to
replace fossil fuels has led to the development of a strong anaerobic digestion
(AD) infrastructure worldwide (Vasco-Correa et al. 2018). AD allows for recovering
energy (i.e. biogas, a CH4/CO2 mixture) from wastes while stabilizing the residual
solid/liquid organic fraction (Bharathiraja et al. 2018). AD involves different com-
munities of microorganisms in four stages: hydrolysis, acidogenesis, acetogenesis,
and methanogenesis (Chen et al. 2008; Zhang et al. 2014). During the first stage,
hydrolytic bacteria secrete enzymes able to hydrolise the organic matter and decom-
pose the complex organic polymers (i.e. lipids, carbohydrates, and proteins) into
soluble monomers (i.e. monomeric sugars, long-chain fatty acids, and amino acids)
and hydrogen. Acidogenesis is the second stage, wherein the soluble monomers are
fermented into volatile fatty acids and other products, which, during the subsequent
acetogenesis stage, are converted to acetate, carbon dioxide and hydrogen by
acetogenic bacteria. In the final phase, methanogenesis, acetic acid, and hydrogen
are converted into methane by methanogenic archaea (Bianco et al. 2021a; Luo et al.
2019).
Many waste materials from several activities are lignocellulosic materials (LMs).
The disposal of these LMs is often difficult due to the enormous volumes produced,
especially during the harvesting season (Barbu et al. 2020; Oh et al. 2018). AD is a
viable and green alternative to landfill disposal or combustion for these LMs
(Alonso-Fariñas et al. 2020). The use of LMs for AD is, however, still limited by
their resistance to biological and chemical degradation. The main issue of AD of
LMs is the complex and resistant structure, mainly consisting of cellulose, hemicel-
lulose, and lignin (Bhatia et al. 2020). Various strategies have been explored to
increase the biodegradability of LMs (Bianco et al. 2021b; Kohli et al. 2020;
Mancini et al. 2018c; Papirio 2020; Yao et al. 2018). Pretreatments of LMs aim to
increase the efficacy of lignocellulose hydrolysis by improving the accessibility of
microorganisms to the sugar fraction (cellulose and hemicellulose) of LMs. This can
be achieved by removing lignin and/or hemicellulose or by decreasing the degree of
polymerization and crystallinity of the cellulosic component of the biomass (Kumar
and Sharma 2017).
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 87

LMs may be converted to various forms of energy, including heat (via burning),
steam, electricity, hydrogen, ethanol, methanol, or methane. The choice depends on
multiple factors, such as conversion efficiencies, transport of energy, need for heat or
steam, economies of scale, and environmental impact. Under most circumstances,
methane is an ideal fuel. Firstly, compared to other fossil fuels, methane produces
few atmospheric pollutants and generates less carbon dioxide per unit of energy.
Therefore, its use for appliances, vehicles, industrial applications, and power gener-
ation is increasing. Furthermore, methane can be used at various stages of purity, and
its transport cost and energy conversion efficiency are comparable to those of
electricity (Chynoweth et al. 2000; Gür 2016). Besides, an extensive pipeline
distribution system is already in place worldwide for methane use. In contrast,
other fuels such as methanol and hydrogen are not well developed commercially
for production, distribution and usage (Chynoweth et al. 2000).
This chapter aims to overview the characteristics and potential of LMs for AD,
with a specific focus on three emerging pretreatment technologies to enhance the
conversion of LMs into methane: organosolv, N-methylmorpholine N-oxide
(NMMO), and ultrasound pretreatment. The principles of each pretreatment will
be thoroughly discussed, pointing out advantages and drawbacks. Particular atten-
tion will be given to the effect of the three pretreatments on the physical character-
istics and chemical composition of LMs, focusing on the subsequent valorisation of
the pretreated solid residues through the AD process.

4.2 Lignocellulosic Materials: Structure and Potential

Biomass is one of the most abundant resources on the planet, with a global
production of 2
1011 tons per year (Reddy and Yang 2005). Biomass can be
converted into fuels and provide renewable materials at the same time, offering a
viable alternative to fossil fuels. Biomass is produced via photosynthesis, by fixing
atmospheric carbon dioxide and converting solar energy to chemical energy to build
up the carbon backbone of plant cells (Zhang 2008). LMs represent more than 60%
of the global biomass and serve as a cheap and abundant feedstock (Bilal et al. 2017).
Agricultural, municipal, and industrial activities generate LMs as waste, generally at
low cost (Table 4.1). Also, the use of LMs for biofuel production does not create
conflicts between land use for food and energy production (Kucharska et al. 2018).
Depending on the origin, LMs are classified as forest residues, municipal solid
waste, waste paper, or crop residue resources (Balat 2011).
The structural and chemical composition of LMs is extremely variable (Table 4.1)
due to various genetic and environmental factors. The chemical composition of LMs
includes mainly cellulose, hemicellulose, and lignin, arranged in a three-dimensional
and complex structure (Fig. 4.1) (Zhang et al. 2019). Depending on the specific
substrate, a significant part of LMs may consist of non-structural compounds. The
most common extractives present in LMs are free sugars (sucrose, glucose, and
88 A. Oliva et al.

Table 4.1 Chemical composition (in terms of cellulose, hemicellulose, and lignin content) of the
most employed lignocellulosic materials for methane production
Biomass Cellulosea Hemicelluloseb Ligninc
origin Substrate (%) (%) (%) Reference
Agricultural Maize straw 38.3 29.8 3.8 Khatri et al. (2015)
residues Wheat straw 31.0 18.4 18.3 Mancini et al. (2018a)
Rice straw 28.6 19.5 17.3 Mancini et al.
(2018b)
Barley straw 39.1 25.7 15.2 Duque et al. (2013)
Sweet sor- 37.7 28.1 21.5 Dong et al. (2019)
ghum straw
Oat straw 35.0 28.2 4.1 Gomez-Tovar et al.
(2012)
Rye straw 42.1 23.8 19.5 Ingram et al. (2011)
Triticale straw 33.0 23.0 29.0 Teghammar et al.
(2012)
Sugarcane 47.6 22.6 27.6 Hashemi et al.
bagasse (2019a)
Sunflower 34.1 26.2 26.8 Hesami et al. (2015)
stalks
Nuts Peanut shell 23.6 12.2 40.0 Shen et al. (2018)
residues Almond shell 23.4 21.9 30.6 Oliva et al. (2021)
Walnut shell 25.6 23.0 46.7 Şenol (2021)
Pistachio shell 20.1 23.2 24.3 Shen et al. (2018)
Chestnut shell 26.8 24.5 36.8 Bianco et al. (2021b)
Hazelnut shell 18.0 17.2 39.1 Shen et al. (2018)
Hazelnut skin 10.2 3.6 39.7 Oliva et al. (2021)
Industrial Spent coffee 8.8 33.6 20.3 Oliva et al. (2021)
wastes grounds
Brewery spent 19.2 26.9 30.5 Ravindran et al.
grain (2018)
Cocoa bean 13.5 7.0 29.9 Mancini et al.
shell (2018b)
Rubber wood 43.6 8.3 31.0 Tongbuekeaw et al.
waste (2020)
Oil palm 36.1 22.4 26.4 Tang et al. (2018)
empty fruit
bunch
Olive pomace 12.3 8.9 34.0 Elalami et al. (2020)
Grape pomace 15.8 8.6 35.4 Bordiga et al. (2019)
Forest Switch grass 42.0 19.0 24.0 Larnaudie et al.
residues (2019)
Spruce wood 42.0 20.0 27.0 Teghammar et al.
(2012)
Poplar wood 49.0 23.0 27.0 Rego et al. (2019)
Birch wood 40.1 26.8 24.5 Goshadrou et al.
(2013)
(continued)
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 89

Table 4.1 (continued)

Biomass Cellulosea Hemicelluloseb Ligninc
origin Substrate (%) (%) (%) Reference
Pine wood 44.5 28.0 26.8 Mirmohamadsadeghi
et al. (2014)
Elm wood 46.4 26.3 26.2 Mirmohamadsadeghi
et al. (2014)
a
Cellulose content (g/g dry matter) was considered equal to the glucan content (Mussatto et al.
2011)
b
Hemicellulose content (g/g dry matter) is reported as the sum of xylan, mannan, galactan, arabinan,
galactan, and rhamnan (Mussatto et al. 2011)
c
Lignin content (g/g dry matter) is reported as the sum of acid soluble and acid-insoluble lignin
(Sluiter et al. 2008)

fructose), phenolic compounds, proteins, lipids, waxes, chlorophyll, essential oils,

starches, and fatty acids (Tajmirriahi et al. 2021a, b).
The complex structure of lignocelluloses results in its resistance to biological and
chemical degradation, with hydrolysis being the limiting step (Kainthola et al.
2019a). Hydrolysis of lignocellulose biodegradation requires several enzymes to
work together, including cellulases, hemicellulases, and lignin-degrading enzymes
(Xu et al. 2019). The main reason for biomass recalcitrance is the low accessibility of
crystalline cellulose fibers, which prevents cellulases from working efficiently.
Equally, the presence of lignin and hemicellulose prevents cellulase from accessing
the substrate efficiently (Mancini et al. 2016a; Xu et al. 2019; Zoghlami and Paës
2019).

4.2.1 Cellulose

Cellulose is a linear polysaccharide consisting of a repeated unit called cellobiose

consisting of D-glucose subunits linked to one another by β-(1,4)-glycosidic bonds
(Su et al. 2018). The cellobiose units are composed of long-chain cellulose poly-
mers, linked together by van der Waals and hydrogen bonds. Cellulose is packed
into microfibrils, which reduce the access of the enzymes and complicate cellulose
degradation (Kumar et al. 2009; Zhang et al. 2019). Cellulose alternates crystalline
and amorphous regions, with the latter being the weakness for chemical and biolog-
ical attack. Cellulose degradation aims to decompose the polysaccharide into free
sugar molecules. The resulting product is glucose, a six-carbon sugar (Singhvi and
Gokhale 2019).
Cellulose crystallinity presents several structures (i.e. Iα, Iβ, II, III, and IV),
depending on the crystallites disposition (Blanco et al. 2018). Native cellulose has
a parallel chain disposition and exists in nature as cellulose Iα and Iβ. Cellulose Iα
shows one-chain triclinic cells and abounds in bacterial cellulose and algae
(Nishiyama 2009). Cellulose Iβ is present in cotton and wood materials and reveals
 90

Fig. 4.1 Schematic representation of the structure of lignocellulosic materials containing cellulose, hemicellulose, and lignin
A. Oliva et al.
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 91

two-chain monoclinic cells (Blanco et al. 2018). Cellulose Iα conversion to cellulose

Iβ is a non-reversible process requiring high temperatures (260–280  C) (Matthews
et al. 2012). Cellulose II is obtained by maceration or dissolution and subsequent
regeneration of cellulose I using solvent-based processes (Corrêa et al. 2010).
Cellulose II presents an antiparallel chain disposition, making it more appreciated
for textile applications and easily accessible for enzymes and microorganisms
(Wikandari et al. 2016). Only very low or high pH allows cellulose solubilisation
in water, whereas solvents like NMMO or ionic liquids dissolve cellulose at neutral
pH (Baruah et al. 2018). Cellulose III is obtained by treating cellulose I and II with
an ammonia solution, whereas cellulose IV derives from cellulose III treatment in
glycerol over 260  C (Corrêa et al. 2010). The transformation to cellulose III and IV
can be reverted using thermal or chemical processes (Isogai et al. 1989; Wada et al.
2006). In contrast, cellulose II is non-reversible to cellulose I (Nagarajan et al. 2017).

4.2.2 Hemicellulose

Hemicellulose does not have a fixed structure. Its backbone can be either a homo-
polymer or a hetero-polymer with short branches linked by β-1,4-glucan bonds and,
occasionally, by β-1,3-glucan bonds (Zhang et al. 2019). These branches consist of
five-carbon sugars (i.e. xylose, rhamnose, and arabinose), six-carbon sugars
(i.e. glucose, mannose, and galactose), and uronic acids. Generally, xylose is the
dominant sugar for hardwoods and agricultural residues, while mannose prevails in
softwoods (Baruah et al. 2018; Singhvi and Gokhale 2019). Contrary to cellulose,
the polymers present in hemicelluloses are easily degradable, due to the amorphous
(non-crystalline) structure and the lower degree of polymerisation. Hemicellulose,
together with lignin, represents a barrier around the cellulose (Fig. 4.1), reducing the
access of cellulases enzymes (Baruah et al. 2018; Zoghlami and Paës 2019). The
most common pretreatments for hemicellulose hydrolysis are dilute acid or alkaline
compounds, steam explosion or enzymes (Zoghlami and Paës 2019).

4.2.3 Lignin

Lignin is the third most abundant polymer in nature, after cellulose and hemicellu-
loses, and generally represents 10–25% of the total feedstock dry matter (Balat
2011). Lignin is an aromatic, complex, three-dimensional cross-linked polymer
synthesized from phenylpropanoid precursors (Fig. 4.1). The lignin structure con-
sists of phenyl propane structural units, liked by aryl ether linkages. The structural
variableness is given by the substitution of the methoxyl groups present in the
aromatic rings (Baruah et al. 2018). The lignin content ranges from 10 to 20% in
various LMs, such as straws, hulls, bagasse, and stalks, and can increase to 30–40%
for nut shells and pinewood (Ponnusamy et al. 2019). The three most common
92 A. Oliva et al.

monomers present in lignin are sinapyl alcohol, coniferyl alcohol, and p-coumaryl
alcohol, corresponding to the three main structural units, which are syringyl,
guaiacyl, and hydroxyphenyl, respectively (Ralph et al. 2019).
Lignin protects the plants from microbial attack and oxidation and gives rigidity
and impermeability to their structure (Ponnusamy et al. 2019). The presence of
lignin is one of the main drawbacks of using LMs in fermentation and AD, as it
makes lignocelluloses resistant to chemical and biological degradation by reducing
the hydrolysis rate (Reddy and Yang 2005). Organic solvents, thermal, and fungal
pretreatment are generally suggested for efficient lignin removal (Amin et al. 2017;
Singhvi and Gokhale 2019).

4.3 Parameters Affecting Lignocellulose Conversion

to Biofuels

The biodegradation of LMs is influenced by four main factors, i.e. (1) accessible
surface, (2) crystallinity and degree of polymerization of cellulose, as well as the
(3) lignin and (4) hemicellulose content (Mancini et al. 2016a). Cellulose accessi-
bility is a key factor in the bioconversion of LMs to fermentable sugars. Thus, the
contact between cellulose and cellulase is one of the most critical factors affecting
the enzymatic hydrolysis yield and rate. The contact area depends on biomass
porosity and particle size (Meng and Ragauskas 2014; Xu et al. 2019). Cellulase
accessibility to cellulose mainly depends on porosity, rather than the external surface
of the substrate (Siqueira et al. 2017). Over 90% of the substrate enzymatic digest-
ibility depends on the substrate porosity (Wang et al. 2012). In AD, a limited access
to cellulose results in a scarce contact between biomass and hydrolytic bacteria,
which reduces the release of fermentable sugars for the subsequent degradation
steps. The accessible surface increases along the AD process proportionally with
the degradation of the cell wall components (Xu et al. 2019). The microorganisms-
substrate contact controls the hydrolysis efficiency, especially during the first days of
AD. On the contrary, other factors prevail later, such as the compact structure and the
degree of crystallinity of the remaining cellulose (Oliva et al. 2021; Xu et al. 2019).
The ordered structure and high crystallinity of cellulose are the main deterrents to
convert it to biofuels. Nevertheless, amorphous regions, which are more accessible
to enzymatic attack, are randomly present in the cellulose structure (Zoghlami and
Paës 2019). The cellulose becomes more accessible by decreasing the crystallinity
degree, enhancing the biofuel production from cellulose-rich materials (Jeihanipour
et al. 2011). As described in Sect. 4.2.1, cellulose is a linear homopolymer composed
of microfibrils, joint together to form fibrils and finally fibers. The degree of
polymerization refers to the average length of the polysaccharide chains. An increase
in the degree of polymerization is reflected in a higher density and tensile strength of
the cellulosic component of LMs, making cellulose hydrolysis more difficult (Hallac
and Ragauskas 2011; Mattonai et al. 2018).
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 93

A further factor affecting the hydrolysis of LMs is the presence of hemicellulose

and lignin. Hemicellulose and lignin form a physical barrier around cellulose. It is
important to remove or alter them, while avoiding the degradation of the hemicel-
lulose sugars to obtain a high sugar yield (Singhvi and Gokhale 2019; Xu et al. 2019;
Zoghlami and Paës 2019). Hemicellulose contributes to the resistance of the plant
cell wall and reduces the overall hydrolysis rate of LMs (Xu et al. 2019). Neverthe-
less, hydrolysis and acidification of hemicellulose alone are faster than those of
cellulose, suggesting that the methane production from hemicellulose can be
optimised by controlling the organic loading rate (Li et al. 2018). Other studies
reported that the hemicellulose-cellulose linkages contribute to reducing the crystal-
linity of cellulose, enhancing the hydrolysis step (Li et al. 2015; Xu et al. 2012). On
the other hand, lignin is well known to negatively affect the AD process by reducing
the biodegradability of LMs and the overall methane yield (Li et al. 2018). Lignin
consolidates the cell wall structure and prevents contact of hydrolytic enzymes with
carbohydrates. In addition, lignin is capable of adsorbing cellulase enzymes, further
protecting the cellulosic component of LMs (Lu et al. 2016).

4.4 Pretreatment Methods to Enhance Methane Production

from Lignocellulosic Materials

The use of LMs for methane production is limited due to their resistance to the
enzymatic attack (Mahmood et al. 2019). Therefore, many studies have focused on
developing cost-effective pretreatments to reduce the recalcitrance of LMs
(Table 4.2) (Lee et al. 2021; Matsakas et al. 2020; Oliva et al. 2021). Pretreatments
aim to increase the efficacy of lignocellulose hydrolysis by improving the accessi-
bility to cellulose. This scope can be achieved by removing or altering the lignin and
hemicellulose fraction of LMs (Fig. 4.2) (Ali et al. 2020a; Haldar and Purkait 2021).
To be efficient and economically advantageous, pretreatment methods should
meet the following features: (1) high recovery of carbohydrates, (2) high digestibility
of the cellulose in the subsequent enzymatic hydrolysis, (3) high solid concentrations
as well as a high concentration of free sugars in the liquid fraction, (4) no destruction
of hemicelluloses and cellulose, (5) no formation of possible inhibitors for hydrolytic
enzymes and fermenting microorganisms, (6) cost-effectiveness and low consump-
tion of chemicals, (7) low generation of residues, and (8) low capital and operational
costs (Kumari and Singh 2018; Taherzadeh and Karimi 2008).
Pretreatment techniques can be differently classified. A first classification con-
cerns the pH maintained during the process, with pretreatments grouped in acidic,
neutral and alkaline (Ravindran and Jaiswal 2016). Nevertheless, the most common
classification categorises pretreatments into physical, chemical, physicochemical
and biological (Table 4.2) (Singh et al. 2015).
94 A. Oliva et al.

Table 4.2 Effectiveness of different pretreatment methods on the enhancement of the methane
potential of lignocellulosic materials
Category Pretreatment Substrate Δ CH4 Reference
Physical Ball milling Wheat straw +49% Dell’Omo and
Spena (2020)
Microwave Energy crop (Sida +39% Zieliński et al.
hermaphrodita) (2019)
Extrusion Rice straw +72% (Chen et al. 2014)
Chemical Alkaline (NaOH) Wheat straw +15% Mancini et al.
(2018a)
Acid (H2SO4) Cassava residues +57% Zhang et al.
(2011)
Ionic liquid Rice straw +137% Gao et al. (2013)
([C4mim]Cl/DMSO)
Physicochemical Steam explosion Rubber wood waste +670% Eom et al. (2019)
Liquid hot water Sunflower residues +173% Lee and Park
(2020)
Biological Fungal (white-rot) Rice straw +114% Kainthola et al.
(2019b)
Microbial consortia Napier grass +49% Wen et al. (2015)
(WSD-5)
Microbial consortia Catalpa sawdust +76% Ali et al. (2020b)
(CS-5)

Fig. 4.2 Schematisation of the effect of pretreatment on the lignocellulosic structure

4.4.1 Physical Pretreatments

The most common physical pretreatments include different milling (e.g. ball, col-
loid, vibro-energy, roller, and hammer), extrusion and irradiation, with the primary
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 95

objective of increasing the accessible surface area and decreasing cellulose crystal-
linity and degree of polymerisation (Amin et al. 2017).
Milling is a size reduction technique employed to increase the surface/volume
ratio and alter the structure and the degree of crystallinity of LMs, making pretreated
substrates more amenable to cellulase attack (Ravindran and Jaiswal 2016).
Irradiation pretreatments involve gamma rays, electron beam, ultrasounds (see
Sect. 4.7) and microwaves, intending to improve the enzymatic hydrolysis of
lignocelluloses (Taherzadeh and Karimi 2008). The pretreatment effectiveness is
proportional to the lignin content, resulting in a lower efficiency on less recalcitrant
(i.e. low lignin) substrates (Keikhosor et al. 2013).
Extrusion pretreatment relies on the spinning of a single or twin screw into a
temperature-controlled barrel. The mechanical action causes strong shearing forces
between the substrate, the screw, and the barrel, locally increasing pressure and
temperature. Apart from the particle size reduction, those forces alter also the
biomass structure and change the crystallinity of the cellulosic component of the
biomass (Duque et al. 2017; Zheng and Rehmann 2014).

4.4.2 Chemical Pretreatments

Chemical pretreatments act directly on the main components of the biomass, remov-
ing lignin and hemicellulose or decreasing the crystallinity degree of the cellulose
(Ponnusamy et al. 2019). The chemical agents employed are divided into four main
categories: alkali, acids, salts, and organic solvents.
Alkaline pretreatment involves base solutions, e.g. NaOH, KOH, and Ca(OH)2,
aiming to enhance the digestibility of LMs. The main effects on LMs are lignin and
hemicellulose removal, an increase in porosity and the reduction of polymerisation
and crystallinity degree of the cellulose (Tu and Hallett 2019). Alkaline pretreat-
ments occur at mild conditions, i.e. ambient pressure and temperature, but generally
last over 24 h (Amin et al. 2017). NaOH and KOH are the most employed basic
solutions for alkaline pretreatment, having particular effectiveness for lignin removal
from low lignin content LMs (Baruah et al. 2018). Nevertheless, the recycling of
alkaline solutions is challenging, and Na+ and K+ ions can inhibit the subsequent AD
process (Bianco et al. 2021b). On the other hand, a Ca(OH)2 solution can be more
easily recovered but is less effective than NaOH and KOH based solutions (Amin
et al. 2017).
Acid pretreatment is performed with diluted or concentrated solutions. Organic
acids, such as formic acid, as well as inorganic acids, i.e. sulfuric, nitric, phosphoric,
and hydrochloric, are widely employed (Baruah et al. 2018). Dilute acid
pretreatment (0.1–5%) aims to remove the hemicellulosic component of the biomass
and is effective at high temperatures (100–250  C). On the other hand, acids
concentrated at 30–70% hydrolise both cellulose and hemicellulose and require
temperatures below 100  C (Solarte-Toro et al. 2019). Acid solutions are unable to
dissolve lignin but alter the cellulose-hemicellulose-lignin linkages, which increases
96 A. Oliva et al.

the biodegradability of the solid residues (Amin et al. 2017). Nevertheless, the
formation of inhibitory compounds can occur. Phenolic compounds, aldehydes
and furfurals are the most know inhibitory compounds observed after acid pre-
treatments (Ali et al. 2020a). Acid-pretreated solid residues of LMs require abundant
washing to be ready for AD or other biological processes (Rajan and Carrier 2014).
On the other hand, before undergoing fermentation processes, the pH of the hydro-
lysate has to be neutralised with alkaline solutions (Gonzales et al. 2017), which
increases the overall process costs (Castilla-Archilla et al. 2021) and can create
further inhibition (Bianco et al. 2021b).
Ionic liquids and NMMO (see Sect. 4.6) act on the cellulosic component of LMs
(Halder et al. 2019; Mancini et al. 2016b). Ionic liquids are salts in the liquid state at
room temperature in which isolated ions and cations interact by Coulomb forces.
NMMO is a zwitterion containing localised positive and negative charges in a single
molecule (Böhmdorfer et al. 2017). Ionic liquids and NMMO dissolve cellulose,
which can then be regenerated using an anti-solvent (Mancini et al. 2016a). The
regenerated cellulose shows a lower crystallinity, which is a critical factor for the
bioconversion of LMs (Xu et al. 2019). NMMO is effective at different concentra-
tions in aqueous solutions (Wikandari et al. 2016), the mechanisms of which are
thoroughly discussed in Sect. 4.6.
Organosolv pretreatment (see Sect. 4.5) involves organic solvents such as etha-
nol, methanol, and acetic acid, heated to high temperature to remove lignin and
reduce the recalcitrance of LMs. Lignin is a valuable product and can be recovered at
good purity levels after organosolv pretreatment (Ferreira and Taherzadeh 2020).

4.4.3 Physicochemical Pretreatments

Physicochemical pretreatment methods combine chemical and physical approaches.

This category includes several pretreatment methods such as liquid hot water, wet
oxidation, ammonia fiber explosion, steam explosion, and CO2 explosion. Steam
explosion and liquid hot water are the two most studied strategies.
Steam explosion combines thermal and pressure effects to hydrolyse the
hemicellulosic component of the biomass (Jacquet et al. 2015). LMs undergo
high-pressure (5–50 atm) saturated steam at temperatures between 160 and
260  C. The pretreatment time is generally short (1–10 min) and inversely related
to temperature (Amin et al. 2017). Water molecules firstly penetrate LMs and
explosively escape once pressure is released, causing cell wall disruption (Ravindran
and Jaiswal 2016). Higher temperatures lead to substantial hemicellulose hydrolysis
into glucose and xylose monomers, liberating acetic acid, which acts as a catalyst to
hydrolyse the remaining sugars (Baruah et al. 2018). However, harsh pretreatment
conditions can generate inhibitors such as phenolic compounds, formic and levulinic
acid (Cantarella et al. 2004; Martín et al. 2018). Alternatively, an external catalyst
(e.g. H2SO4, SO2, H3PO4, and CO2) allows lowering the pretreatment temperature
while maintaining a high hemicellulose hydrolysis rate (Duque et al. 2016).
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 97

Liquid hot water pretreatment, similarly to steam explosion, requires high tem-
perature and pressure to remove hemicellulose and disrupt lignin bonds making the
remaining cellulose more available for AD (Hashemi et al. 2019b; Qiao et al. 2011).
Contrary to steam explosion pretreatment, water remains in the liquid state, and
pressure allows maintaining this status at high temperatures (Ruiz et al. 2020). The
hemicellulose sugars, together with other hydrolysable compounds, are hydrolysed,
making liquid hot water pretreatment ideal for LMs rich in non-structural sugars
(Ravindran and Jaiswal 2016). pH has to be controlled to avoid the formation of
inhibitory compounds (Yang et al. 2018). Liquid hot water pretreatment does not
need particle size comminution but demands a large amount of water (Baruah et al.
2018).

4.4.4 Biological Pretreatments

Biological pretreatments include enzymes, microbial consortia, and fungal strains

(Baruah et al. 2018). Compared to physical and chemical methods, a biological
pretreatment has various advantages, such as no requirement for chemicals and a
lower energy input (Taherzadeh and Karimi 2008). Biological pretreatment methods
are performed under mild environmental conditions, which reduce the risk of
generating inhibitory compounds (Ravindran and Jaiswal 2016). Nevertheless, the
long pretreatment time and the competition for carbohydrates between organisms
carrying out pretreatment and biogas production limit biological pretreatments in
commercial applications (Tu and Hallett 2019).
Several wood-decay fungi have been studied, and white-rot strains are most
interesting for the biological pretreatment of LMs, since they can selectively
metabolise lignin from LMs with low carbohydrate consumption (Amin et al.
2017). In contrast, soft-rot and brown-rot fungi use enzymes to degrade cellulose
and hemicellulose with minimal lignin removal (Ravindran and Jaiswal 2016).
White rot fungi have two enzyme systems: the oxidative ligninolytic system and
the hydrolytic system. The first one makes use of three enzymes, i.e. lignin peroxide,
manganese peroxide and laccase, which attack the phenyl rings in lignin. The second
one degrades cellulose and hemicellulose to release fermentable sugars using cellu-
lase and hemicellulase enzymes (Nadir et al. 2019). White-rot fungi degrade lignin
with two modes of action, namely selective and non-selective decay. In selective
decay mode, fungi selectively degrade the lignin and hemicellulose fractions, while
the cellulose fraction is essentially unaffected. Non-selective degradation consumes
similar amounts of cellulose, hemicellulose, and lignin for fungal growth (Baruah
et al. 2018).
Microbial consortia employ mixed cultures to increase the total sugar yield and
reduce the lignin content of LMs (Ali et al. 2020b; Wen et al. 2015; Zhong et al.
2016). Similarly to fungal pretreatment, enzymatic pretreatment uses pure enzymes
(e.g. laccase, manganese peroxidase, cellulase, and xylanase) to achieve the same
goals (Koupaie et al. 2019). Nevertheless, microbial consortia and enzymatic
98 A. Oliva et al.

pretreatment are still attempting to meet a cost-effective balance beyond the labora-
tory scale (Koupaie et al. 2019; Wen et al. 2015).

4.5 Organosolv Pretreatment

4.5.1 Mechanism and Process Parameters of Organosolv

Pretreatment

Organosolv pretreatment is the most efficient pretreatment method to remove lignin,

which protects the polysaccharides against degradation (Ostovareh et al. 2015).
Organosolv is a chemical pretreatment in which the LMs are mixed with an organic
or aqueous organic solvent and heated to dissolve the lignin component. Besides,
depending on the operating conditions, partial hemicellulose hydrolysis can occur
(Oliva et al. 2021). After pretreatment, lignin can be extracted from the solvent by
precipitation, membrane filtration (Arkell et al. 2014), and water electrolysis (Jin
et al. 2013). Precipitation is the most employed strategy and is performed via
acidification of the lignin-rich liquor (Mussatto et al. 2007).
A wide range of organic or aqueous organic solvents has been explored to pretreat
LMs, with or without the addition of inorganic or organic acid catalysts (Table 4.3).
The optimal process temperature depends on the type of biomass, solvent, and
catalyst, and usually ranges from 150 to 200  C (Taherzadeh and Karimi 2008).
Organic solvents are classified into low boiling point alcohols (e.g. methanol and
ethanol), higher boiling point alcohols (e.g. ethylene glycol and glycerol), and other
classes of organic compounds (e.g. dimethyl sulfoxide, ethers and ketones) (Borand
and Karaosmanoǧlu 2018). In the choice of the solvent, the price and easiness of
recovery should also be considered. The solvent should be separated and reused to
reduce the operational costs of the process (Zhou et al. 2018). Also, solvent residues
must be removed from the pretreated material to avoid the inhibition of the subse-
quent AD process (Behera et al. 2014; Harmsen et al. 2010). Due to the high cost of
the solvents, ethanol and methanol are preferred over alcohols with a higher boiling
point. The most employed catalysts to enhance the pretreatment effectiveness are
hydrochloric, sulfuric, and phosphoric acid (Ferreira and Taherzadeh 2020), but
organic acids such as acetic and formic acid have also been investigated (Borand and
Karaosmanoǧlu 2018).
After organosolv pretreatment, three separate components are obtained: a pure
cellulose fraction, an aqueous hemicellulose stream, and a highly pure lignin fraction
(Meng et al. 2020). Further, similarly to other pretreatments, organosolv causes a
decrease in the crystallinity of LMs and enhances the accessibility of carbohydrates
for microbial degradation (Mancini et al. 2016a). At higher temperatures, the
cellulose fraction of the biomass is also degraded. Furthermore, to be effective, the
pretreatment time varies between 0.5 and 2 h. Under these process conditions,
organosolv is well suitable to dissolve hemicellulose, recover cellulose, and make
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 99

Table 4.3 Effectiveness of organosolv pretreatment for lignin removal (Δlignin) and increment of
the methane potential (ΔCH4) of different lignocellulosic materials
Optimal pretreatment Pretreatment
Substrate condition effectivenessa Reference
Rice straw 50% EtOH, 180  C, 1 h Δlignin: 18% Mancini et al. (2018b)
ΔCH4: +41%
Rice straw 75% EtOH, 150  C, 1 h, Δlignin: 22% Mirmohamadsadeghi
catalyst ΔCH4: +32% et al. (2014)
Wheat straw 50% EtOH, 180  C, 1 h Δlignin: 14% Mancini et al. (2018a)
ΔCH4: +15%
Sugarcane 25% EtOH +10% Ammonia, Δlignin: 49% Hashemi et al. (2019a)
bagasse 70  C, 12 h ΔCH4: +135%
Sweet sorghum 50% PrOH, 160  C, 0.5 h, Δlignin: 25% Ostovareh et al. (2015)
stalks catalyst ΔCH4: +107%
Sunflower 50% EtOH, 160  C, 0.5 h Δlignin: 26% Hesami et al. (2015)
stalks ΔCH4: +124%
Hazelnut skin 50% MeOH, 130  C, 1 h, Δlignin: 9% Oliva et al. (2021)
catalyst ΔCH4: +1700%
Hazelnut skin 50% EtOH, 180  C, 1 h Δlignin: N.O. Mancini et al. (2018b)
ΔCH4: +10%
Almond shell 50% MeOH, 200  C, 1 h Δlignin: N.O. Oliva et al. (2021)
ΔCH4: +7%
Cocoa bean 50% EtOH, 180  C, 1 h Δlignin: N.O. Mancini et al. (2018b)
shell ΔCH4: N.O.
Spent coffee 50% MeOH, 200  C, 1 h, Δlignin: N.O. Oliva et al. (2021)
grounds catalyst ΔCH4: +10%
Forest residues 50% MeOH, 190  C, 1 h, Δlignin: 4% Kabir et al. (2015)
catalyst ΔCH4: +320%
Elmwood 75% EtOH, 180  C, 1 h, Δlignin: 27% Mirmohamadsadeghi
catalyst ΔCH4: +73% et al. (2014)
Pinewood 75% EtOH, 150  C, 0.5 h, Δlignin: N.O. Mirmohamadsadeghi
catalyst ΔCH4: +84% et al. (2014)
Rubber wood 75% EtOH, 210  C, 0.5 h Δlignin: 74% Tongbuekeaw et al.
waste ΔCH4: +179% (2020)
a
N.O. means that no significant effect on the specific parameter was observed

it more susceptible to enzymatic hydrolysis (Ferreira and Taherzadeh 2020). Typi-

cally, an uncatalysed organosolv pretreatment is efficient only towards low-density
hardwoods and agricultural residues. Softwoods or high-density hardwoods require
more severe pretreatment conditions (Harmsen et al. 2010).
100 A. Oliva et al.

4.5.2 Benefits and Drawbacks

The benefits of organosolv pretreatment include: (1) production of high-quality

lignin, which can be used for several applications, (2) reduced amount of waste
produced, (3) lower energy use, (4) increase in porosity, (5) removal of lignin and the
reduction of the hemicellulose fraction, which may shorten the hydrolysis stage,
(6) low formation of inhibitory compounds, compared to the industrially most
employed pretreatments, and (7) easy solvent recovery. On the contrary, potential
drawbacks of the organosolv pretreatment are the high operational and investment
costs, as well as the risk of explosion due to the use of organic solvents and the high
temperatures employed (Ferreira and Taherzadeh 2020; Meng et al. 2020; Zhou et al.
2018).

4.5.3 Effectiveness of Organosolv Pretreatment on Different

Lignocellulosic Materials

Organosolv pretreatment, performed with 75% ethanol using sulfuric acid as a

catalyst, improves the methane production from hardwood elm, softwood pine,
and rice straw (Mirmohamadsadeghi et al. 2014). The optimal process parameters
vary depending on the substrate. Rice straw increases its methane potential by 32%
when pretreated at 150  C for 60 min. Instead, 30 min pretreatment is sufficient to
enhance the methane production from pinewood by 84%. On the other hand, a
higher pretreatment temperature (i.e. 180  C) has a positive effect on elmwood
(Mirmohamadsadeghi et al. 2014). Ostovareh et al. (2015) improved the bioconver-
sion of sweet sorghum stalks to ethanol and biogas by organosolv pretreatment.
Nevertheless, the use of the acid catalyst improves the methane yield only at lower
pretreatment temperatures.
The organic solvent selection plays a key role when pretreating hazelnut skin
(Table 4.3). The use of methanol and catalysed-methanol is more efficient than
ethanol organosolv pretreatment (Mancini et al. 2018b; Oliva et al. 2021). In
contrast, methanol-organosolv pretreatment is not strong enough to overcome the
recalcitrance of nut shells, such as almond shell, and causes a loss of biodegradable
matter from spent coffee grounds, decreasing the methane production (Oliva et al.
2021). On the other hand, ethanol-organosolv pretreatment is particularly effective
on rice straw and wheat straw, raising the methane potential of the two LMs to
332 and 316 mL CH4/g VS, respectively (Mancini et al. 2018a, b).
Organosolv pretreatment was effective on forest residues using three different
organic solvents (i.e. ethanol, methanol, and acetic acid) with or without 1% w/w
sulfuric acid, acetic acid, and hydrochloric acid as catalysts (Kabir et al. 2015). The
methane potential of catalyst-free pretreated forest residues achieves 300, 230, and
330 mL CH4/g VS for ethanol, methanol, and acetic acid pretreated LM, respec-
tively, rather than the untreated biomass that reaches only 50 mL CH4/g VS. The use
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 101

of catalysts coupled to ethanol and methanol improved methane production from

forest residues, but the methane yield decreased in the case of acetic acid catalysed
pretreatment (Kabir et al. 2015). The economic analysis performed in that study
showed that the methanol supply and recovery were cheaper than for the other two
solvents (Kabir et al. 2015). Ethanol-organosolv pretreatment can also be combined
with steam explosion to enhance the AD of birch and spruce woodchips (Matsakas
et al. 2020).
Recent studies have focused on rubber wood waste valorisation using organosolv
pretreatment under a biorefinery approach (Charnnok et al. 2020; Tongbuekeaw
et al. 2020). Organosolv pretreatment (75% ethanol) significantly increases the
methane production from rubber wood waste from 59 to 166 mL CH4/g VS,
reducing the lignin content by 74% (Tongbuekeaw et al. 2020). Sequential
ethanol-organosolv and enzymatic pretreatments enhance the methane potential of
rubber wood waste. The organosolv step removes almost 50% of the lignin,
obtaining a glucose-rich hydrolysate for the subsequent enzyme-assisted AD
(Charnnok et al. 2020).

4.6 N-Methylmorpholine N-Oxide Pretreatment

4.6.1 Mechanisms and Process Parameters of the NMMO

Pretreatment

NMMO is a cyclic, tertiary amine, aliphatic oxide capable of dissolving cellulose by

disrupting the original hydrogen bonds and creating new linkages with the dissolved
polymer (Sari and Budiyono 2014; Satari et al. 2019). The effect on cellulose
depends on the NMMO hydration (Fig. 4.3). A water content lower than 17%
completely dissolves cellulose, while an NMMO content of 76–82% leads to the

Fig. 4.3 Effect of NMMO

pretreatment on cellulose
fibers
102 A. Oliva et al.

swelling of the cellulose fibers by creating balloons, in which the cellulose starts to
dissolve. A further increase of the water content (25–30%) reduces the cellulose
dissolution and mainly results in the swelling of the cellulose fibers. An NMMO
content lower than 65% proportionally decreases cellulose swelling, with no disso-
lution observed (Cuissinat and Navard 2006). The ability of NMMO in dissolving
cellulose is attributed to its chemical structure, presenting weak polar N-O bonds
with a negative charge on oxygen and positively charged on nitrogen. NMMO tends
to form new hydrogen bonds with both water and cellulose, preferentially with
cellulose until the water content of the aqueous solution is below 17% (Mancini
et al. 2016a; Wikandari et al. 2016).
The interest in NMMO started in the early twentieth century with applications in
the textile industry and culminating with its usage in the Lyocell process (Sayyed
et al. 2019). Recently, the effectiveness of NMMO on cellulose has allowed its use
as a pretreatment for LMs (Table 4.4) (Khoshnevisan et al. 2016; Shafiei et al. 2011;
Sołowski et al. 2020). The dissolution mode (85% NMMO) is recommended for
ethanol production, while a lower NMMO concentration results in a better improve-
ment in terms of methane production (Jeihanipour et al. 2010). The dissolution mode
foresees the complete solubilization and subsequent regeneration of the cellulosic
component of the biomass. The rate of cellulose dissolution is inversely related to the
cellulose concentration and degree of polymerization (Wikandari et al. 2016). The
regeneration of cellulose occurs by adding an anti-solvent, such as boiling water
(Cuissinat and Navard 2006). The regenerated cellulose shows a lower total crystal-
linity index (TCI) and lateral order index (LOI), which facilitate the microbial attack.
On the other hand, NMMO pretreatment in swelling/ballooning mode allows a
higher increase in porosity, with a lower decrease of the crystallinity indexes
(Jeihanipour et al. 2010).
TCI and LOI of cellulose-based materials can be estimated by Fourier-transform
infrared spectroscopy. TCI is expressed as the ratio of the absorbance value at 1375
and 2902 cm1 and is proportional to the entire crystallinity of the sample. LOI is
representative of the ratio between cellulose I and cellulose II, and is calculated as
the infrared spectral ratio 1420/893 cm1. The LOI increases with the crystallinity of
cellulose I and decreases with the increasing crystallinity of cellulose II (Carrillo
et al. 2004; Nelson and O’Connor 1964a, b).

4.6.2 Benefits and Drawbacks

NMMO is often recommended as the most advantageous cellulose solvent compared

to the well-known phosphoric acid, alkaline solutions, and other ionic liquids.
NMMO offers the advantage of acting directly on the cellulosic component of the
biomass, reducing the risk of losing carbohydrates, in contrast with other pretreat-
ments such as steam explosion, alkaline, phosphoric acid, and biological pretreat-
ments (Wikandari et al. 2016). Depending on the stage of hydration, NMMO acts
differently on the cellulosic component of the biomass, by increasing the porosity or
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 103

Table 4.4 Effectiveness of NMMO pretreatment on glucan or total carbohydrates content

(Δglucan, Δcarbo), crystallinity index (ΔLOI), water swelling capacity (ΔWSC), and increment
of the methane potential (ΔCH4) of various lignocellulosic materials
Optimal pretreatment Pretreatment
Substrate condition effectivenessa Reference
Cotton linters 73% NMMO, 90  C, 1 h ΔLOI: 22% Jeihanipour et al.
ΔWSC: 23% (2010)
ΔCH4: +17%
Rice straw 85% NMMO, 130  C, Δglucan: +16% Teghammar et al.
1h ΔCH4: +629% (2012)
Rice straw 85% NMMO, 120  C, ΔLOI: N.O. Mancini et al.
3h ΔCH4: +81% (2016b)
Triticale straw 85% NMMO, 130  C, Δglucan: +35% Teghammar et al.
15 h ΔCH4: +583% (2012)
Wheat straw 85% NMMO, 120  C, Δglucan: N.O. Mancini et al.
3h ΔWSC: +46% (2018a)
ΔCH4: +11%
Barley straw 85% NMMO, 90  C, 7 h Δcarbo: +5% Kabir et al. (2014)
ΔCH4: +92%
Hazelnut skin 85% NMMO, 120  C, ΔLOI: 9% Mancini et al.
3h ΔCH4: N.O. (2016b)
Cocoa bean shell 85% NMMO, 120  C, ΔLOI: +15% Mancini et al.
3h ΔCH4: +14% (2016b)
Oil palm empty fruit 85% NMMO, 120  C, Δglucan: N.O. Purwandari et al.
bunch 3h ΔLOI: 76% (2013)
ΔCH4: +48%
Forest residues 85% NMMO, 90  C, Δcarbo: +7% Kabir et al. (2014)
30 h ΔCH4: +114%
Forest residues 85% NMMO, 120  C, Δcarbo: +10% Aslanzadeh et al.
3h ΔCH4: +162% (2014)
Birch wood (milled) 85% NMMO, 130  C, Δglucan: +8% Goshadrou et al.
3h ΔWSC: +57% (2013)
ΔLOI: 18%
ΔCH4: +48%
Softwood spruce 85% NMMO, 130  C, Δglucan: +12% Teghammar et al.
chips 15 h ΔCH4: +1088% (2012)
Pinewood chips 85% NMMO, 120  C, Δglucan: +12% Shafiei et al. (2014)
30 h ΔWSC: +280%
ΔLOI: 6%
ΔCH4: +580%
Cassava residues 85% NMMO, 90  C, 2 h ΔLOI: 19% Cheng et al. (2017)
ΔCH4: +28%
a
N.O. means that no significant effect on the specific parameter was observed
104 A. Oliva et al.

reducing the crystallinity of LMs (Jeihanipour et al. 2010). NMMO pretreatment can
be operated in relatively mild conditions, with temperatures ranging from 90 to
130  C and pretreatment times between 20 min and 30 h (Mancini et al. 2016a).
The pretreatment feasibility strongly depends on solvent recovery and recycling.
The solvent recovery takes place by treating the liquid stream of the NMMO
pretreatment with ion-exchange resins to remove contaminants and subsequent
evaporation of the water, obtaining the monohydrate form of NMMO (Satari et al.
2019). NMMO pretreatment non-recyclable liquid waste streams can be treated with
ozone, with ozonation products being easily biodegradable at neutral pH (Stockinger
et al. 1996). The loss of NMMO during recovery is <2% (Sari and Budiyono 2014).
Nevertheless, the use of NMMO as a pretreatment for LMs on a large scale is still
limited because of the considerable amount of water required for the washing step
(Mancini et al. 2016a).
The efficacy of recovered NMMO depends on the chemical composition of the
LMs. In particular, the pretreatment of lignin-rich LMs reduces the efficiency of
recovered NMMO (Millati et al. 2020), most likely due to negative side reactions
and release of by-products such as tannins, resin acids, and phenolic compounds
(Kabir et al. 2014). The NMMO action during pretreatment does not produce furans,
reducing the risk of inhibition in the subsequent biofuel production processes
(Wikandari et al. 2016). However, leftover NMMO after pretreatment can inhibit
the AD process, even at low (0.5–1%) concentrations (Millati et al. 2020).

4.6.3 Effectiveness of NMMO Pretreatment on Different

Lignocellulosic Materials

Recent studies show the effects of 85% NMMO pretreatment at 120  C on rice straw,
wheat straw, and hazelnut skin (Mancini et al. 2016b, 2018a). The pretreatment is
particularly effective on rice straw, increasing the methane production by 82%, even
though no significant effect on the LOI was observed. On the other hand, NMMO
pretreatment significantly reduces the crystallinity index of pretreated hazelnut skin,
resulting in a higher methane production during the first days of AD (Mancini et al.
2016b). Similarly to hazelnut skin, NMMO pretreatment increases the porosity of
wheat straw, enhancing the specific rate constant Rm from 21 to 32 mL CH4/g VS/d
(Mancini et al. 2018a).
Teghammar et al. (2012) investigated the effect of 85% NMMO pretreatment on
spruce wood, rice straw and triticale straw, observing that only for rice straw a longer
pretreatment time (i.e. 15 h) leads to an inhibition of the AD process. On the other
hand, 15 h NMMO pretreatment is particularly efficient for spruce wood and triticale
straw, especially when no comminution is performed. The negative effect of a longer
pretreatment time is due to the loss of glucan and xylan during pretreatment
(Teghammar et al. 2012). On the contrary, all investigated pretreatment times
(i.e. 1, 3, and 15 h) can positively affect the AD of pinewood, confirming that a
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 105

longer time is required when larger particle size substrates undergo NMMO
pretreatment (Shafiei et al. 2014). The dissolution mode pretreatment (i.e. 85%
NMMO) increases the porosity and reduces the crystallinity of birch wood after
3 h pretreatment, enhancing the methane potential by 47% (Goshadrou et al. 2013).
To the authors’ knowledge, only Purwandari et al. (2013) studied the effect of the
NMMO concentration on pretreatment of LMs, although it is reported to be a key
factor for cellulose hydrolysis and subsequent conversion to methane (Jeihanipour
et al. 2010). The authors investigated 73, 79, and 85% NMMO pretreatment at
90 and 120  C. A longer pretreatment time at 90  C positively affects the subsequent
AD of oil palm empty fruit bunches. In contrast, increasing the pretreatment
temperature to 120  C, the optimal pretreatment time is 3 h, and the significance
of the NMMO concentration is attenuated (Purwandari et al. 2013).

4.7 Ultrasound Pretreatment

4.7.1 Mechanism and Process Parameters of Ultrasound

Pretreatment

Ultrasound, or sonication, techniques have been widely employed in medical appli-

cations as well as chemical and food processing for decades using different frequen-
cies depending on the purpose (Chemat et al. 2011; Miller et al. 2012; Suslick 1999).
Recently, ultrasounds are getting attention as pretreatment method to enhance the
bioconversion of lignocellulosic and other waste materials due to their environmen-
tal friendly approach (Table 4.5) (Bussemaker and Zhang 2013). Ultrasound
pretreatment relies on diffusion of sound waves (> 20 kHz) in liquid media, creating
alternations of compression and rarefaction phenomena (Fig. 4.4a) (Yang et al.
2017). The alternation of high and low pressurised zones gives rise to gas bubbles
that grow up with the sound waves diffusion until imploding for cavitation
(Fig. 4.4b). The bubbles collapse releases a considerable amount of energy, creating
zones with high temperature (2000–5000 K) and pressure (up to 1800 atm) (Hassan
et al. 2018; Luo et al. 2014).
Ultrasonic cavitation is affected by several parameters. Firstly, the frequency
(f) emitted by the ultrasonic device plays a key role. It is reported that a higher
intensity is required to obtain cavitation at high sonic frequencies, overwhelming the
cohesive forces of the medium (Luo et al. 2014; Santos et al. 2009). The intensity of
sonication is proportional to the amplitude of vibration of the ultrasonic source.
Generally, high amplitudes are not recommended, since they can easily lead to
deterioration of the ultrasonic transducer. The optimal amplitude also depends on
the viscosity of the medium. Higher amplitudes are required when the resistance of
the sample to the movement of the ultrasonic device increases (Santos et al. 2009).
Another important parameter to take into account when performing ultrasound
pretreatment is the power density (Pd) (Zou et al. 2016b), calculated as:
106 A. Oliva et al.

Table 4.5 Effectiveness of ultrasound pretreatment on reducing sugars (Δsugars), crystallinity

(ΔCrI), lignin removal (Δlignin), and increment of methane potential (ΔCH4) for different ligno-
cellulosic materials
Optimal pretreatment
Substrate condition Effectivenessa Reference
Wheat straw (co-digestion with f: 40 kHz, P: 200 W, t: 20 min Δsugars: Zou et al.
dairy manure) +24% (2016a)
ΔCH4:
+80%
Maize straw (co-digestion with f: 50 kHz, P: 250 W, t: 30 min Δsugars: Zou et al.
dairy manure) +16% (2016b)
ΔCH4:
+70%
Wheat straw f: 20 kHz, P: 450 W, t: 10 min ΔCH4: +47% Korai and Li
(ultrasound assisted with (2020)
KOH)
Cannabis straw f: 40 kHz, P: 100 W, t: 30 min ΔCrI: +21% Qi et al.
ΔCH4: (2021)
+77%
Corn stover f: 40 kHz, P: 200 W, t: 90 min ΔCH4: +15% Hassan et al.
(2017)
Grape pomace f: 50 kHz, P: 60 W, t: 70 min Δlignin: El Achkar
18% et al. (2018)
ΔCH4:
+10%
Olive pomace f: 20 kHz, P: 450 W, t: 10 min Δlignin: Elalami
10% et al. (2020)
ΔCH4: N.O.
Sida hermaphrodita (L.) f: 25 kHz, P: 300 W, t: 10 min ΔCH4: Kisielewska
Rusby (codigestion with cattle +127% et al. (2020)
manure)
Vinegar residues (grinded) f: 40 kHz, P: 50 W, t: 60 min ΔCH4: +30% Kong et al.
(2021)
a
N.O. means that no significant effect on the specific parameter was observed

P∙t
Pd ¼
V ∙ TS0

where P is the ultrasonic power (W), t is the pretreatment time (t), V is the volume
(L) of the slurry undergoing ultra-sonication, and TS0 is the initial total solid content
(kg) of the slurry.
Apart from the characteristics of the ultrasonic wave, the operating conditions
also impact the cavitation phenomenon. Temperature has a contrasting effect during
ultrasound pretreatment. High temperatures break down the interactions between the
solute and matrix, such as hydrogen bonds, Van der Waals forces, and dipole
attractions. Nevertheless, the vapour pressure increases with the temperature of the
solvent, with the solvent vapour filling the cavitation bubbles, thus reducing their
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 107

Fig. 4.4 Mechanisms of ultrasound pretreatment: (a) alternation of compression and rarefaction
zones in the liquid media, (b) effect of ultrasonic waves on gas bubbles, (c) effect of ultrasounds on
lignocellulosic structure

power of collapse (Bussemaker and Zhang 2013). While water is the most employed
medium for ultrasound pretreatment, other liquids with a lower polarity (e.g. organic
liquids) are also expected to be efficient. On the other hand, viscous liquids (e.g. oils)
make cavitation harder (Santos et al. 2009).

4.7.2 Benefits and Drawbacks

Cavitation has both thermal and physical effects on the solid particles present in the
media, contributing to the fractionation of the lignocellulosic structure and lysing of
the membranes and cell walls of the LMs (Fig. 4.4c) (Rehman et al. 2013; Santos
et al. 2009). Ultrasound pretreatment reduces the crystallinity and degree of poly-
merisation of cellulose, enhancing its hydrolysis and the solubilisation of the overall
organic matter. Besides, ultrasound pretreatment modifies the surface of LMs and
disrupts the lignin linkages, separating the lignin fraction from the cellulose and
hemicellulose sugars (Bundhoo and Mohee 2018; Bussemaker and Zhang 2013;
Hassan et al. 2018).
The main concern on ultrasounds techniques regards cost end energy aspects.
Although some researchers reported the unfeasibility of ultrasound pretreatment on a
laboratory scale (Bundhoo and Mohee 2018), other studies show its energetic
convenience when employing commercial technologies on a larger scale (Cano
et al. 2015). Apart from the energy and economic aspect, the possible degradation
of cellulose and hemicellulose sugars should be considered when performing ultra-
sound pretreatment for LMs valorisation. Nevertheless, this aspect highly depends
on the lignocellulosic substrate (Bussemaker and Zhang 2013).
108 A. Oliva et al.

4.7.3 Effectiveness of Ultrasound Pretreatment on Different

Substrates

Several authors report that ultrasounds can reduce the particle size and increase the
surface area of sewage sludge (Bougrier et al. 2006; Cho et al. 2013; Chu et al. 2001;
Na et al. 2007) and manures (Elbeshbishy et al. 2011). Recently, the same effect has
been observed on LMs, showing that the pretreatment duration is proportional to the
substrate disruption. Scanning electron microscopic images show that the damages
to the external surface of wheat straw and maize straw increase with the pretreatment
time. Therefore, ultrasound pretreatment improves the vulnerability of LMs, thus
increasing the available contact surface for the subsequent AD process (Zou et al.
2016a, b).
Ultrasound pretreatment also acts on the chemical composition of LMs. The
cavitation phenomena lead to the formation of radicals that contribute to increased
oxidative stress during pretreatment (Santos et al. 2009). This aspect, together with
high pressure and temperature, reduces the lignin content of LMs, generally leaving
the cellulosic component of the biomass unaffected. On the other hand, the removal
of hemicellulose sugars is observed, most likely due to their amorphous structure and
weak linkages (Perrone et al. 2016). Therefore, ultrasonic waves enable to obtain
cellulose-rich materials, increasing the digestability of LMs during AD. Imam and
Capareda (2012) investigated the effect of hot water and ultrasound-assisted hot
water pretreatment on sweet sorghum residues. Hot water pretreatment alone is not
effective on lignin removal. On the other hand, the ultrasonic implementation
reduced the lignin content by 48%, compared to the untreated sweet sorghum.
Similarly, ultrasound pretreatment of sugarcane bagasse reduced the lignin content
from 27 to 21%, with minor effects on the cellulose and hemicellulose content
(Ramadoss and Muthukumar 2014). Ultrasound-ammonia pretreatment removed
70% of the lignin from sugarcane bagasse (Velmurugan and Incharoensakdi 2016).
While ultrasound pretreatment generally does not change the cellulose content,
ultrasonic waves may affect the cellulosic hydrogen bonds (Nakashima et al. 2016).
In particular, ultrasounds reduce the crystallinity of the cellulosic part of LMs by
rearranging the cellulose structure, leaving a more amorphous polymer, vulnerable
to enzymatic attack (Bussemaker and Zhang 2013). On the other hand, some studies
report an increase in the crystallinity index after ultrasound pretreatment. This is
likely a consequence of the high lignin removal, resulting in a higher cellulose
content remaining in the solid phase after the pretreatment.
The methane potential increases as a consequence of the changed structural and
chemical characteristics of the ultrasound pretreated LMs (Subhedar and Gogate
2014). Qi et al. (2021) investigated the effectiveness of ultrasound pretreatment on
the co-digestion of cannabis straw and municipal wastewater, obtaining a 77%
increment of the methane production after 30 min pretreatment at 100 W. Zou
et al. (2016a, b) observed a positive effect on methane production from wheat and
maize straw co-digested with dairy manure after ultrasound pretreatment.
4 Pretreatment of Lignocellulosic Materials to Enhance their Methane Potential 109

Interestingly, ultrasound pretreatment shows a great potential when combined

with alkaline pretreatment (Hassan et al. 2017). The combination can occur using an
alkaline solution as the medium of the ultrasound pretreatment or, simply, when
ultrasonic irradiation follows the previously performed alkaline pretreatment
(Hassan et al. 2017; Korai and Li 2020). Sequential NaOH-ultrasound pretreatment
enhances the methane potential of corn stover from 148 up to 320 mL CH4/g VS
(Hassan et al. 2017). Similarly, ultrasound pretreatment of wheat straw using 2, 5
and 6% KOH as the medium allows a 47% increment of methane production (Korai
and Li 2020). Despite the promising effects, other studies did not observe a signif-
icant effect on methane production after ultrasound pretreatment, most likely due to
the low specific energy applied, which lowers the rate of sugar solubilisation
(Elalami et al. 2020; Passos et al. 2015).

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Chapter 5
Biogas Production from Dairy Cattle
Residues: Definition of the Pretreatment
Approach Through a Bibliometric Analysis
of Publications and Patents

Ricardo Müller, Marcio Antonio Vilas Boas, Mônica Sarolli S. M. Costa,

Felipe Souza Marques, Douglas Alves Santos, Günther Bochmann,
Marcelo Bevilacqua Remor, and Daiana Gotardo Martinez

Abstract The use of bedding in livestock, such as wood shavings, is a technique

that brings economic advantages, as it reduces the demand for water to clean the
facilities and provides better animal welfare. On the other hand, the material used as
litter is a lignocellulosic material which requires a pretreatment strategy for success-
ful anaerobic digestion to produce biogas. This work examines the advances in
science (articles) and technology (patents) to delimit pretreatment technology routes
for lignocellulosic residues using a bibliometric approach. To identify the main
articles and patents, search strategies were defined in such a way that all relevant
documents were grouped and organized into four pretreatment routes (physical,
biological, chemical, and combined). The number of published articles (2941) was
35% higher than patents (1923), and 70% of all the documents have been published
in the last 10 years. The bibliometric analysis revealed that the chemical technolog-
ical route has a higher technological maturity, as well as more research and devel-
opment (R&D) effort applied. Articles using sodium hydroxide (NaOH) as an
alkaline catalyst were analyzed and compared to judge its suitability. The work

R. Müller (*)
United Nation Industrial Development Organization (UNIDO), Foz do Iguacu, Brazil
M. A. Vilas Boas · M. S. S. M. Costa · M. B. Remor
Agricultural Engineering Graduate Program, Western Parana State University (UNIOESTE),
Cascavel, Brazil
F. S. Marques · D. G. Martinez
International Center for Renewable Energy – Biogas (CIBiogas), Foz do Iguaçu, Brazil
D. A. Santos
National Institute of Industrial Property (INPI), Curitiba, Brazil
G. Bochmann
Institute for Environmental Biotechnology, University of Natural Resources and Life Science
(BOKU), Vienna, Austria

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 121
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_5
122 R. Müller et al.

concludes that the combination of physical and alkaline pretreatment could provide
degradation of cattle dairy residue and the costs and time in new R&D investigations
can be reduced through the analysis of the technological maturity stage.

Keywords Biomass lignocellulosic · Alkaline hydrolysis · Search strategies ·

Technological routes · Technological maturity · Bovine manure · Renewable
energy · Methane production

5.1 Introduction

Biogas as a renewable energy source can replace conventional fuels to produce heat,
power, and vehicular energy. However, in anaerobic digestion the challenge is to
degrade and convert biomass with high fiber content or lignocellulosic biomass, such
as corn stover, rice straw, sugarcane bagasse, and some animal residue such as
poultry litter and dairy cattle litter into biogas.
A considerable amount of dairy cattle rearing activity occurs in intensive or semi-
intensive confined systems to increase the animal production capacity. In the inten-
sive system, the lactating animal spends practically it is whole time in confinement,
and in the semi-intensive system, the confinement time is 4 h. In both confinement
methods, the environmental conditions in the housing area are controlled for
increasing productivity (Perissinotto et al. 2009).
In intensive confinement, usually the animal is located in a shed. The number of
animals is equivalent to the number of bays, from where it only goes out to feed
itself, spending the rest of the time lying down ruminating. Traditionally, agriculture
and wood residues such as sawdust, corn stover, rice straw, and wood shavings are
used as litter for animals (Natzke et al. 1982). These substrates are rich in organic
matter, which makes it interesting for the production of biogas through anaerobic
digestion after their use is over.
The major challenge in the production of biogas from dairy cattle residues and
cattle litter (manure + wood chips) is that they are difficult to degrade by anaerobic
treatment due to the high lignin content. Wood residues (dust, sawdust, or wood
shavings) and agricultural residues (wheat and corn stover, rice straw, among others)
have high levels of cellulose, hemicellulose, and lignin when compared to animal
waste. For non-residual biomass, the cellulose, hemicellulose and lignin concentra-
tions vary between 51–31%, 17–7%, and 36–30%, respectively (Olsson and Hahn-
Hägerdal 1996).
A possible solution for lignin removal from the dairy cattle residues is to
implement a physical, chemical, or biological pretreatment system or a combination
of these, allowing the anaerobic microorganisms to degrade cellulose and hemicel-
lulose (Mosier 2005).
Data collection and analysis of patent databases from the literature can be used to
set up the suitable pretreatment technology. This strategy has been employed in
many areas of research recently, such as in the field of wind energy (Dubarić et al.
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 123

Fig. 5.1 Strategy steps for defining the technological pathway through bibliometric analysis

2011), solar energy (Zhao et al. 2015), ethanol production from lignocellulosic
biomasses (Silva Schlittler et al. 2012), or in the field of lignocellulosic fuels
(Toivanen and Novotny 2017), demonstrating that such information can be used to
analyze the evolution and level of technology maturity in those respective fields.
However, there are few similar studies in the field of biogas production, more
particularly on pretreatment technologies for dairy cattle residue for biogas
production.
Academic research is directly related to the technological development (demon-
strated by patent registration) in the field of biogas production (Lora Grando et al.
2017). This is because companies, researchers, and universities around the world
actively document their most relevant discoveries through patents to gain a techno-
logical and commercial advantage before engaging in any scientific publication.
In this context, this work aimed to analyze the stage of maturity and R&D efforts
based on patent data and research articles to suggest which is the most promising and
developed technological route, as demonstrated in Fig. 5.1. The case study used was
the market challenges of dairy cattle litter residues for biogas production.

5.2 Main Routes of Pretreatments

There are many promising pretreatment technologies available that are carried out
with some level of commercial success for marketable products and easily
implemented on an industrial scale in the biogas production process. The
Table 5.1 compares the main pretreatment processes and their effects on cellulose,
hemicellulose and lignin.
The combination of alkaline and physical pretreatments stands out among the
different technologies for promoting mainly the increase in surface area (ISA),
decrystallization of cellulose (CD) and low formation of inhibitors (IF). These
aspects are very relevant for the anaerobic process. Such pretreatment technologies
124 R. Müller et al.

Table 5.1 Effect of different pretreatments on lignocellulosic substrates (Hendriks and Zeeman
2009)
Effect
Pretreatment ISA CD SH LS ALS IF
Mechanical High High N/D N/D N/D N/D
Irradiation High Low Low N/D N/D Low
Steam explosion High N/D High Low High High
Hot water High No effect High Low Low Low
Acid High N/D High Low High High
Alkaline High N/D Low Moderate High Low
Thermal + acid High No effect High N/D N/D Low
Thermal + alkaline High No effect Low Moderate High Low
Explosion of ammonia High High Low High High Low
Biological High N/D High High High N/D
ISA Increase of surface area, CD Cellulose decrystallization, SH Solubilization of hemicellulose, LS
Solubilization of lignin, ALS alteration of the lignin structure, IF inhibitor formation, N/D Not
determined

can be classified into three technological routes: physical (including mechanical

shear, heat, pressure, and electric fields), chemical (acids, bases, and solvents),
biological (microbial and enzymatic), and also the combinations of these processes
(Bochmann and Montgomery 2013; Chen et al. 2017).
Since the potential of lignocellulosic biomass could be interesting for large-scale
biogas production and the three main pretreatment technologies have a primary role
in achieving this, there is need for relevant investigations to compare them. Many of
the previous investigations have presented technical problems during large-scale
operation, which often resulted from a limited understanding of the reality in an
actual biogas plant. According to Achinas et al. (2017), there is a technological gap
between universities and industry research, indicating that less mature technologies
require more R&D efforts and resources.
Research achievements at universities are usually recorded through articles,
easily found in scientific databases. While in the industry most of the scientific
achievements are registered in patent documents in patent offices around the world
and can be found in patent databases. Therefore, the joint analysis of these two
databases could provide essential information for the review of research priorities for
the technological development of problems still little studied (Russo and Ladisch
2008), such as the anaerobic digestion of dairy cattle litter residue, increasing the
success of research and reducing the time and investments in R&D in this area of
research.
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 125

5.3 Material and Methods

Considering the technological routes previously delimited, seven search strategies

(set of keywords) were structured to collect patent and scientific articles data so that
all relevant documents have been organized for further analysis of each technolog-
ical route. The “Lens.org” platform, which has over 117 million patent applications
from 95 different jurisdictions, has been used for the acquisition of patent data and
“Scopus”, which is the largest scientific database, with over 5 thousand journals and
70 million documents in its library, was used to acquire data from scientific articles.
The keywords used were delimited based on the works of Lora Grando et al. (2017)
and Magrí et al. (2017).
The search interval was carried out in the period 1998–2018, considering the
“period of silence” of 18 months (2019–2020), the period between the request and its
public release (i.e. the search time interval is defined according to the priority date).
For patent searches, specific technical terms have been defined for each technology,
applying them in the search fields delimited to “title”, “abstract” and “claims”; in the
scientific article’s cases, technical terms similar to the patent search have been used,
delimiting them to “title”, “abstract” and “keywords”. Both search platforms (“Lens.
org” and “Scopus”) have an automated truncation system and boolean operators
(AND, OR, or NOT), as well as search delimiters (“ ”). These were used to restrict
the fields of interest. It is important to note that the search process develops in an
exploratory manner, so the current patent searches have not focused solely on
specific IPC (International Patent Classification) codes. The searches were
conducted based on strategic compositions between keywords and specific IPC
codes following the proposal presented in Lora Grando et al. (2017).
The following is a list of keyword groups and IPC used in both platforms:
P: Patent—“Lens.org” Plataform
P1—(pretreatment OR treating);
P2—(biomass OR “raw material” OR “organic matter”);
P3—(lignin OR lignocellulosic OR cellulose OR hemicellulose OR fiber);
P4—(residue OR waste OR manure OR sludge OR slurry OR dung OR sewage);
P5—IPC (B01, B02, B03, B04, B05, B06, B07, B08, B09, B23, B24, B25).
P6—IPC (C1, C2, C7, C8, C11, C12, C13);
P7—(chemical)
P8—(biological OR fungi OR bacteria OR enzymes)
P9—(alkaline OR NaOH OR “sodium hydride”).
P10—(acid)
A: Article—“Scopus” Plataform
A1—(pretreatment)
A2—(biomass OR residue OR waste OR manure OR sludge OR slurry OR dung
OR sewage)
A3—(lignin OR lignocellulosic OR cellulose OR hemicellulose)
A4—(physical)
A5—(biological)
126 R. Müller et al.

A6—(chemical)
A7—(acid)
A8—(alkaline OR NaOH OR “sodium hydride”)
Initially, five search strategies were established (general, physical, biological,
chemical and the combination of these pretreatments). However, since the chemical
route had a higher number of records, two more search strategies were set up for acid
and alkaline chemical pretreatment. The rules for search strategies are listed below:
1. General pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND ((P5) OR (P6))
Article: (A1) AND (A2) AND (A3) AND (A4) AND (A6) AND (A7)
2. Physical pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P5)
Article: (A1) AND (A2) AND (A3) AND (A4)
3. Biological pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P6) NOT (P7 OR P9
OR P10)
Article: (A1) AND (A2) AND (A3) AND (A5)
4. Chemical pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P6) NOT (P8)
Article: (A1) AND (A2) AND (A3) AND (A6)
5. Combination pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P5) AND (P6)
Article: (A1) AND (A2) AND (A3) AND (A4 OR A5 OR A6)
6. Acid chemical pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P6) AND (P9 OR P10)
NOT (P8)
Article: (A1) AND (A2) AND (A3) AND (A6) AND (A7) NOT (A8)
7. Alkaline chemical pretreatment:
Patent: (P1) AND (P2) AND (P3) AND (P4) AND (P6) AND (P10) NOT (P8)
Article: (A1) AND (A2) AND (A3) AND (A4 OR A5 OR A6) AND
(A8) NOT (A7)
The databases were exported and processed with Microsoft Excel software. A
graphical analysis of the level of technological maturity, as recommended by
Achinas et al. (2017) was performed using the methodology proposed by Wang
et al. (2016). The number of patent applications were considered as an indicator of
technological maturity as shown in Fig. 5.2. The purpose of this comparison was to
find out which technology is placed where on this curve, which contains four stages:
germination, growth, maturity and decline.
The technical and scientific efforts in all pretreatment routes were measured and
compared with articles using the Kiviat diagram, considering the total records of
publication in the period from 1998 to 2018. Finally, a descriptive analysis of the
pretreatment effects on lignocellulosic biomasses similar to the dairy cattle residues
(with litter) was done. The observed parameters were: type of biomass, operating
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 127

Fig. 5.2 The technological

maturity prediction model
(Wang et al. 2016)

Articles Patents
450
400
Number of records

350
300
250
200
150
100
50
0
1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018
Year

Fig. 5.3 Number of articles and patents worldwide in the period 1998–2018

conditions (temperature, exposure time and the dose of the catalyst solution),
increase in methane content and biogas.

5.4 Results and Discussion

5.4.1 Analysis of Technological Routes Based on Patent

Documents

There have been 1923 patent applications found worldwide, 50.9% of which were
requested by China, 25.1% by the United States, and only four patents by Brazil.
When correlating the number of articles and patents produced in the last 20 years, it
was noticed that the number of publications of scientific articles (2941 publications)
and patent applications (1923 applications) in the period 1998–2006 did not present
significant changes, varying from 0 to 50 (Fig. 5.3). From 2007 onwards, an increase
128 R. Müller et al.

in the number of records was observed, which was also confirmed by Wang et al.
(2016), who related the effect to the fossil fuel crisis, directly impacting the value of
oil and stimulating investments in other energy sources, particularly on renewable
ones.
Overall, the number of articles published was approximately 35% higher than the
number of patents. Magrí et al. (2017) also observed a higher number of articles,
twice as compared to patent deposits, while investigating the nutrients in the
biodigester effluent. About 70% of patent applications and article publications
occurred in the last 10 years, a convergence of the results was observed by Lora
Grando et al. (2017). It can be assumed that the pretreatment routes are relatively
new and emerging in the race for energy sustainability, especially to replace fossil
fuels in the transportation sector.
Until 2008, the patents number was on average 56% higher than the published
articles, after which the difference decreased, and in 2012 the number of articles
exceeded the number of patents. A possible explanation is the very broad patent
claims where one technology can have several applications in different segments
(Lora Grando et al. 2017). However, as the technologies (patents) are tested in the
laboratory and at the industrial scale with different types of biomass and experimen-
tal conditions, the number of articles is expected to increase more than patents. The
difference between the number of articles and patents supposedly confirms the
technological gap pointed out by Achinas et al. (2017). Conversely, the difference
can represent the level of maturity of research in universities and the lack of
alignment with the market needs.
Another important behavior to be observed is the decrease in the number of
published patents after 2011, which may indicate the transition of maturity level
from the germination to the growth stage. The number of patent documents is an
indicator of the level of technology maturity and is useful to guide economic and
scientific efforts (de Luna and Santos 2017), and can be used as an instrument for
decision-making (Wittfoth et al. 2017). Therefore, the higher is the maturity of a
technological route, the more aligned it will be with the market demand. On the other
hand, the lower the maturity, there is more possibility of a disruptive discovery,
consequently, more effort in R&D and investment.
The technological maturity stage for chemical, physical and biological pretreat-
ments are shown in Figs. 5.4, 5.5, and 5.6, respectively. The chemical and physical
route passed through the first and second stages and reached the third, maturity stage,
where solutions supposedly are more accessible to the market and probably most
competitive. The first stage, germination, was passed in 2011, and the second stage,
growth, in 2016 for both pretreatments.
The biological route showed a conservative behavior in the germination stage,
without any significant leap in the patent number. The analysis shown in Fig. 5.6
reflects the literature review, where biological pretreatment of biomass for biogas
production is not commercially ready due to the low biogas increase or high
operation and implementation costs.
For the combination of pretreatments, the graphical analysis is not representative
due to the low number of patents. This type of combined pretreatment is at the
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 129

Fig. 5.4 Technological maturity analysis for the chemical pretreatment route

Fig. 5.5 Technological maturity analysis for the physical pretreatment route

germination stage. One possible explanation is that each component is generally

intended to be patented in it’s own right, to ensure a more wide-ranging claim.
Once the maturity stage of each pretreatment is understood, it is important to
measure the R&D efforts, bringing together the total articles records and patents. The
130 R. Müller et al.

Fig. 5.6 Technological maturity analysis for the biological pretreatment route

Fig. 5.7 The ration between patent and article documents number

Kiviat diagram was applied to compare different pretreatments and to define which
one has the higher R&D effort (Fig. 5.7).
Considering the main pretreatments, the chemical route showed the best results,
with 40% and 48% of the scientific (articles) and technical (patents) efforts, respec-
tively, followed by biological and physical methods, which had no significant
difference, and the combination of pretreatments with the lowest effort. These data
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 131

confirm the hypothesis that chemical pretreatment is the most studied technique and
has been widely used for the degradation of lignocellulosic biomass.
Acid and alkaline chemical pretreatments totaled 21% and 20% of all articles and
patent documents and 51% and 49% in the chemical route, respectively. The total
number of articles or patents can be different from the individual sum because some
documents appeared in more than one search strategy. Other keywords to delimit the
search strategy were tested without a positive effect or with an excessively restrictive
result. For example, this was the case for alkaline pretreatment, where once the
condition “NOT acid” was added; the results were not representative because most
of the patents included the word acid in the claims to describe the process. The same
was not observed in the case of acid pretreatment.

5.4.2 Chemical Pretreatment Methods

The chemical pretreatment, by acid or alkaline hydrolysis, is the most studied

technique among the main categories of pretreatment, and it has been widely used
for the delignification of different types of lignocellulosic biomasses. The commonly
used chemicals for the acid pretreatment are H2SO4, HCL, HNO3, among others. As
for the alkaline pretreatment NaOH, Ca(OH)2, CaO, KOH and NH4OH are primarily
used. Other chemical pretreatment methods such as use of ionic liquids are less
conventional due to their high costs (Zheng et al. 2014).
The acid pretreatment process provides high hemicellulose solubilization, which
allows better cellulose degradation during the enzymatic process, resulting in high
biogas yield on biomass (Achinas et al. 2017). Another positive factor of the acid
treatment when using HCl or HNO3 is the decrease of the H2S concentration in the
biogas produced due to the lower amount of sulfate present (Hendriks and Zeeman
2009).
However, in this process if a low energy expenditure needs to be maintained, a
high volume of reagent (30–70% v/v) is required. Otherwise, it is not possible to
have satisfactory results in temperatures below 40  C. This is one of the disadvan-
tages of this process. The acid solution also causes corrosion of metal structures and
equipment, has a high environmental risk, high operational cost and formation of
inhibitory compounds during acid treatment and thus has limited its interest on an
industrial scale (Zheng et al. 2014; Achinas et al. 2017).
In contrast to acid pretreatment methods, alkaline pretreatment can be carried out
at ambient temperature and pressure, or even lower temperature conditions. This
results in a lower degradation of sugars present in the biomass and can be beneficial
for biogas production (Rabemanolontsoa and Saka 2016). However, at ambient
temperatures, extended exposure of the fibers to the catalyst is required and may
sometimes take hours or days, depending on the lignin content in the biomass
(Pavlostathis and Gossett 1985; Neves et al. 2006; Pei et al. 2014; Rabemanolontsoa
and Saka 2016). Contrary to the acid pretreatments, alkaline does not allow the
132 R. Müller et al.

recovery of the catalyst, because it is converted into salts, which are unrecoverable or
incorporated into the biomass during the pretreatment reactions (Mosier 2005).
The alkaline pretreatment route showed better results on substrates with low
lignin content such as grasses, agricultural residues, softwood residues, among
others. In this method, the saponification of the intermolecular ester bonds causes
lignin to rupture and separation of the bonds between hemicellulose and lignin
structures leads to the solubilization of lignin and hemicelulose (Sun et al. 2005;
Eggeman and Elander 2005; Achinas et al. 2017).
In most of the previous studies on alkaline pretreatment, NaOH is used as
catalysts, because of its high efficiency, low production inhibitory compounds and
low cost when compared with the other reagents (Amin et al. 2017). Moreover,
NaOH stands out for being non-toxic or non-inhibitory to the subsequent anaerobic
digestion even if there is any residual NaOH left in the treated biomass (Xie et al.
2011).
With this analysis, it can be assumed that the chemical alkaline pretreatment
routes (particularly using NaOH) are more aligned with the market demand due to
the technological maturity and total R&D effort.

5.4.3 Alkaline Hydrolysis with NaOH

Among the chemical pretreatment technologies, alkaline pretreatment using NaOH

can be presented as a promising process with practical advantages such as opera-
tional simplicity and low cost. Besides, alkaline treatment contributes positively to
the anaerobic digestion process. This is due to the fact that any alkaline catalyst
remaining in the substrate can be useful during anaerobic digestion, controlling the
reactor acidification by acting as the pH control agent (Pavlostathis and Gossett
1985). Neves et al. (2006), studying the methane yield from barley residue, con-
cluded that alkaline hydrolysis increases the methane yield, by preventing digester
acidification and reducing the TS (total solids) and VS (volatile solids) content of the
residue. Even recalcitrant biomasses such as residues from the wood industry can
have satisfactory results with alkaline hydrolysis as Mirahmadi et al. (2010)
observed a 84% and 74% higher methane yield for hardwood (spruce) and softwood
(birch) residues, respectively.
The substrate biodegradability also increases, since the structure and the func-
tional groups of lignin, cellulose, and hemicellulose are broken after pretreatment
with NaOH. Furthermore, it contributes to the modification of the chemical structure
because the lignin originally has a high molecular weight and the three-dimensional
network structure is replaced by low molecular weight molecules and becomes
linear. Zhao et al. (2014) studying rice straw pretreatment using NaOH, observed
a 26% increase in the biogas production and 30.4% increase in methane yield,
associated with lignin solubilization and the degradation of cellulose and hemicel-
lulose, which were 32.25% and 36.96%, respectively. NaOH treatment also accel-
erates the degradation activity of the microorganisms, as it expands the pore size of
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 133

biomass (He et al. 2008), allowing the acidogenic bacteria to ferment the biomass
more easily (Neves et al. 2006; Pei et al. 2014). In addition to a larger surface area,
the alkaline hydrolysis releases soluble compounds that can be easily digested by
methanogenic anaerobic bacteria directly, increasing the methane content in the
biogas (Cheng and Liu 2010).
There are some factors that have significant influence on the alkaline pretreatment
efficiency with NaOH; such as reagent concentration, fiber length and pretreatment
(reaction/exposure) time. Selecting a proper dosage is very important, as this decides
the process efficiency without hampering the process economics. Also, it needs to be
mentioned here that just increasing the dosage does not always results in improved
biogas production. At high NaOH dose (>5–9% by weight) along with lignin the
sugars can also be solubilized which ultimately results in low available substrate for
anaerobic digestion and lower biogas yield. But this phenomenon is highly depended
on the biomass type. Moreover, a very high NaOH concentration can also be
detrimental to the anaerobic digestion process due to the inhibitory function of
high amounts Na+ ions in the solution (Monlau et al. 2013; Pang et al. 2008; Pei
et al. 2014; Xie et al. 2011; Zhu et al. 2010).
The length of biomass fibers can be a limiting factor in the pretreatment process,
requiring the adoption of physical pretreatment to reduce the length/size of the
biomass fibers. For example, during studying the efficiency of alkaline pretreatment
of banana stem for biogas production, Pei et al. (2014) found that the shorter the fiber
length, the higher is the biogas yield and the methane content.
The exposure time of the biomass to the alkaline catalyst (NaOH) also has a direct
relationship with the degradation process but the type of biomass plays a critical role
here. Some authors reported satisfactory results in minutes, hours, and days of
pretreatment, depending on the type of biomass and other experimental conditions
such as temperature (Table 5.2). For example, the residue from a vegetal extraction
process treated with NaOH for 15 min can achieve a 55% increase in biogas
production at the ambient temperature in comparison to the biogas produced from
untreated residue. However, using the same pretreatment conditions, only 25% and
37% increase in biogas production were observed from corn stover and rice straw,
respectively. (Zhao et al. 2014; Zhu et al. 2010).
As already mentioned earlier in the introduction that there are rarely any previous
studies on biogas production from dairy cattle litter let alone on its pretreatment for
process improvement. Hence, here previous studies on similar substrates to dairy
cattle litter are compared for understanding the effect of NaOH pretreatment on
biogas production (Table 5.2). From the NaOH pretreatment of different
lingocellulosic biomass depicted in Table 5.2, it can be concluded that dairy cattle
residue consisting of wood shavings as the litter can be treated using NaOH for
enhancing biogas production. NaOH treatment definitely have a positive effect on
the methane content and/or on the biogas production from different types of ligno-
cellulosic biomass. However, depending on the fiber size and the degree of decom-
position of the wood shavings, the use of a physical pretreatment, combined with
NaOH, can help to achieve a more satisfactory results.
134 R. Müller et al.

Table 5.2 Effect of alkaline pretreatment of different lignocellulosic biomass with NaOH along
with the respective experimental conditions
NaOH Increase in Increase
Temperature Treatment dose methane in biogas
Biomass ( C) time (%) (%) (%) Reference
Woods residues
Spruce shavings 15; 0; 5; 2h 7 84 0 Mirahmadi
Birch shavings 50; 80; 100* 2h 7 74 N/D et al. 2010
15; 0; 5; 50;
80; 100*
Banana stem 30–50 1, 2, 3*, 3 50.9–56.2 37.2 Pei et al.
30–50 4, 5 days 6 85.6 2014
30–50 1, 2, 3*, 9 82.2
30–50 4, 5 days
12 58.3
1, 2, 3*,
4, 5 days
1, 2, 3*,
4, 5 days
Agricultural residues
Rice straw Environment 24 h 5 32.8 25.8 Zhao et al.
Rice Environment 24 h 5 28.4 19.7 2014
straw + sewage
sludge
Residue of veg- 37 15 min 8 0 55 Cheng and
etal extraction Liu 2010
Rice straw 20 21 days 6 N/D 64.5 He et al.
2008
37 3h 1,5 70.9 50.0 Sabeeh
et al. 2020
Corn stover 20 21 days 4 N/D 16.6 Pang et al.
20 21 days 6 71 23.8 2008
20 21 days 8 N/D 23.7
20 21 days N/D
10 22.9
Sunflower 55 3, 6, 12, 0,5 N/D N/D Monlau
stalks 55 24*; 36 h 2 N/D N/D et al. 2013
55 3, 6, 12, 4 36 N/D
55 24*; 36 h N/D
6 N/D
55 3, 6, 12, N/D
10 N/D
24*; 36 h
3, 6, 12,
24*; 36 h
3, 6, 12,
24*; 36 h
Corn stover 20 24 h 1 0 0 (Zhu et al.
20 24 h 2,5 0 0 2010)
20 24 h 5 0 37
20 24 h
7,5 ( ) ( )
Energy crops
Grass silage 1 10
(continued)
5 Biogas Production from Dairy Cattle Residues: Definition of the. . . 135

Table 5.2 (continued)

NaOH Increase in Increase
Temperature Treatment dose methane in biogas
Biomass ( C) time (%) (%) (%) Reference
20; 60; 100*; 48 h 2,5 23 N/D Xie et al.
150 48 h 5 38 N/D 2011
20; 60; 100*; 48 h 7,5 39 N/D
150 48 h N/D
20; 60; 100*; N/D
150
20; 60; 100*;
150
Sorghum 55 12 h 4 0 Sambusiti
55 12 h 10 0 0 et al. 2013
(*) Response with significant difference (N/D) Not determined ( ) Negative effect

Acknowledgments To CIBiogas, for the scholarship and the suggestion of the research issue. To
the UNIOESTE, for providing infrastructure and mentoring. To the INPI, for the training to search
and use patent documents. This study was financed in part by the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior—Brasil (CAPES).

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 Part III
AD of Specific Waste-Streams
Chapter 6
Anaerobic Digestion of Dairy Industry
Wastewater

Sridhar Pilli, K. Bella, Y. Manojkumar, Vasam Vinila,

and Venkateswara Rao Polisetty

Abstract Dairy industries are one of the major food industries which generate a
huge amount of wastewater both in terms of volume and strength. The chemical
composition of dairy wastewater ranges from 1000 to 4500 mg/l for COD, 500 to
3000 mg/l for BOD, and 160 to 800 mg/l for TSS. Such wastewaters, if discharged
into the environment without proper treatment will pose serious detrimental effects
on water, land and air. The major portion of the wastewater generated from dairies is
highly organic which depicts its higher degree of biodegradability. Compared to the
aerobic and physicochemical methods employed for treating dairy wastewaters,
anaerobic treatment methods are highly promising, cost-effective, and provide a
sustainable energy generation option. The present manuscript critically evaluates the
effect of various factors like pH, temperature, organic loading rate, hydraulic
retention time, availability of nutrients, and C/N ratio on the process of anaerobic
digestion. Recent studies and older research are considered for this study which
concludes that these factors have a pertinent influence on the performance of an
anaerobic digester. To check the feasibility of adopting an anaerobic digestion
technology in both small and large-scale dairy units, technical and economic anal-
ysis is a must. This chapter also provides a detailed techno-economic analysis (TEA)
framework for biogas production from dairy wastes.

Keywords Dairy wastewater · Anaerobic digestion · Parameters · Techno-

economic analysis (TEA)

S. Pilli · K. Bella · Y. Manojkumar · V. Vinila · V. R. Polisetty (*)

Water and Environment Division, Department of Civil Engineering, National Institute of
Technology - Warangal, Warangal, Telangana, India
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 141
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_6
142 S. Pilli et al.

6.1 Introduction

“Operation Flood” also known as the “White Revolution” in the year 1970
transformed India from a milk deficient nation to the leading producer of milk in
the world. Since 1997, India is the leading milk-producing country in the world.
Dairy products are considered as nutritious food source. Further, it is also considered
as the means for increasing employment opportunities and thereby boosting the
economy. India is also a leading consumer of dairy products and has both private and
cooperative dairies. In 2019, the milk production was 175 billion litres and by 2020
it is expected to increase by 4%. India contributes to about 22% of the global milk
production. With the growing population, there is a tremendous increase in the
demand for dairy products including milk, curd, cheese, butter, ghee (clarified
butter), ice cream, custard and cream. The dairy industry is one of the food
processing industries which consume large volumes of water in every process
(Singh et al. 2014). During milk processing, around 2% of the processed milk
comes out in the waste, and the wastewater generated is 0.2–10 l/l of milk processed
(Mehrotra et al. 2016).
Milk processing in the dairy industry involves several unit operations such as
receiving, storing, clarification, pasteurization, standardization, homogenization,
deodorization, storage and packaging (Burke et al. 2018; Motarjemi et al. 2014).
For cleaning the various units, different alkaline solutions, detergents, emulsifiers,
and sanitizers are used. In the dairy industry, the wastewater produced is mainly
categorised into three types (Britz et al. 2006):
(i) Processing wastewater: i.e. the wastewater formed while cooling the milk in
coolers and condensers for evaporating milk or whey.
(ii) Cleaning wastewater or sanitary wastewater: The cleaning wastewater evolves
during the washing of process units, storage tanks, and milk cans, in contact
with milk and other milk products. Spillages, losses in packaging, breakdown
of equipment, and by-products (such as whey and brine) spillages/losses are
considered in this category. Cleaning wastewater is around 90% organic as it
comes from milk and milk products manufacturing residues: whey, cream,
cheese pieces, water from clarification and separation (Kolev Slavov 2017).
(iii) Industrial wastewater: includes water used in clean in point (CIP) of equipment
in contact with milk products.
The composition and concentration of wastewater depend on the operation
method, production process and design of the process plant. This wastewater is
highly unstable and biodegradable (Sharma et al. 2008).

6.1.1 Composition of Dairy Wastewater

Water used in the dairy industry is generally contaminated by milk and various milk
products produced in the processes. The characteristics of the effluents generated in a
6 Anaerobic Digestion of Dairy Industry Wastewater 143

Table 6.1 Composition of dairy industry effluents

Milk Characteristic (mg/l except pH)
processing
effluents COD BOD pH TSS TN TP FOG Reference
Mixed dairy 2300 1250 6.04 2000 60 75 – Porwal et al.
(2015)
Yogurt 1500 1000 7.2 650 93 – – Bhavsar et al.
(2012)
Whey (cheese 71,526 20,000 4.1 22,050 – – – Deshpande et al.
effluent) (2012)
Butter and 3218 1377 7.1 2240 – 2 1320 Mohanrao and
Ghee unit Subrahmanyam
(1972)
Milk 2580 1139 6.93 1233 – – – Singh et al.
processing (2014)
Skim milk 88,000 62,000 6.6 34,904 70–80 10–15 40–50 Mehrotra et al.
powder and (2016)
whey effluent
Butter 1498 650 9.55 260 – – – Shivsharan et al.
washing (2013)

dairy industry are summarised in Table 6.1. Wastewater from the dairy industry
consists of milk solids, detergents, milk wastes and sanitizers. The organic fraction
in the dairy wastewater is because of lactose, glucose, fat and protein present in the
milk products (Hassan and Nelson 2012). The characteristics of dairy wastewater
and its composition depend on the size of the dairy industry and the type of products
produced in the industry. A typical dairy industry processing milk, curd, ice cream,
butter and buttermilk generates wastewater having COD, BOD, TSS in the range of
2500–3000, 1300–1600, 72,000–80,000 mg/l, respectively and a pH of 7.2–7.5
(Raghunath et al. 2016). Whereas, the wastewater from fluid milk processing has a
COD, BOD, and TSS in the range of 500–1300, 950–2400, and 95–450 mg/l,
respectively, and the pH 5–9.5 (Demirel et al. 2005). Further, the COD, BOD, and
TSS of the wastewater produced at the milk reception due to spillage and cleaning of
the milk cans are 2540, 800, and 650 mg/l, respectively and the pH is 7.18
(Janczukowicz et al. 2008). Wastewater from the washing of equipment such as
boilers has a COD, BOD, and TSS concentration of 14,640, 3470 and 3820 mg/l,
respectively and a pH of 10.37 (Janczukowicz et al. 2008).
In many countries, large amounts of dairy products in the form of cheese and
butter are consumed. Cheese whey streams contain protein, lactose and other mineral
elements that can be utilized in the manufacture of different products such as lactic
acid, vitamins, fermented whey drinks, baker’s yeast and antibiotics. However,
recovery of whey or whey protein results in new wastewater streams that also
need to be treated (Malaspina et al. 1995; Oreopoulou and Russ 2007). The waste-
water produced from the cheese whey processing is 60–80 times more concentrated
than domestic sewage. The whey stream has a COD and TSS concentration in the
144 S. Pilli et al.

range of 80,000–90,000 mg/l and 8000–11,000 mg/l, respectively and a pH of

6 (Baroudi et al. 2012).
During yogurt and buttermilk production, the wash water produced contains a
large amount of fat and curd solids. The pH of the effluent produced from these units
is around 7.2 and the concentration of COD, BOD, TSS is 1500, 1000, 190 mg/l,
respectively (Turan 2004). Due to higher temperature, i.e. 17–25  C the dairy
wastewater has faster biodegradability compared to domestic wastewater. Wash
water and cooling water from processing units have an average temperature of
18  C in winter and 23  C in summer (Tsachev 1982). The BOD and COD of the
dairy wastewater can be expressed in terms of the milk constituents as 1 kg of lactose
is equivalent to 1.13 kg of COD, 1 kg protein is equivalent to 1.36 kg COD and 1 kg
milk fat is equivalent to 3 kg COD (Singh et al. 2014).
In India, on average the wastewater generated from a dairy industry constitutes
COD, BOD, TS, and SS in the range of 1000–4500, 500–3000, 900–1340, and
160–810 mg/l, respectively. The pH is 7.5–8, nitrogen concentration is about 6% of
BOD, phosphorus 10–100 mg/l and COD is 1.5 times the level of BOD.

6.1.2 Environmental Effects of Dairy Wastewater

From the diary wastewater characteristics, it is evident that it contains a substantial

amount of organic matter and nutrients leading to the deterioration of receiving water
bodies and the environment. Figure 6.1 presents the pollutants from the milk
processing unit and dairy wastewater.

6.1.2.1 Effects on Receiving Water Streams

Due to the high organic content and biodegradability, the dairy wastewater depletes
the dissolved oxygen content of the receiving stream and creates anaerobic condi-
tions resulting in foul odour. A higher concentration of COD and BOD is toxic to
aquatic life. Suspended solids, soluble organics and trace organics in dairy waste-
water lead to eutrophication of the receiving streams. Even if the BOD is low but the
phosphorus and nitrogen content is too high, this could trigger the increased pro-
duction of algae. Wastewater disposed into the streams causes the growth of algae
and bacteria that consume oxygen and suffocate the aquatic life (Deshpande et al.
2012). Lactose, a low molecular weight organic compound present in dairy waste-
water promotes the growth of sewage fungi.
Bacteria convert the nitrogen in protein to the inorganic forms of nitrogen as
nitrate, nitrite, ammonia and ammonium ions. Different forms of nitrogen are
harmful to both humans and livestock. Nitrate ions converted to nitrite ions in the
bloodstream and haemoglobin converted to methemoglobin does not allow oxygen
to be taken up by the blood. Methemoglobinemia is a disease observed in infants due
to the presence of methemoglobin in the bloodstream. To avoid such effects, the
6 Anaerobic Digestion of Dairy Industry Wastewater 145

Fig. 6.1 Pollutants from the milk processing unit and dairy wastewater

nitrite-nitrogen concentration in drinking water must be below 10 g/m3. All the

inorganic forms of nitrogen and inorganic phosphorus act as nutrients for plant
growth. To reduce the undesirable plant growth in the receiving streams, the
inorganic nitrogen and inorganic phosphorus concentrations must be limited to
30–100 mg/m3 and 15–30 mg/m3, respectively (Shete and Shinkar 2013).

6.1.2.2 Effects on Land

Disposal of dairy wastewater on land is one of the treatment techniques. The

nutrients present in the wastewater are removed through:
(a) Plant uptake
(b) Adsorption and immobilization in the soil
(c) Atmospheric losses
(d) Leaching into groundwater
Nitrogen and phosphorus uptake by plants account for 500 and 30 kg/ha/year,
respectively. Up to 90% of nitrogen and phosphorus is recycled to pasture if the
146 S. Pilli et al.

animals subsequently consume the pasture. Groundwater contaminated by nitrate is

one of the limitations for treating dairy wastewater by disposing on soils. Ground-
water when further used by humans can affect human health (Barnett et al. 1998;
Shete and Shinkar 2013). Sodium, magnesium, potassium and calcium present in the
dairy wastewater are immobilised by soils and occupy cation exchange sites on clay
and soil collides. The pH of dairy wastewater can affect the pH of soil and nutrient
availability, which may further affect the microorganisms present in soil and may
alter the fertility of the soil.

6.1.2.3 Effects on Atmosphere

Anaerobic decomposition of dairy wastewater contributes to methane emissions.

Further using the dairy wastewater for irrigation can add to nitrous oxide emissions
(Milani et al. 2011). Moreover, the boilers used in the dairy plants account for carbon
dioxide, sulfur oxides and nitrogen oxides into the atmosphere. Thus, carbon
dioxide, methane and nitrous oxide are the greenhouse gas emissions from the
dairy industry. Besides, dairy effluents have a very foul odour that is not pleasant.
Volatile dust particles and particulate matter emitted from the powder driers and
boiler stacks will settle on the surrounding buildings and can be corrosive
(Deshpande et al. 2012; Shete and Shinkar 2013).

6.1.3 Treatment of Dairy Wastewater

The concentration of the dairy wastewater is rich in nutrients and organic matter thus
conventional treatment techniques are not effective in removing them. Mechanical
treatment followed by chemical or biological treatment are generally carried out
(Birwal et al. 2017; Yonar et al. 2018). In a conventional dairy wastewater treatment
system, the preliminary stage includes screens, grit chambers and a skimming tank to
remove floating materials, inorganic soil material and oil and grease from the dairy
effluents respectively. Solids removed from dairy wastewater, such as fats, oils and
greases (FOGs), increase the biodegradability and methane production, but at the
same time decrease the methane production potential by decreasing the BOD
concentration. Figure 6.2 shows the possible treatment ways of dairy wastewater
through mechanical, physico-chemical and biological methods.
Treatment of dairy wastewater through the biological process is promising since
dairy wastewater effluent is mainly organic. Aerobic treatment processes are less
efficient due to the growth of filamentous substances and acidification (high lactose
and low buffer) (Nadais et al. 2010; Prazeres et al. 2012). Bulking and foaming,
additional biomass production, as well as poor activity at low temperature, are the
other disadvantages of aerobic treatment processes (Britz et al. 2006). Anaerobic
treatment followed by aerobic treatment is the most commonly used process for
treating dairy wastewater.
6 Anaerobic Digestion of Dairy Industry Wastewater 147

Fig. 6.2 Process diagram of the treatment of dairy wastewater

6.2 Anaerobic Digestion of Dairy Wastewater

Anaerobic systems used for the treatment of dairy industry effluent are anaerobic
lagoon, contact digester, upflow anaerobic sludge blanket reactor, stirred tank
reactor, fixed-film digester, anaerobic filter reactor, expanded bed digester, mem-
brane anaerobic digester, separated phase digester and hybrid digesters (Hassan and
148 S. Pilli et al.

Nelson 2012). Anaerobic digestion of dairy wastewater produces organic acids such
as lactic acid, propionic acid and acetic acid, and biogas with hydrogen, methane and
carbon dioxide. In some cases, methanogenesis is inhibited for the production of
other products such as hydrogen, propionic and acetic acid, which is called dark
fermentation (Kasmi 2018).
There are many studies focusing on anaerobic treatment of dairy wastewater with
very high treatment efficiency. The COD removal of various dairy wastewaters and
biogas production presented in the literature is summarised in Table 6.2. Gavala
et al. (1999) stated that an up-flow anaerobic sludge blanket (UASB) reactor under
steady-state conditions removed 90% of COD from the influent having a COD of
2050 mg/l and organic loading rate of 0.031 kg of COD/m3 day. An anaerobic
UASB reactor followed by an aerobic reactor resulted in 90% and 85% removal of
BOD and COD respectively, in a cheese manufacturing industry with a biogas
production rate of 0.40 l biogas/g of COD removed (Malaspina et al. 1995). At an
organic loading rate of 10 g/day, an anaerobic downflow-upflow hybrid reactor
(DUHR) removed 98% COD followed by an anaerobic sludge blanket reactor
(SBR) that achieved 90% removal of both nutrients and COD (Derramadero and
Guyot 1995).
A combination of an anaerobic filter reactor and UASB reactor to treat dairy
wastewater was evaluated by Calli and Yukselen (2002), and it was concluded that
the digester generated 0.354 m3 methane/kg of COD (Calli and Yukselen 2002). The
performance of an anaerobic fixed film reactor treating dairy wastewater at 3 and
2 days HRT was evaluated by Koshta (2010) and observed a BOD removal
efficiency of 87.69% and 89.42%, respectively. The performance of anaerobic
sequence batch reactors (ASBR) was evaluated at an HRT of 6 h and a temperature
of 58  C and observed a removal efficiency of 62% and 75% of COD and BOD5,
respectively (Dugba and Zhang 1999). Further, Nadais et al. (2006) evaluated an
intermittent feeding cycle, i.e. 48 h feed + 48 h without feeding, to the UASB reactor
and observed that intermittent operation has a higher COD removal efficiency (22 g
COD/l/day) compared with continuous feeding (3–6 g COD/l/day). Dębowski et al.
(2018) treated the simulated dairy wastewater in an anaerobic biofilm reactor with
magneto active microporous media. At a suitable organic loading rate ranging from
6 to 8 kg COD/m3 day, the anaerobic reactor removed 80% of chemical oxygen
demand, 70% of organic matter and 80% of phosphorous. The methane production
was in the range of 420.6–557.1 l/day. Anaerobic digestion of the dairy wastewater
as a substrate was performed by Karthiyayini et al. (2017) and observed 36% COD
and 33.5% of volatile solids removal. Similarly, many recent studies suggest that
anaerobic digestion is a more energy-efficient process for dairy wastewater treatment
with increased biogas production (Shete and Shinkar 2017; Meegoda et al. 2018;
Pilli et al. 2020).
The anaerobic digestion of milk fat (triglycerides) in dairy wastewater by micro-
organisms (Clostridia and Micrococci sp.) results in the products such as acetate,
hydrogen and CO2. But, the presence of long-chain fatty acids (LCFAs) in fats, oils
and greases (FOGs) can cause inhibition of methanogenesis and digester instability.
In anaerobic digestion, lactose generates acetate, lactate, formate, propionate and
Table 6.2 Process performance of the anaerobic digestion of dairy wastewater
OLR COD Methane
HRT (kg COD/ Temperature removal production
Dairy waste type Reactor (Days) m3 day) pH ( C) (%) (m3/kg COD) Reference
Ice cream Anaerobic filter 0.5 6 – – 85 0.32–0.34 Ince (1998)
Acid whey CSTR 5 – 6.5 – – 0.3 Saddoud et al.
(2007)
Raw milk Anaerobic filter 0.5 5–6 – – 90 – Omil et al. (2003)
Cheese plant CSTR 5.7 60 7.0 - - 0.28 Hwang (1997)
waste
Cheese whey Downflow fixed-film 6.6 8.3 – 30 76 0.34 Van den Berg and
Kennedy (1983)
Deproteinized Downflow fixed bed 5 2.8 7.25 – – 27 De Haast et al.
whey (1986)
Effluent of inte- UASB 0.2083 8.5 – 30 87 – Ozturk et al.
grated plant (1993)
6 Anaerobic Digestion of Dairy Industry Wastewater

Synthetic dairy Hybrid 1.7 0.82–6.11 – 35 90–97 0.354 Ramasamy et al.

effluent (2004)
Pre-fermented Up-flow anaerobic filter 2 3 7.2 – – 0.28 Gannoun et al.
whey (2008)
Cheese whey Downflow fixed-film 4.9 13 – – 75 0.28 Van den Berg and
Kennedy (1983)
Whey No mix anaerobic digester 6 10 7.0 – – 0.6 Yang et al. (2003)
Cheese produc- UASB 10–20 2.3–4.5 – 35 79–91 – Gavala et al.
tion wastewater (1999)
Cheese whey Rotating biological con- 3 – – 37 85 – Mawson (1994)
tact reactor
Synthetic CSTR 7.45 – – 35 98 – Ramasamy and
ice-cream Abbasi (2000)
wastewater
149

(continued)
Table 6.2 (continued)
150

OLR COD Methane

HRT (kg COD/ Temperature removal production
Dairy waste type Reactor (Days) m3 day) pH ( C) (%) (m3/kg COD) Reference
Dairy – – – 7.52–8.3 – 83.75 – Asha (2014)
wastewater
Cheese Semi-continuous digester 9 0.633 – 35 78–90 – Lebrato et al.
wastewater (1990)
Synthetic dairy Anaerobic diphasic fixed- – 1.265 – – 70.4 0.33 Srinivasan et al.
wastewater film fixed bed digester (2009)
Dairy Hybrid UASB – 2.5–3.5 – – 88 – Thenmozhi and
wastewater Uma (2013)
Dairy Anaerobic biofilm reactor – 0.0726 – – 84 – Sivakumar and
wastewater Asha (2012)
Dairy effluent Up-flow anaerobic fixed 0.5 5.4 – – 87 – Deshannavar et al.
bed reactor (2012)
Dairy Up-flow anaerobic packed 56 – – – 59 – Deshpande et al.
wastewater bed bioreactor (2012)
Dairy Fixed bed fixed-film – – – – 80 0.318 Ramesh and
wastewater anaerobic reactor Kumar (2007)
Dairy Fixed Film and Fixed Bed – 0.004 – – 80.88 0.265 Ramesh et al.
wastewater Anaerobic Reactor (2012)
Dairy Anaerobic Sludge Blanket – – – – 87.06 179.35 m3/day Gotmare et al.
wastewater reactor (2011)
Dairy Combination of CSTR 1.6 1.25–4.5 – – 82 0.26 Jürgensen et al.
wastewater and baffled reactor (2018)
Dairy Horizontal flow reactor – 1–2 85 0.33 Dębowski et al.
wastewater (2020)
Dairy Internal circulation – – 5–6 – 80 69.8 Charalambous
wastewater bioreactor et al. (2020)
S. Pilli et al.
6 Anaerobic Digestion of Dairy Industry Wastewater 151

ethanol as intermediate metabolites. The major products of lactose digestion by

microorganisms (Clostridium, Streptococci, Acetobacter sp.) are methane, carbon
dioxide, hydrogen, cellular carbon and ethanol. Milk proteins in dairy wastewater
are digested by microorganisms (Fusobacterium, Selenomonas, Eubacterium sp.) to
produce organic acids, carbon dioxide, ammonia and hydrogen (Hassan and Nelson
2012; Kasmi 2018).

6.3 Parameters Effecting the Anaerobic Digestion of Dairy

Wastewater

There are many important process parameters in anaerobic digestion and by opti-
mizing them, an increase in biogas yield and treatment efficiency can be achieved.
The main parameters include pH, temperature, organic loading rate, HRT, nutrients
and mixing (Abdelgadir et al. 2014), which are discussed in detail below.

6.3.1 pH

The pH value is the negative logarithm of hydrogen ion concentration. It is the

expression of acidic or basic conditions of a solution (Shete and Shinkar 2017). In
anaerobic digesters, the buffering system is required for the interaction of volatile
fatty acids, bicarbonates and ammonia. Lactose present in dairy wastewater breaks
down into short-chain fatty acids such as acetic, propionic, butyric and other acids by
bacteria and cause a rapid decrease of pH in the digester. Low pH of dairy
wastewater inhibits the growth of methanogens and reduces gas quality, quantity,
and COD removal. The base (calcium, sodium) added to control the pH might affect
the gas production when applied in exceeding amount. The optimum pH range for
acidogenesis is around 5.5–6.5. The most suitable pH for methanogens is around 7.0
and the acceptable range for methanogenesis is 6.5–7.5 (Hassan and Nelson 2012;
Abdelgadir et al. 2014). Therefore, maintaining the pH range within 6.6–7.4 is
suitable in anaerobic digesters. Controlling the pH in digesters increases the diges-
tion efficiency, but it increases the operation cost in anaerobic digestion of dairy
wastewater as well due to the requirement of additional chemicals (Hassan and
Nelson 2012; Nyaki and Njau 2016).

6.3.2 Temperature

Anaerobic digestion is a natural process that occurs in different temperature ranges

such as psychrophilic (5–25  C), mesophilic (30–37  C), thermophilic (50–60  C)
152 S. Pilli et al.

and hyperthermophilic (>65  C). Industrially, anaerobic digestion occurs in

mesophilic (30–37  C) and thermophilic (50–60  C) temperatures (Hassan and
Nelson 2012; Shete and Shinkar 2017). The rate of reaction and pathogens destruc-
tion occurs faster in thermophilic digesters, but it requires a lot of energy input to
maintain elevated temperature. Thermophilic digesters are better capable to produce
high methane gas with dairy wastewater to make the digester economically feasible.
Thermophilic digesters facilitate treatment of a wastewater with higher organic
loading rate. On the other hand, mesophilic digesters require less operating costs.
An increase in wastewater temperature enhances turbulence, mixing, adsorption and
sedimentation, thereby increasing biogas production (Meegoda et al. 2018). For
example, the methane production gradually increased with an increase in tempera-
ture from 20 to 40  C using cheese whey and animal waste as the substrate
(Tabatabaei et al. 2011). With the decrease in temperature from 35 to 20  C in the
anaerobic digester the COD removal efficiency decreased by 40%. Some digesters
do not require heat energy and rely on ambient temperature, but seasonal fluctuations
affect the performance of these digesters. For example, the production of biogas in
summer is faster when compared to winter (Tabatabaei et al. 2011; Abdelgadir et al.
2014).

6.3.3 Organic Loading Rate

The organic loading rate (OLR) of the anaerobic digester is the total amount of COD
in kg/m3/day of digester volume supplied to the digester. It is the total amount of
organics fed into the digester in a day (Shete and Shinkar 2017). An increase in OLR
beyond the optimum level decreases the methane production in the digester and the
COD removal efficiency from the dairy wastewater. Overloading the digester causes
wastewater to quickly hydrolyze and acidify thus creating over-accumulation of
volatile fatty acids, which has the potential of inhibiting methanogenesis (Meegoda
et al. 2018). A higher organic loading rate often causes inhibition of methanogens in
the digester and causes reactor failure. Some reactors like fixed film, fluidized bed
and expanded bed reactors can withstand high organic loading rates. The desired
OLR of the digesters is the provision of the possible highest substrate concentrations
to methanogens for methane production while compensating the inhibitory com-
pounds (Tabatabaei et al. 2011).

6.3.4 Hydraulic Retention Time

Hydraulic retention time (HRT) is the mean length of time that liquids remain in a
digester. A higher loading rate reduces the hydraulic retention time, which may
affect the efficiency of digestion (Meegoda et al. 2018). Accumulation of volatile
fatty acids increases with a decrease in hydraulic retention time. Long hydraulic
6 Anaerobic Digestion of Dairy Industry Wastewater 153

retention is required in the digester to avoid cell wash out of slow-growing

methanogens. Dairy wastewater requires less retention time when compared to the
digestion of more complex compounds such as lignocellulosic waste. The HRT has
great influence on the process economics as shorter retention time increases biogas
productivity and reduces the running costs. The optimum hydraulic retention for
mesophilic digestion ranges from 15 to 30 days (Abdelgadir et al. 2014; Hassan and
Nelson 2012).

6.3.5 Nutrients

In the anaerobic digester, along with the substrate and inoculum, (micro-)nutrients
are also essential for anaerobic digestion. Inorganic nutrients such as nickel, iron,
cobalt and other trace minerals are added to the digester to enhance the anaerobic
digestion by enhancing the microbial population growth (Shete and Shinkar 2017).
In case of dairy wastewater, macronutrients such as nitrogen and phosphorous are
already present. Calcium increases the granulation, which helps in retaining slow-
growing methanogens in the digester and improve the treatment efficiency (Hassan
and Nelson 2012; Demirel and Yenigun 2004).

6.3.6 C:N ratio

Carbon to nitrogen is another critical parameter influencing biogas production from

wastewater. They are major nutrients present in wastewater that need to be removed
before discharge into a water body. The optimum carbon-nitrogen ratio is in the
range of 20–30. A higher C:N ratio reduces gas production and a lower C:N ratio
causes ammonia accumulation (Hassan and Nelson 2012). Free ammonia accumu-
lation in the reactor reduces the growth of microorganisms and highly inhibits the
methanogenic process. A low amount of nitrogen can cause acidification in the
digester and inhibit methanogens (Xue et al. 2020). Along with carbon and nitrogen,
microbes require phosphorous and other micronutrients for their survival. Apart
from the methane production, the percentage of biogas obtained also depends upon
the C:N ratio (Shete and Shinkar 2017).

6.4 Techno-economic Analysis of Anaerobic Digestion

of Dairy Wastewater

Biomass and various industrial wastes have been used as sources of renewable
energy production through anaerobic digestion for biogas production. The biogas
produced through anaerobic digestion has been used in ranges of 1–6 m3 for
154 S. Pilli et al.

household purposes and more than 1000 m3 for industrial purposes (Deublein and
Steinhauser 2011). Dairy industries have huge potential in producing biogas from
the highly organic effluents it generates. But many dairies see the biogas generation
strategy as a high-risk investment due to the high sensitivity of the process, varia-
tions in feedstock characteristics, segregation of wastes and operational problems
(Schmidt 2014). Many governments have put several policy initiatives and off-take
agreements to solve these issues. For this, at first, it is important to conduct an
integrated analysis to assess the technical and economic feasibility and environmen-
tal impacts of employing anaerobic digestion technologies.
A techno-economic analysis (TEA) indicates the technical and economic analysis
of a process/technology through various simulation approaches through software
like Aspen plus and Intelligen Superpro Designer. The results provide an insight into
the feasibility of a project. Also in certain cases, Life Cycle Assessments (LCA) are
used to compare the impacts of various processes/products quantitatively on the
environment (Li and Khanal 2016). The whole process of anaerobic digestion can be
divided into three areas: collection and transportation of feedstock, pre-treatment and
anaerobic digestion, and post-processing of biogas/co-product handling. Similarly,
in TEA for the preparation of TEA framework, three basic sections can be identified
namely (a) the unit operations involved in choosing, collecting and transporting
feedstock to the digestion site, (b) processing stage of feedstock to produce biogas,
and (c) final stage of upgrading the biogas generated for electricity generation,
heating, liquid methane and house-hold purposes (Rajendran and Murthy 2019).
Large dairy industries can utilise the wastes generated for electricity generation and
heating purposes. by installing an anaerobic digestion plant within the industry,
because the wastes generated may be large enough to generate the energy required
for heating. Mostly large dairies can afford a one-time capital on setting up a
treatment plant within the industrial premises. This will also help in reducing the
collection and transportation cost in transferring wastes from the source to the
digestion site. Hence, the first section in a TEA framework pertaining into operations
involving the transportation of wastes can be neglected and this will help in reducing
the complexity. Figure 6.3 represents a typical TEA framework for biogas produc-
tion from dairy wastes.
The technical goals include maximising dairy waste utilization for AD,
maximising biogas yield, and process stabilisation. The economical objective is to
reduce the costs, i.e. capital, operational and maintenance costs. The whole waste
processing value chain is determined by the TEA framework and through experi-
mentally validated process specifics. The economic analysis involves cost assess-
ments and investment analysis. The techno-economic analysis is conducted to
estimate the cost required and energy generated over the value chain from feedstock
digestion to energy production by various means like combined heat and power
(CHP), electricity, or vehicle fuel. Studies on the use of biogas, as vehicle fuel are
one of the leading technological advancements in the application of biogas.
Table 6.3 shows a summary of AD studies on dairy wastes, which types of wastes
used, OLR, other operational parameters, biogas yield and expenditures encountered
are summarised.
 6 Anaerobic Digestion of Dairy Industry Wastewater

Fig. 6.3 TEA framework for biogas production from dairy wastes
155
 156

Table 6.3 Techno-economic analysis of anaerobic digestion of dairy wastes

Type of dairy Hydraulic retention T Payback Capital
waste Organic loading rate time ( C) Biogas yield period cost Reference
Dairy manure 2.29 kg DM/m3 day 33 days 40 30 m3/tDM 3.4 years 11.5M$ Akbulut (2012)
Cheese whey 2.27–2.74 kg VS/m3 25 days 35 437.3 N mL
CH4/g NA NA Mainardis et al.
day VSadded (2019)
Dairy manure NA 20 days 35 97.3 m3/t TS 6 years 2.26M$ Chen et al. (2015)
Dairy manure 2.6 kg VS/m3 day 27 days 37 235 lbiogas/kg VSadded 11 years NA Imeni et al. (2020)
Cheese whey NA 35 days 37 0.60 l CH4/g VSadded 7 years US$3860 Escalante et al.
(2018)
S. Pilli et al.
6 Anaerobic Digestion of Dairy Industry Wastewater 157

6.5 Case Studies on Techno-economic Analysis

of Anaerobic Digestion of Dairy Wastewater

Pilot-scale studies are conducted more in number than field scale/industrial scale
set-ups. Some of the few recent anaerobic digestion studies on dairy wastes includ-
ing their techno-economic analysis are discussed below.

6.5.1 Case Study 1

6.5.1.1 Case Description

Mainardis et al. (2019) has studied the techno-economic feasibility of diffused AD

implementation in some selected dairies. Different kinds of cheese whey are
analysed and their respective methane yield was evaluated using the biochemical
methane potential (BMP) test. Results showed that digestion of cheese whey alone
from a dairy could cover that dairy’s electrical and thermal demand. Based on the
results, pilot-scale anaerobic digesters were constructed. The study was carried out in
the Friuli-Venezia Giulia region, located in the North-eastern part of Italy. This place
is characterised by several small to medium dairy plants. A series of dairies present
in the middle plain area of the Friuli-Venezia Giulia region is considered. The study
used a territory-oriented based approach, and different varieties of cheese whey like
fat, skimmed and mozzarella are considered. The anaerobic sludge is collected from
a full-scale anaerobic digester of the wastewater treatment plant is used as an
inoculum for all studies. Three varieties of cheese whey were collected from five
local dairies depended upon their specific availability. Samples were sonicated for a
treatment time of 5 and 10 min with 20 kHz frequency at 40 and 80 W. A
biochemical Methane Potential (BMP) test was performed for the sonicated samples.
Fifteen individual reactors having 650 ml volume each, inoculum and substrate
volume up to 400 ml and equipped with stirrers were used. To overcome rapid
acidification, a high inoculum to substrate (I/S) ratio of 6.0 was maintained. The
temperature maintained in the reactors was 35  C. The mixing regime adopted for
the reactors was 30s off and 30s on.

6.5.1.2 Energy Analysis

Data regarding electricity, diesel, natural gas and water consumption by the five
dairies are furnished. Heat and electricity that can be derived were calculated by
burning biogas in a combined heat and power unit (CHP) having 50% electricity and
35% heat. Energy consumption of the five dairies is collected. Thermal consumption
rates were high (0.247–0.557 MJ/kg milk) compared to the values presented in the
literature. Among the five dairies, methane yield was high for dairies 2 and
158 S. Pilli et al.

4, i.e. 307.7 Sm3/day and 317.8 Sm3/day, respectively. Based on the values obtained,
an appropriate digester size that can be installed in each dairy-based on cheese yield
was proposed. Results showed that each dairy gets some surplus energy after
covering most of the plant energy needs if they start digesting all the whey produced
by themselves. Thus, the anaerobic digestion of dairy waste/wastewater approach is
a way for sustainable development. Further, the transportation and management
costs are significantly minimised along with renewable energy production and
improved energy balance.

6.5.2 Case Study 2

6.5.2.1 Case Description

Tan et al. (2021) has conducted anaerobic digestion studies for determining the
suitability of using an attached biofilm reactor with cattle manure as the substrate.
Techno-economic evaluation of the biogas system was also done. The cow manure
collected from a dairy at Ladang (Malaysia) was cultured overnight and then
acclimatised using a sequencing batch reactor for use as an inoculum. The digester
was set to operate at 37  C, HRT of 7 days and pH 7. Granular Activated Carbon
(GAC) was used as a support carrier on which the biofilm layer was formed. The
percentage of GAC used was 25% (v/v) which means, 25% of the total working
volume was attached over the support carrier, which helps in the accumulation of
microorganisms on the biofilm layer formed. The maximum methane production of
934.54 mL/g VS was observed at 14 days HRT. Using the modified Gompertz
equation methane production was estimated to be the maximum on 20 days HRT
period.

6.5.2.2 Energy Analysis

An energy economic analysis was conducted for the study. The lifespan of the
digester system is estimated to be 20 years and the rate of electricity is 0.35
Malaysian Ringgit (RM) /kWh. The cost spent on the digester set up was divided
into fixed capital and working capital, where fixed capital is determined based on
some factors (equipment cost, purchase cost, piping and site development cost) and
working capital is 5% of the fixed capital. Also, operational cost is the sum of fixed
operating cost and variable cost. The generated biogas was used for heating and
electricity generation purposes. A combined heat and power (CHP) unit having 48%
thermal efficiency and 38% electrical efficiency was operated using the methane
generated. The revenue obtained from the sale of electricity generated using biogas
was around RM 236.88 and RM 2842.56 in a year. This electricity can be effectively
utilised within the farm which will reduce the electricity bills enormously. However,
this alone cannot help in covering the total operational cost. Thus, fertiliser formed in
6 Anaerobic Digestion of Dairy Industry Wastewater 159

the digester can also be supplied to the farmers which will help in expanding the
revenue. At the initial stages, the cash flow was a little down as the profit was used to
pay off the debts. Later, from 2 to 7 years the revenue from the biogas plant showed
increment.

6.5.3 Case Study 3

6.5.3.1 Case Description

Fantozzi et al. (2015) studied the potential of energy production through anaerobic
digestion of spoiled milk using a pilot-scale anaerobic plant. Spoiled milk comprises
a major portion of waste generated from the dairy industry. Milk gets spilt out during
many practices like collection, transferring milk from cans, processing units and
packaging. The whole fresh milk that got expired and stored in warehouses are used
for this study. The digestate from a secondary anaerobic digester fed with corn waste
and sorghum silage was used as inoculum. The experimentation involves
biomethanation (BM) tests in bottles and a pilot-scale set up. The substrate to
inoculum ratio of 1:3 fixed from BM tests is used for the pilot-scale study. The
study was carried out at a temperature of 35  C for 40 days. Usage of the optimum
amount of organic matter has avoided the rapid acidification problem and attained a
maximum methane production of 0.362 Nm3/kg VS.

6.5.3.2 Energy Analysis

The anaerobic digester plant consists of a CSTR reactor with a pre-tank load, a
cogeneration unit to produce electricity and heat and a biogas storage unit. The
power unit is in the micro-scale range (below 200 Kw). The net electric output power
is around 105 kW and thermal power of 127 kW. The small to medium dairies
located near the plant process around 15,000 tons of milk every year and 10% of the
product normally becomes waste which is utilised for producing biogas. The diges-
tion plant is operated and maintained by a dairy plant which collects raw material
from the three other dairies. Since the dairies are located with a 50 km distance,
transportation cost is not much high. Also, the benefit of supplier companies is their
avoided transportation costs to landfills. The revenue from the incentives was
calculated considering that about 8% of the electric power is consumed for plant
operation. Disposal costs are imposed on other dairies which will also add to the
revenue earned. Around 8% of the electric power generated is used for operating the
plant.
160 S. Pilli et al.

6.6 Future Perspectives

From the bioreactor point of view, the two-stage and high-rate anaerobic digestion
systems for dairy wastewater are attractive for achieving process energy efficiency.
The production of hydrogen from dairy wastewater along with methane could also
be a promising strategy (Demirel and Yenigun 2004; Karadag et al. 2014; Murari
et al. 2019). In addition, highly polluting dairy wastes like whey wastes can be
processed further to derive several value added byproducts (Asunis et al. 2020). The
two-stage anaerobic digester consists of two reactors, an acidogenesis or
hydrogenogenesis reactor and a methanogenesis reactor. In high-rate anaerobic
digesters, the substrate is completely mixed, operated at elevated temperature and
the retention time is lower. Such high-rate systems are more beneficial from an
economic point of view. Further, techno-economic analysis is critical for
establishing the full-scale process. Nutritional, chemical, mechanical and energy
requirements and the innovative design of anaerobic digesters is essential to improve
methane production and to reduce costs. Another strategy is co-digestion which
utilizes multiple substrates for methane production in anaerobic digestion.
Co-digestion of dairy wastewater with other organic feedstocks is advantageous to
enhance methane production and to achieve a waste sustainable process. Demand for
dairy products are increasing worldwide and as a result increase in dairy industries
and the use of various chemicals (hormones, antibiotics and other drugs) are
becoming more prevalent. Such new chemicals in dairy wastewater may seriously
affect the anaerobic digestion process and the treated water quality. Hence, special
care should be taken to develop innovative research ideas to tackle this problem in
order to prevent toxicity in the anaerobic digestion of dairy wastewater and prevent
environmental degradation.

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Chapter 7
Solid State Anaerobic Digestion
of Agricultural Waste for Bioenergy
Production

Kunwar Paritosh, Himanshi Singh, Nupur Kesharwani, Nidhi Pareek,

and Vivekanand Vivekanand

Abstract Huge quantities of agricultural residues and stubbles are mainly disposed
by burning on site causing air pollution. The organic matter present in the residues
and stubble can be utilized in a planned manner, subsequently reducing the emission
(greenhouse gases) caused by burning. These agricultural stubbles are an attractive
feedstock for clean energy production through anaerobic digestion (AD). Conven-
tional liquid anaerobic digestion systems may be profitable but have a high-water
footprint. Solid-state anaerobic digestion (SSAD) not only helps to reduce water
consumption, but it also allows for a high organic loading rate and prevent nutrient
loss in the digestate. Nevertheless, process stability of an anaerobic digestion system
running on high solid concentrations may have several constraints such as limited
mass transfer and process inhibitors like ammonia, p-cresol and D-limonene if
present in the feedstock for SSAD. In the case of lignocellulosic biomass, its
recalcitrant nature may hinder the methane production under the SSAD. Apart
from these, the high total solid (TS) content may inhibit the process stability by
producing excess total volatile fatty acids (TVFAs) during SSAD.

Keywords Solid state anaerobic digestion · Lignocellulosic biomass · Agricultural

residues · Biogas · Bioenergy

K. Paritosh · V. Vivekanand (*)

Centre for Energy and Environment, Malaviya National Institute of Technology, Jaipur, India
e-mail: [email protected]
H. Singh
Centre for Converging Technology, University of Rajasthan, Jaipur, India
N. Kesharwani
Department of Civil Engineering, National Institute of Technology, Raipur, India
N. Pareek
Department of Microbiology, School of Life Sciences, Central University of Rajasthan, Ajmer,
India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 165
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_7
166 K. Paritosh et al.

7.1 Introduction

The modern world’s economic status and growth rate are based on energy and its
consumption (Cantarero 2020; Paritosh et al. 2020a). Depleting fossil fuel reserves,
geo-political issues over crude oil reserves, greenhouse gas (GHG) emissions and its
socio-environmental impacts are detrimental factors for a sustainable world. In the
last decades, global energy demand has increased manifold and mankind has been
forced to explore other alternative forms of energy from sustainable sources.
Renewable sources like wind, solar, ocean, small hydro, geothermal and biomass
have been accepted as major players for ensuring energy supply under a sustainable
development goal (MNRE 2019). Keeping this in mind, many countries including
the developing ones are investing in renewable energy sources. For example, total
installed grid connected renewable energy capacity in India has increased to
85.9 GW at the end of 2019 as per Ministry of New and Renewable Energy,
Government of India (MNRE 2019).
Bioenergy derived from biomass, i.e. biofuel, can be classified as liquid or
gaseous biofuels. Liquid biofuels encompass bioethanol, biodiesel and biobutanol
while example of gaseous biofuels is biomethane, biohydrogen and syngas. The
biofuels are also classified as first, second, third and fourth generation biofuels based
on the substrate used for their production. In first generation biofuels, food crops and
grains are used for biofuel production while for second generation, crop residues
such as rice straw, wheat straw, corn stover and millet straw are used. The third
generation biofuels are derived from algae and fourth generation biofuels are those
obtained from genetically modified microorganism. First generation biofuel is less
desirables as it competes with food whereas the other three biofuels are attractive to
the investors and stakeholder as they utilise renewable and waste biomass.
India produces around 634 million tonnes of agricultural stubbles on yearly basis
(Kumar et al. 2018). Organic carbon present in the agricultural stubbles and residues
may be processed for fuels and energy production. Due to the lack of effective and
efficient technology, farmers are compelled to burn these stubbles on site as to clean
it before the next crop season. This direct burning of the agricultural stubble may
produce around 1600 kg of CO2, 112 kg of CO, 9.2 kg of CH4, 5 kg of particulate
matter and 6 kg of hydrocarbons per ha land (Guo et al. 2020). Theoretically,
burning of these stubbles not only contributes to high GHG emissions, but also
causes immediate problems in the surrounding areas in the form of severe deterio-
ration of air quality (smog formation), and hence crop burning is not at all a
sustainable approach for its disposal.
Agricultural stubbles have huge energy potential and may substitute fossils for
fuel or electricity and are a promising alternative to meet future energy demands
(Hansen et al. 2020). Presently, bio-based energy has approximately 15% share in
the Worlds’ total energy use which is almost 45 EJ. Numerous studies have
suggested that the potential market for bioenergy may increase up to 50% of the
total energy use by the year 2050 (Perea-Moreno et al. 2019). As per a study,
220 billion tonnes of dry biomass are produced worldwide annually (Dahunsi and
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 167

Enyinnaya 2019). This biomass is equivalent to 4500 EJ of solar energy obtained

every year and has the ability to support an annual market of 270 EJ. Because of the
huge energy potential and sustainable nature of it, biomass seems to be an attractive
substitute to fossil fuels (Maletta and Díaz-Ambrona 2020).

7.2 Anaerobic Digestion

Anaerobic digestion (AD) is a biological process, practiced extensively for conver-

sion of biodegradable waste to renewable bioenergy (biomethane) using anaerobic
microorganisms (Caposciutti et al. 2020). This method has capability of utilizing
different organic wastes such as forest woods, lignocellulosic materials, agricultural
crops, food waste and municipal solid waste with high efficiency and minimum
by-product generation. Biogas produced from the AD process can be utilized as fuel
having a high calorific value of 30–35 MJ/m3 and has the potential of replacing other
fuel sources like liquid petroleum gas and natural gas (Sheets et al. 2015). Other high
energy requiring technologies and methods such as landfilling, pyrolysis and incin-
eration are utilized to handle biodegradable waste, but AD is preferred due to its
biological nature which is a lost cost and low energy operation. Also, AD can utilize
various feedstocks at large or small scale and further provide help in the reduction of
waste sludge, killing of pathogens, and provide essential nutrients in the form of
digestate (Xu et al. 2018).
AD is a microbe driven, multi-phase and complex bio-chemical process. The AD
process comprises of mainly four different biochemical phases namely hydrolysis,
acidogenesis, acetogenesis and lastly, methanogenesis. These biological phases
include application of microorganisms in order to decompose organic matter and
produce biogas consisting of primarily methane (CH4) and carbon dioxide (CO2).
However, the efficiency of AD process depends on different factors such as type of
biomass feedstock provided and operational parameters such as temperature, pH,
alkalinity, mass transfer rate, volatile fatty acid accumulation, carbon to nitrogen
(C/N) ratio, recalcitrant nature of lignocellulosic residues, low concentration of
micronutrient and ammonia inhibition. Operating bioreactor under non-optimal
condition or imbalance of any of these factors can cause inhibition to the microor-
ganisms and that can result in the deterioration of the methanogenesis performance
(Thanh et al. 2016).
Various methods have been developed to resolve these issues such as solid
concentration optimization in anaerobic digester for better mass transfer, buffering
agent addition in the reactor to balance pH, substrate co-digestion to stabilize the
C/N ratio, pre-treatment of biomass (particularly the lignocellosic ones) to disrupt
the lignin complex for enhancement of methanogenesis (Jain et al. 2015). Other than
that, to achieve good process stability and performance, many material supplemen-
tation such as carbon-based additives are added to anaerobic reactors for improving
its performance and to enhance its economic feasibility (Paritosh et al. 2021). The
carbon-based additives reinforce direct interspecies electron transfer in the system
168 K. Paritosh et al.

and improve syntrophic relations in the reactor. Moreover, the presence of materials
such as biochar or activated carbon accelerate the utilization of volatile fatty acids
(VFA) and ensure availability of substrate to methanogens. Whereas, addition of
elements such as cobalt (Co), nickel (Ni), iron (Fe) and zinc (Zn). in the AD acts as
micronutrients and accelerate the metabolic activities of methanogens which pro-
vides a better yield of methane (Paritosh et al. 2020b). Nanoparticles of the above
mentioned trace elements have also been supplied by various researches to enhance
anaerobic digestion of biomass and biogas production (Lee and Lee 2019).

7.3 Solid State Anaerobic Digestion

Anaerobic digestion can be categorized into two distinct forms based on their total
solid (TS) content in the reactor medium. The first one is liquid state anaerobic
digestion with a solid content <15%, whereas the other one being solid state
anaerobic digestion (SSAD) with a TS content >15%. SSAD has the following
advantages over liquid anaerobic digestion (LAD): feasibility of using higher
organic loading rate (OLR), less energy requirement, smaller reactor volume and
increased volumetric methane yield (Brown et al. 2012; Rico et al. 2015; Panjičko
et al. 2017). Beside, pathogen inactivation may also be achieved in SSAD of
biodegradable waste (Jiang et al. 2018).
However, SSAD has a few challenges which include slow mass transfer, process
instability, end product needs additional treatment and lower biogas production
(Karthikeyan and Visvanathan 2013; Carlos-Pinedo et al. 2019). These issues need
to be addressed in order to enhance process efficiency, and to further ensure its
feasibility at a larger scale for successful commercialization of this technology.

7.4 Feedstock Identification for SS–AD

Several types of feedstocks including various wastes such as the organic fraction of
municipal solid waste (OFMSW), food waste, forest waste, agricultural waste,
animal waste, solid manure, energy crops, industrial waste, residual lignocellulosic
biomass, paper and pulp waste have been established as good substrates for biogas
generation using the SSAD process (Fig. 7.1). The physical and chemical

SSAD
Feedstock

Agro-
Crop
Food waste Energy crops OFMSW industry
residues
waste

Fig. 7.1 Feedstock for solid state anaerobic digestion

7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 169

composition of the substrate are very important and directly affect the process
efficiency of AD system. For example, the presence of a high amount of recalcitrant
compounds (such as lignin) in the biomass lower the biogas production whereas a
high amount of easily utilizable compounds (such as sugars) enhances methanogenic
activity (Paritosh et al. 2019). Hence, even before starting the process the suitability
of any substrate, including lignocellulosic biomass for SSAD, should be determined
by biomass characterization.

7.4.1 Organic Fraction of Municipal Solid Waste (OFMSW)

The organic fraction of municipal solid waste (OFMSW) comprises of yard trim-
mings, grass clippings, vegetable wastes, food wastes and fruit peel wastes
(Kesharwani and Bajpai 2020). The approach for waste collection and transportation
plays a major role in the SSAD process performance. Also, the seasonal variations
and environment conditions can affect the composition of the OFMSW which in turn
affects the SSAD process performance (Forster-Carneiro et al. 2007). Several studies
have demonstrated that both physical as well as chemical properties of OFMSW
have significant impact on biogas production.
Michele et al. (2015) performed SSAD of OFMSW by recirculation of the
digestate. This liquid digestate flushing helped in removing fermentative products
(such as volatile fatty acids (VFA)) inhibiting methanogenesis. The ratio of solid
waste to digestate was in between 1:1.18–1:0.9 on w/w basis. The total solids
removal was 36.9%, however the loss of organic matter was attributed to the
washout with the percolate from the reactor. Hence, the percolate which was high
in organic content was subjected to AD for biogas production in a second AD reactor
(LAD). The mass balance showed that the methane content from the dry AD and the
percolate were 18.4% and 49.7%, respectively, at a 21 d hydraulic retention time
(HRT). However, only 20.4% and 25.7% of potential producible methane was
generated by adopting 15 and 20 d of HRT using LAD of the same waste.
Food waste is also considered a part of OFMSW and contains organic materials
which are transformed into simple molecules that are readily digested in the AD
process. However, accumulation of VFAs caused by high soluble organic contents
act as inhibitor by decreasing the pH of the system leading to reduction in methane
yield of the AD process (Micolucci et al. 2018). Co-digestion of OFMSW with
lignocellulosic biomass can be a beneficial approach for enhancing the process
efficiency. Brown and Li (2013) examined the effect of feedstock to inoculum
(F/I) ratio (1, 2 and 3) and substrate concentration (0, 10 and 20%) on co-digestion
of food waste (FW) with yard waste on biogas production using SSAD. A high
volumetric biogas production rate (8.6 L per L reactor volume) was achieved with
10% FW concentration and a F/I value of 2.
In another study conducted by Wang et al. (2012), the effect of different ratios of
FW to distiller’s grain on biogas production using SSAD was investigated. A 75.7%
increase in the biogas production was observed with co-digestion compared with
170 K. Paritosh et al.

mono-digestion. Favourable synergistic effects were shown on the VFA/alkalinity

ratio and propionate/acetate ratio when, distiller’s grain and FW were co-digested.
The optimum ratio for FW to distiller’s grain was 8:1 with 20% TS in this study.
Zhu et al. (2014) examined co-digestion of soybean processing waste with
addition of hay through SSAD for methane production. The authors studied the
effect of the F/I ratio, leachate recirculation and pre-mixing of inoculum with
substrate on biogas production. Maximum methane production was achieved at a
F/I ratio of 3 (256 L/kg VS) and soybean processing waste and hay ratio of 75:25.
The methane production during co-digestion was 148% and 50% higher as com-
pared to mono-digestion of soybean processing waste and hay individually. The
leachate recirculation accelerated the SSAD process, however no effect of premixing
on the biogas production was observed.
Million tonnes of yard trimmings, grasses and leaves waste are generated in urban
centres, and can be considered as a major component of OFMSW. These green
wastes largely consist of hemicellulose and cellulose which are beneficial substrates
for higher biogas production in the AD process. Xu et al. (2016) conducted research
on yard trimmings by comparing SSAD digestate and dewatered LAD finished
material as inoculum. The F/I ratio was varied from 0.2 to 2 whereas the TS content
selected for the study was in between 20 and 35%. The highest methane production
of 244 L/kg VS was obtained at a F/I ratio of 0.2 and TS content of 20%. The
dewatered effluent at 24% TS and F/I ratio of 0.6 showed an increased volumetric
methane yield compared with other experimental conditions.

7.4.2 Lignocellulosic Biomass and Residues

Lignocellulosic biomass is derived from plant based wastes such as agricultural

residues, wastes generated from municipal parks and forests, and is one of the main
sources of renewable energy production. Lignocellulosic material mainly constitutes
of three main complex components which are cellulose, hemicelluloses and lignin.
The carbohydrate part, i.e. cellulose (9–80%) and hemicelluloses (10–50%), is
fermentable, whereas lignin presence is 5–35% in the biomass and is considered
as inhibitory compound in the AD process (Fig. 7.2) (Yadav et al. 2019). The
characteristic of lignocellulosic materials such as structural and chemical properties
vary greatly depending on its source (biomass type). These properties are the main
deciding factor for successful microbial degradation of the biomass and sometimes
can cause complications for biogas production due to the higher presence of inhib-
itory substances.
Cellulose is a linear polysaccharide polymer of cellobiose which is connected by
β-1, 4-glycosidic bonds. When the cellulose chain is linked by hydrogen bonds or
van der Waals forces, high tensile strength microfibrils are produced. Cellulose is
further comprised of two components, the first one is amorphous cellulose which is
readily digestible and the other is crystalline form which is difficult to hydrolyse.
Hemicellulose is more amorphous in nature and constitutes of pentoses
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 171

Fig. 7.2 Lignocellulosic biomass structure

(e.g. arabinose, xylose), hexoses (e.g. glucose, rhamnose) and acids

(e.g. galacturonic acid). Lignin is a complex polymer consisting of sinapyl, coumaryl
and coniferyl alcohol which is inert and insoluble in nature. These features of lignin
make it recalcitrant and difficult parts of biomass to digest during AD process.
Brown et al. (2012) conducted a comparative study of a variety of lignocellulosic
materials namely switch grass, yard waste, leaves, waste paper, wheat straw, corn
stover, maple and pine biomass for biogas production using LAD and SSAD
methods. For most of the biomass, there was no significant difference in methane
production by SSAD and LAD method, except for pine and waste paper. However,
due to the small volume used in SSAD systems, the volumetric methane productivity
was higher in the SSAD reactor for all the feedstocks. Corn stover, wheat straw and
switch grass produced comparatively more methane (2–5 times) than yard waste,
maple and pine biomass.
Xu et al. (2013) studied biogas production by the SSAD process using corn stover
as feedstock along with digestate of LAD from three distinct anaerobic systems
under mesophilic conditions. Anaerobic digestate was collected from three LAD
reactors that were fed with dairy waste, FW and sewage sludge, respectively. The
anaerobic digestate to corn stover was mixed in such a manner that the F/I ratios will
vary in between 2 and 6. The SSAD reactor at the F/I ratio of 2 showed the highest
methane yield (238.5 L/kg VS) with digestate from a LAD reactor treating dairy
waste as the inoculum. In case of the SSAD reactor inoculated with digested FW, the
maximum methane production of 199.6 L/kg VS was at F/I ratio of 4. In a similar
172 K. Paritosh et al.

study, Liew et al. (2012) compared the biogas production potential of corn stover
with yard waste, leaves and wheat straw using SSAD at F/I ratio of 2. The maximum
methane yield of 81.2 L/kg VS was for corn stover as feedstock, while yard waste,
leaves and wheat straw yielded 40.8, 55.4 and 66.9 L/kg VS of methane,
respectively.
Methane production from albizia plant biomass was examined in two different
anaerobic digestion systems, i.e. SSAD and LAD rectors (Ge et al. 2014). The study
found higher methane production using LAD where the methane yield from albizia
leaves and wood chips were 161 and 113 L/kg VS, respectively. The methane
production from albizia leaves using SSAD was comparable (156.8 L/kg VS) to
the LAD system, however, it was much lower (59.6 L/kg VS) in case of albizia wood
chips using SSAD. Similar to other previously mentioned studies, the volumetric
methane production was much higher (five times more) in the SSAD system in
comparison to the LAD reactor.
Cui et al. (2011) compared raw wheat straw and spent wheat straw from horse
stall for biogas production in a SSAD system. The experimental conditions used
during the study were 20% TS, F/I ratio of 2, 4 and 6, and the inoculum used was
digestate collected from a LAD reactor. The maximum daily methane yield from
spent wheat straw was observed 8 and 3 days earlier in comparison to raw wheat
straw with a F/I ratio of 2 and 4, respectively, indicating improved degradation rate
for spent wheat straw. The maximum methane generation of 150 L/kg was with
spent wheat straw when the F/I ratio was 4 and it was 56.2% higher than that of raw
straw. Cellulose and hemicellulose digestibility was also, respectively, 24.1 and
49.4% higher in spent wheat straw compared with raw straw.
Yan et al. (2015) investigated the effects of different parameters such as solid
concentration, temperature and C/N ratio on the digestion of rice straw employed for
biogas production using a SSAD system. Maximum biogas production (447.4 mL/g
VS) was observed with an initial TS of 20% and C/N ratio of 29.6 at 35.6 oC.
Sheets et al. (2015) investigated the influence of different factors, namely TS
concentration (20 and 30%), temperature (36 and 55
C) and controlled air exposure,
on biogas production using switch grass as the substrate in a SSAD system. The air
exposure did not show any positive effect on the methane production from switch
grass. The biogas generation increased from 102 and 145 L CH4/kg of VS with
increase in TS concentration from 20 and 30% in mesophilic conditions. Under
thermophilic conditions, the methane yields were 88 and 113 L CH4/kg VS for
20 and 30% of solid concentrations, respectively.
Contrary to this previous study, biogas production decreased with increase in
initial TS concentration from 8 to 38% during biogas production from giant reed
biomass (Yang and Li 2014). The inhibitory effect was attributed to the high
accumulation of VFA at high solids concentration. The maximum methane produc-
tion (129.7 L CH4/kg VS) was at a F/I ratio of 2 and TS content of 20–23% using the
SSAD process (Table 7.1).
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 173

Table 7.1 Methane production from lignocellulosic waste using SSAD process
TS T CH4
Feedstock (%) (oC) yield Remarks Reference
Rice straw 20 37 263 Incubation time and moisture significantly Mustafa
L/kg affected the lignin degradation et al.
VS (2016)
Wheat 18 37 254 Fungal treatment facilitated faster start-up of Rouches
straw L/kg SS–AD reactor et al.
VS (2019)
Rice straw 20 37 258 Fungal treatment showed linear relation Mustafa
L/kg between methane yield and lignin et al.
VS degradation (2017)
Palm fruit 20 40 73.3 Straw mushroom cultivation reduced the Mamimin
bunches m3 / recalcitrance et al.
tonne (2021)
Sugarcane 15 35 143 Lignin droplets formed during thermal Lima et al.
bagasse L/kg treatment hindered the hydrolysis (2018)
VS
Rice husk 21 – 18 Optimization of enzyme concentration is Nugraha
L/kg required et al.
TS (2018)
Distilled 20 52 212 Methanoculleus and Methanosarcina were Wang
grain L/kg detected in abundance et al.
VS (2018)
Rice straw 21 37 190 68% higher glucose yield was obtained at Momayez
L/kg 60 min treatment et al.
VS (2018)
Wheat 25 35 1.2 Startup time of SS–AD digester was reduced Zhu et al.
straw m3 / by 10 days (2020)
m3d
Rice straw 15 35 357 Gas productivity was improved by 2.85– Qian et al.
+ manure L/kg 5.88% per unit TS after treatment (2019)
TS
Rose stalk 12.1 55 117 Treatment facilitated higher VS removal and Liang et al.
L/kg lower digestion time (2016)
VS

7.5 Factors Affecting SSAD Process

7.5.1 Solid Concentration

Solid concentration is one of the most important parameters for the SSAD process
and significantly affects the process efficiency. Hence, many of the previous studies
have focused on optimizing the solids concentration in the digester. A very high
solid concentration in the SSAD process contributes to reduced biogas production by
limiting microbial access to the substrate (Bollon et al. 2013). The water content in
the system is also relevant in this regard as it facilitates mass transfer and low water
174 K. Paritosh et al.

content can suppress the digestion process in the SSAD system (Le Hyaric et al.
2012).
Anaerobic digestion of municipal solid waste (MSW) was studied at two different
solid concentrations of 20% and 30% under mesophilic conditions (Fernández et al.
2010). The dissolved organic carbon and VFAs removal was higher at low TS
concentration of 20%, whereas at high TS concentration (30%) digestion of organic
waste compounds decreased by 17%. Abbassi-Guendouz et al. (2012) investigated
digestion of cardboard at various solid concentrations (10–30%). The results dem-
onstrated that increase in the solid concentration was detrimental to the methane
production rate. The threshold value for TS was 30% in this study, and beyond this
methanogenic activity gets inhibited. In another similar study on methane production
from organic wastes obtained from the palm oil industry (oil farm fronds, oil palm
trunks and empty fruit branches) at three different solid contents (16, 25 and 35%)
observed a negative correlation with increase in solid concentration in the AD
process (Suksong et al. 2016). The maximum methane production (72 L/kg biomass)
and total solids removal was at 16% solid concentration, whereas the methane yield
decreased for the other two solid contents.
Hence, from the above studies it can be summarized that methane yield and
methanogenic activity tends to decrease with an increase in solid concentration. The
reason behind this trend is mainly related to the dysfunction of mass transfer at high
solid content (Abbassi-Guendouz et al. 2012; Fernández et al. 2010). For example,
Bollon et al. (2013) found that when solid concentration increased from 10% to 25%,
the medium solutes diffusion coefficient reduced by 3.7 times.

7.5.2 Inoculum

Inoculum is another important factor as it provides the microbes, the main catalyst in
the AD process (Cui et al. 2011; Shi et al. 2014). LAD effluents and digestate from
the SSAD process are generally better inocula than activated sludge, rumen fluid and
manure because the digestate from anaerobic processes provides high numbers of
active methanogens that are more suited to the AD process. For example, Xu et al.
(2016) established in their study that effluent from the LAD process is a better
inoculum source than manure, rumen fluid, lake sediments and sewage sludge for
initiating the SSAD process. In another study, Forster-Carneiro et al. (2007) noted
that the lag phase in the SSAD process reduced from 20–30 days to 2–5 days when
LAD effluent was used as inoculum instead of fresh manure. Suksong et al. (2019)
reported a twofold increase in methane yield using LAD effluent as inoculum in
comparison to SSAD finished materials. The LAD effluent used had high alkalinity
(5.9 g/kg) and low VFA concentration (0.05 g/kg) which may have contributed to
the better performance of the system (Suksong et al. 2019).
Often recalcitrant components in biomass prevent efficient utilization of the
biomass for biogas production. In such cases, different process improvement strat-
egies are applied, one among them is the use of hydrolytic microorganisms. Weiß
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 175

et al. (2010) used enriched hydrolytic microbes for enhanced degradation of ligno-
cellulosic biomass rich in hemicellulose. The study found an increase in xylanase
activity by 1.62% as well as 53% increase in methane yield with supplementation of
hemicellulolytic bacteria to the AD process. According to Ma et al. (2013) the
optimal ratio of hydrolytic microbes to methanogens was recommended to be
24 in AD process, the hydrolysis process becomes the rate limiting step at a ratio
below 24, while a ratio higher than 24 makes methanogenesis the rate limiting step.
Similarly, enhancement in biogas production from corn stover due to the addition of
dairy manure as inocula was attribute to the activity of hydrolytic microbes in the
AD process (Xu et al. 2013). The biogas yield from corn stover using dairy manure
was 30% and 100% higher than those using sewage sludge finished material and
food waste as inoculum.
Gu et al. (2014) compared different inoculum sources such as digestates from
dairy manure, chicken manure, municipal sludge, swine manure, paper mill sludge
and anaerobic granular sludge for biogas production with rice straw as the substrate.
Compared to sludge, digested manure as inoculum demonstrated significantly
improved lignocellulose degradation and methane production due to the high
enzyme activity (mainly cellulase and xylanase) in animal manure digestates.
The inoculation size in SSAD is another aspect which has the ability to increase
methanogenic activity. The optimized concentration of inoculum can give a good
start to the SSAD process and may as well reduce the lag phase of the AD process
significantly (Yang et al. 2015). The inoculum size in AD is often described as food
to inoculum (F/I) ratio. At mesophilic conditions, inoculation size as F/I ratio of 2–3
on VS basis is recommended for the AD process of lignocellulosic biomass (Zhu
et al. 2014; Liew et al. 2012; Ma et al. 2013). Under thermophilic range, the optimal
F/I ratio should be in the range of 4–6 when the experiment was performed on corn
stover. This difference in optimum F/I ratio under different temperature conditions
was also confirmed by Li et al., where the maximum methane yield for mesophilic
and thermophilic conditions was at F/I ratio of 2.43 and 4.58, respectively. Lin et al.
(2015) investigated SSAD of yard trimmings comprised of wood chips, maple leaves
and lawn grass as substrate for biogas production and found a F/I ratio of 4–6 to be
better for the digestion process under thermophilic conditions (55 oC). In another
study, the F/I ratio of 1 showed best results for methane production under mesophilic
temperature (Brown and Li 2013).
Mixing of inoculum with the substrate is another important aspect of the SSAD
process. In this regard, mixing of inoculum with the substrate is required prior to the
loading in the SSAD reactors. This pre-mixing is particularly needed in case of
processes with high solid content. In large or pilot scale SSAD bioreactors, the
interaction between microbes and feedstock sometimes fails due to improper mixing.
Two different scenarios were created by Zhu et al. (2014) for analysing the effect of
premixing and partial mixing on SSAD process stability and net methane yield. In
the first scenario, the whole inoculum was completely mixed with the substrate at the
start of the process. In the second scenario, half of the inoculum was mixed with
substrate, following which the rest of the 50% inoculum was poured onto the top.
Although, the methane yield was the same in both scenarios, the start-up time was
176 K. Paritosh et al.

less in the premixed SSAD reactor. In another study, three premixing strategies were
employed to digest corn stover anaerobically in a SSAD reactor (Zhu et al. 2014).
Comparison of the completely mixed scenario with partially mixed in one layer and
two layers was performed. The reactor with two layered partial mixing of inoculum
yielded the highest methane at F/I ratio of 4 to 6.

7.5.3 Temperature

Temperature is one of the most important determining factors for the growth and
survival of microbes in the AD process at both laboratory and industrial scale
systems (de Diego-Díaz et al. 2018). Reactor temperature can selectively enrich
microbes and has the capacity to enhance the rate of biochemical reactions in the
bioreactor. The temperature ranges used for the AD process are as follows: thermo-
philic (55–70 oC), mesophilic (20–45 oC) and psychrophilic (0–20
C). Among
these temperature ranges thermophilic and mesophilic conditions have been exten-
sively practiced for the degradation process of lignocellulosic biomass (LCB) and
OFMSW in SSAD. The mesophilic temperature range is more preferred when
compared to thermophilic temperatures due to greater process stability as well as
better growth of methanogens. Although the thermophilic temperature zone has its
own benefits in the AD process, it requires more energy input in the process, making
the process economics unsustainable. However, Sheets et al. (2015) during SSAD of
switch grass concluded that under thermophilic conditions, net energy input can be
decreased with the increase in methane production rate.
Furthermore, thermophilic temperature accelerates the process at initial level and
drives the hydrolysis faster, but often methanogenic conversion is not satisfactory
(Yang et al. 2015). Hydrolysis of substrate can be accelerated in thermophilic
conditions due to enrichment of hydrolytic microorganisms inside the SSAD biore-
actor. But faster hydrolysis of biomass often results in volatile fatty acids (VFAs)
accumulation in the system, causing acidification of the reactor (Shi et al. 2014).
This acidification further reduces methanogenesis, decreasing biogas production and
also reducing stability of the SSAD system (Yan et al. 2015).
Shi et al. (2014) reported that the degradation rate of cellulose and hemicellulose
was higher under thermophilic conditions in contrast to the mesophilic temperature
range. In another study, a total 6–41% of cellulose and 2–34% of hemicelluloses
digestion was observed during thermophilic SSAD of lignocellulosic biomass.
These improved results were attributed to the increased (10–50 times) presence of
cellulolytic and xylanolytic microorganisms in the thermophilic SSAD bioreactor
(Fernández-Rodríguez et al. 2013).
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 177

7.5.4 Inhibition

There are many factors that can cause inhibition in the methanogenesis process in
SSAD. For example, excess VFA accumulation can greatly affect methanogens,
causing instability in the bioreactor (Carlos-Pinedo et al. 2019). Acidification results
in decreased pH values, thus inhibiting methanogens which are most susceptible to
the environmental conditions (Rocamora et al. 2020). The significant reason behind
the increment in VFA accumulation in anaerobic digestion reactors is feedstock
overloading (Eko and Chaiprasert 2020). Zhang et al. demonstrated that the use of
alternative feedstock can avoid VFA accumulation for better stability of the AD
process. The addition of packaging waste along with FW can avoid VFA accumu-
lation during the SSAD process. The study suggested that choice of heterogenous
waste as feedstock may permit high loading of substrate during the digestion. The
ratio of VFA to alkalinity can assist to regulate digester stability. A VFA/alkalinity
ratio within 0.3–0.4 is generally observed in AD plants, but a ratio in the range
0.4–0.6 can provide a stable and safe operation when high organic containing
substrates are used (Lossie and Pütz 2008).
Besides VFA accumulation and alkalinity, the ammonia nitrogen content can also
bring instability in the AD process. A study conducted by Duan et al. (2012) on
sewage sludge found reduced methane generation even at a VFA/alkalinity ratio of
0.2 due to excessive ammonia nitrogen concentrations. This demonstrates that
measuring the VFA/alkalinity ratio to monitor reactor condition could be deceptive
in the long term operation of SSAD. A suitable knowledge of ammonium inhibition
is required to predict the process steadiness.
Free ammonia (NH3) and ammonium ion (NH4+) are available during the diges-
tion of nitrogenous matter and feedstocks rich in protein (FW and OFMSW). The
concentration of the ionic form as well as the non-ionic form of ammonia is
influenced by both temperature and pH of the SSAD system as described by the
following equations (7.1) and (7.2) (Calli et al. 2005).
 
2729:92
pK a ¼ 0:09018 ð7:1Þ
T þ 273:15
TAN
FAN ¼ ð7:2Þ
1 þ 10ðpK a pH Þ

where, pKa is the dissociation constant of ammonium ions, T is temperature (oC),

FAN is free ammonia nitrogen and TAN is total ammonia nitrogen.
During the ammonification process, about 60–80% of nitrogen in the substrate
gets transformed into ammonium or ammonia ion (Yabu et al. 2011). Among these,
free ammonia (FAN) is the major reason behind inhibition: when present in higher
concentrations, it can cause potassium deficiency and proton imbalance within cells
(Yang et al. 2015). Threshold values of the non-ionic form of ammonia is suggested
in the range of 300–800 mg/L (Duan et al. 2012; Yabu et al. 2011).
178 K. Paritosh et al.

Production of inhibitory compounds due to pretreatment of substrate is another

concern, which has negative impact on the biogas production. During pretreatment
of lignocellulosic feedstocks, furan derived compounds such as 5-hydroxyl methyl
furfural and furfurals are produced which negatively affects the AD process (Barakat
et al. 2012). According to Atelge et al. (2020), the inhibitory concentration of furan
and 5-hydroxymethylfurfural on anaerobic digestion process is 1 mg/L and 3 mg/L,
respectively, beyond which they can reduce the methane production rate.
Apart from these inhibitory substances, certain compounds present in specific
substrates are also reported in the literature for their negative effect on the AD
process. For example, a compound named D-limonene, found in citrus fruits peel-
ings and processed fruits waste, has been described to be inhibitory to the
methanogenesis process (Ruiz and Flotats 2014). D-limonene is a colourless and
aqueous secondary plant metabolite that contains cyclic terpenes. It is inhibitory to
methanogens and can destroy the microbial cell membrane. Hence, this compound
needs to be removed in order to successfully utilize citrus fruit waste for methane
production in SSAD. D-limonene can be removed by steam distillation and solvent
extraction methods, but this will increase the process step and can make the process
more energy and cost intensive (Calabrò et al. 2020). Another such inhibitory
compound is p-cresol, present as degradation product in brewery spent grains.
However, two stage SSAD utilizing granular biomass has shown capability to reduce
the negative effect of p-cresol in the methanogenic reactor (Panjičko et al. 2017).

7.6 Approaches for Enhancing SSAD Performance

Lignin present in lignocellulosic biomass is inhibitory to the SSAD process due to its
recalcitrant nature. In order to increase the production of biogas and reduce inhibi-
tion, different pretreatment methods can be applied (Kumar et al. 2018; Saha et al.
2018). Chemical pretreatment involves acid, alkali, ionic liquids (ILs) and organic
solvents to disrupt linkage between complexes in the lignocellulosic matrix (Kumar
et al. 2018). Whereas, physiochemical pretreatment involves usage of carbon diox-
ide explosion, ammonia fibre explosion (AFEX) and wet oxidation. AFEX treatment
includes pressurized ammonia given to biomass with rapid decompression (Stoklosa
et al. 2017). As a result, hydrolysis and ammonolysis reactions break the ester cross
links in the cell wall biopolymers. With the help of biomass pretreatment, various
advantages can be achieved such as lignin removal, decrystallization of cellulose,
increase accessible surface area, alteration of inter-linkage of hemicelluloses and
cellulose in biomass structure (Rouches et al. 2019). The cellulose decrystallization
causes cellulose to become more porous and readily available to the microbes, which
enhances its bioconversion efficiency (Paritosh et al. 2021; Yadav et al. 2019).
Pretreatment for decrystallization of cellulose before digestion can be carried out
with the help of acids. Inorganic acids such as hydrochloric acid (HCl), sulphuric
acids (H2SO4) and phosphoric acid (H3PO4) are commonly employed for this
purpose. However, in the recent times ionic liquids (ILs) have also been used for
7 Solid State Anaerobic Digestion of Agricultural Waste for Bioenergy Production 179

biomass pretreatment. ILs are less corrosive in nature, connect with the hydroxyl
group of cellulose by breaking hydrogen bonds and this ensures dissolution of
cellulose (Han et al. 2020). The pretreatment process using ionic liquids is efficient
in recovering decrystallized cellulose with the help of anti-solvents such as metha-
nol, acetone, ethanol or water and also, the ILs can be recovered to a very high extent
(even 100% in some cases) (Han et al. 2020).
The most preferred ionic liquid used for pretreatment of lignin containing bio-
mass is N-methyl morpholine-N-oxide monohydrate (NMMO). Akhand and
Méndez Blancas (2012) reported a total of 47% increase in methane yield when
rice straw biomass was subjected to NMMO pretreatment. The pretreatment
increased the substrate surface area which facilitated increased microbial degrada-
tion of the feedstock to produce biogas.
Physical pretreatment such as size reduction was applied for methane production
from napier grass with three sizes of 6, 10 and 20 mm (biomass passing through
respective size sieves) (Surendra and Khanal 2015). A higher methane yield was
found for the smallest biomass size of 6 mm as compared to the two other biomass
sizes (10 and 20 mm). This improved results is again attributed to the increase in
specific surface area for microbial degradation of biomass.
Various pretreatment methods such as steam explosion, irradiation, dilute acid
application and liquid hot water have been developed to enhance biogas production
and reduce inhibition (Kumar et al. 2018). In addition, other methods such as wet
oxidation, alkaline treatment and biological methods (fungal or enzymatic) can be
applied for lignin removal (Kumar et al. 2018). Zhao et al. (2014) investigated
pretreatment of yard trimmings using white rot fungi (Ceriporiopsis subvermispora)
for improving the SSAD process. Ceriporiopsis subvermispora pretreatment at 40%
solid concentration showed the highest methane production (44.6 L/kg VS) which
was 154% higher than methane produced from raw yard trimmings. Similarly, when
albizia chips were pretreated with the same fungal strain of Ceriporiopsis
subvermispora, 370% increase in biogas yield was reported (Ge et al. 2015).
Pretreatment of rice straw with combined physical (milling) and biological (fungal)
methods for improved biodegradability of feedstock in the SSAD system was
studied (Mustafa et al. 2017). A 1 month long incubation with Pleurotus ostreatus
and subsequent milling of the rice straw achieved 30.4% lignin removal and 165%
higher methane production in comparison to the experiments with untreated rice
straw.
However, to degrade a higher lignin content in feedstocks such as spruce (29%
lignin content), the alkaline pretreatment method is more suited. In a study by
Mohsenzadeh et al. (2012), birch and spruce biomass was pretreated with different
alkaline reagent combinations (NaOH/urea, NaOH/thiourea, NaOH/urea/thiourea,
and NaOH/polyethylene glycol) at four different temperatures (15, 0, 22 and
80 oC). The pretreament with combinations of NaOH/thiourea at 15 oC showed
the best results in terms of 59.9% and 45.3% increase in yield using birch and spruce
biomass, respectively. Although lignin removal was not maximum at this
pretreatment condition, product yield was the highest, indicating other factors such
as crystallinity of sugars in the biomass have more significance. According to the
180 K. Paritosh et al.

authors, a decrease in crystallinity index has positive correlation with the

hydrolysis rate.
Zhu et al. (2010) studied alkali (NaOH) pretreatment of corn stover at different
concentrations (1–7.5% w/w) in order to increase methane production. The lignin
removal increased from 9.1 to 46.2% by increasing the NaOH concentration from
1 to 7.5% and at optimum condition, a high biogas production of 372.4 L/kg VS was
realised. Pretreatment of poplar waste with NaOH showed improved lignin reduction
by 19.2% and a high methane production (98.2 L/kg VS) from the resulting biomass
by SSAD process (Yao et al. 2017).

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Chapter 8
Food Waste Biorefinery for Bioenergy
and Value Added Products

Miguel Ladero, Jesús Esteban, Juan Manuel Bolívar, Victoria E. Santos,

Víctor Martín-Domínguez, Alberto García-Martín, Álvaro Lorente,
and Itziar A. Escanciano

Abstract Food loss and waste (FLW) is becoming a general environmental and
societal problem as well as an opportunity for its valorisation to a plethora of energy
vectors, chemicals and bio-based materials. Food loss is related to the primary and
industrial sectors (i.e., farms and fish farms, factories), while food waste is produced
by retailers and consumers. This leads not only to direct FLW but also indirect loss
of energy and resources devoted to food production. While societal and political
awareness is rising, with the subsequent actions resulting in an efficiency boost
along the food supply chain, unavoidable FLW amounting to more than 1000 Mtons/
year exists due to personal preferences, safety issues and supply inefficiencies.
Likewise, huge amounts of plant biomass by-products (pomace, bagasse, straw,
stover, peels and pulp) over 5000 Mtons/year are generated. First, second and
third-generation biorefineries can be built based on such biomass as well as that
from forest, cattle, fish and algae. Biorefineries are based on thermal, physical,
chemical and biological treatments and can produce a great variety of energy
vectors, namely hydrogen, biogas, bioethanol, biokerosene, biodiesel and biochar;
chemicals (similar to petrochemicals), materials (biomonomers and biopolymers)
and energy (heat). Even feed and food products could be considered as biorefinery
products, ultimately.

Keywords Food Waste · Sustainability · Biorefinery · Biofuel · Biopolymer

M. Ladero (*) · J. M. Bolívar (*) · V. E. Santos (*) · V. Martín-Domínguez ·

A. García-Martín · Á. Lorente · I. A. Escanciano
Department of Chemical Engineering and Materials, School of Chemical Sciences, Complutense
University, Madrid, Spain
e-mail: [email protected]; [email protected]; [email protected]; [email protected];
[email protected]; [email protected]; [email protected]
J. Esteban (*)
Department of Chemical Engineering and Analytical Science, University of Manchester,
Manchester, UK
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 185
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_8
186 M. Ladero et al.

8.1 Introduction

Food Losses and Food Waste (FLW) are considered a major burden to achieve
Sustainable Development on a global basis. Together with other inefficiencies in the
production, distribution and consumption chains of other basic commodities, such as
energy and water, FLW affect the so-called food-energy-water nexus (Slorach et al.
2020). Process efficacy in the production and distribution of food, drinking water
and energy must be promoted to achieve several of the Sustainable Development
Goals (SDGs) included in the United Nations (UN) Agenda 2030. In particular, one
can think of the following SDGs as the most directly affected goals: Zero Hunger (2),
Good Health and Well-Being (3), Clean Water and Sanitization (5), Affordable and
Clean Energy (7), Decent Work and Economic Growth (8), Build resilient infra-
structure, promote sustainable industrialization and foster innovation (9), Ensure
sustainable consumption and production patterns (12) and Conserve and sustainably
use the oceans, seas and marine resources (14). However, all 17 UN SDGs are
affected by the non-optimal extraction, distribution and consumption of these critical
basic commodities (United Nations 2021).
According to the Food and Agriculture Organization (FAO), about 1300 Mtons
of food was wasted or lost in 2013, with an impact on the global economy above US
$750 million (Xue et al. 2017). In fact, when considering the UN SDG 12, by 2030
Responsible Production and Consumption strategies aim to half the current FLW
(Target 12.3) by increasing loss/waste prevention, reduction, recycling and reuse
(Target 12.5). In this sense, there are several indicators proposed and monitored by
FAO. In particular, indicator 12.3.1. is named “Global Food Loss and Waste” and
focuses on FL from production to retail level, but not including the latter
(subindicator 12.3.1.a) and on FW at the retail and consumer levels (subindicator
12.3.1.b). While the former is being applied through 10 main food commodities by
country and annual period, the latter is less amenable to monitoring and only a
proposal for measurement has been created. If we look at the first subindicator,
worldwide FL in 2016 almost reached 14%, with values as low as 5.8% (FAO 2021).
From this perspective, with FL reaching almost 16% of produced food, Europe and
North American countries are far from the less than 6% FL attained in Australia and
New Zealand. FL in the Asia-Pacific region were, in general, low in 2016, less than
8%, which points to a relatively efficient food supply chain to retailer level.
However, these figures could be misleading, most probably due to lack of monitor-
ing or rapid changes in food consumption (India, China). Ample efforts have been
taken in industrialized countries by international agencies (WRAP, FAO, World
Resources Institute and more) to promote adequate monitoring of FLW, defining a
first global standard as recently as June 2016. Nevertheless, several major pitfalls
remain, indicating that global FLW is notably underestimated, even if it is highly
probable than FLW figures for Europe and Northern America are more reliable (Xue
et al. 2017).
The trends of the World population, GDP per inhabitant, total crop production,
and consumption of fossil and biomass-based fuels are compiled in Fig. 8.1a. In
8 Food Waste Biorefinery for Bioenergy and Value Added Products 187

Fig. 8.1 (a) Several key parameters regarding the World population, economic growth and
resource production in the 2000–2019 period (FAOSTAT 2021; World Bank 2021, IEA 2021);
(b) Production of critical crops from 2000 to 2019 (FAOSTAT 2021)
188 M. Ladero et al.

addition, Fig. 8.1b shows the worldwide trends in the main crops during four 5-year
periods starting in 2000. Although the steady linear growth in the World population
results in an increase of all other variables, it is evident that trends are relatively
different. Some are linear, such as population and crops, whilst other are asymptotic
(GDP/cap; fossil fuels). A closer look at the evolution of different crops (Fig. 8.1b)
indicates that cereal crops, the most abundant, strongly increase (38% growth in the
2000–2019 period), while roots and tubers only experience a growth slightly higher
than 20%. On the contrary, oil crops dramatically increase by 87% in the mentioned
20-year period. Thus, crops maintain the same linear trend as the World population,
but with an increase of 43% in comparison to 19.78%, respectively. This leads to an
economic growth and an increase in the general well-being, as shown by GDP per
inhabitant increments, though the latter seems to be reaching a stable value around
US$12,000 (2010 value) per inhabitant and year. The last 20 years have undergone a
booming hyperbolic economic growth, much in line with the progressive reduction
in consumption of fossil resources. By 2000, the World seemed to reach another
stable value of about US$5400 (2010 value)/cap/year. In this period, there were
important political and economic paradigm shifts (Euro introduction, Gulf crisis,
globalization), which led to a dramatic increment in global wealth growth. As we
reach another plateau, it seems evident that new paradigms are to arrive, possibly
including concepts such as sustainable growth, circular economy, bioeconomy, big
data analysis and industry 4.0 (Dantas et al. 2021). If we considered the exponential
growth in renewable electricity sources (wind and solar) [378% from 2000 to 2018,
reaching a 2% share of the global energy consumption] and the steady increment in
biomass resources for energy, these are called to complement and, ultimately,
substitute fossil energy resources. Notwithstanding, considering an increase of
44% and 52% in gas and coal production in the last 20 years, substitution will
need political action, social perception changes and dramatic technological advance-
ment on energy production, distribution and consumption (LLNL 2021).
European consciousness on the problem posed by food waste (FW) is reflected in
the shift of municipal solid waste (MSW) fate in the last decade. MSW contains up to
50% FW and, by 2005, was mainly disposed of in landfills, while, by 2015, >25% of
it is recycled, with the EU objective to get to 60% in 2025 (Carmona-Cabello et al.
2018). In this valorization strategy, the development of second generation
biorefineries is a must, not only to cope with an important, though, hopefully,
decreasing amount of FW in the future, but also to profit from biomass created
during the food production activities in the primary sector.
Thus, biorefineries are intended to be relevant in an industrial ecology strategy
focused on the use of renewable resources (biomass) through closely integrated
processes in the fashion of existing refinery processes to the plethora of chemicals,
materials, fuels, food and feed needed by mankind. In this sense, both bioeconomy
and circular economy can be established with the aid, among others, of tools such as
life cycle assessment (LCA) and life cycle cost (LCC) in the design of processes and
products (Lam et al. 2018). An integration of human activities in nature, if adequate,
8 Food Waste Biorefinery for Bioenergy and Value Added Products 189

Fig. 8.2 Biorefinery approaches to FLW and food-related biomass valorisation: The thermochem-
ical and the fractionation approximations

can tackle complex problems such as global climate change, resource scarcity and
increasing demands due to growing population and global living standards (Mohan
et al. 2019).
In biorefinery schemes, the chemical composition and physical structure of any
biomass feedstock should be considered to establish its potential for the generation
of products and to obtain positive synergism with other resources of similar or
complementary nature (Carmona-Cabello et al. 2018), including fossil resources
(for example, in the production of hydrotreated vegetable oil (HVO)). Likewise, the
water content can drive towards the need of drying or the selection of fermentation or
hydrothermal processes instead of pure thermal or catalytic ones. In such cases
pyrolysis or other operations common to the thermochemical biorefinery (Kim
et al. 2020) could be undertaken. Additionally, the pretreatment-saccharification-
transformation scheme typical of the fractionation bio-chemical biorefinery (Rastogi
and Shrivastava 2017) could be approached (Fig. 8.2).
In the fractionation biorefinery, carbohydrate-rich biomass has been typically
addressed by a series of pretreatments, saccharification and/or transformation by
chemical and biological means to bioethanol, biokerosene and biodiesel (as a mix-
ture of fatty acid methyl ester (FAME)). Since food-designed resources have been
firstly approached due to their reactivity/accessibility, first-generation biorefineries
have encountered some problems due to the food/fuel controversy. Therefore, due to
its greater availability and non-edible nature, lignocellulosic and algal biomass is
being considered of high interest as feedstock. Lignocellulosic biomass is well-
known for its recalcitrant nature, mainly related to its lignin content. Hence,
190 M. Ladero et al.

pretreatment processes are required to make the carbohydrates accessible for

enzymes and microorganisms. Overall, such processes can be divided into four
general categories: physical or mechanical, chemical, physicochemical and biolog-
ical, and are applied both in the thermochemical and in the fractionation biorefinery
schemes, as explained in Sect. 8.2.1.
Pretreatment and transformation stages (upstream processes) aim to get liquid
streams with high concentrations of monosaccharides (glucose, fructose, galactose,
xylose) and, in general, monomers contained in the biopolymers in lignocellulose or
other biomass. To this end, complex enzymatic mixtures containing cellulases,
amylases, xylanases, arabinases, lipases, and other hydrolytic and oxidative enzymes
(polysaccharide monooxygenases), are applied (Carmona-Cabello et al. 2018). Bio-
logical approaches, called consolidated bioprocess (CBP), combine three successive
processes. These processes are the production of cellulase, the hydrolysis of cellu-
lose and hemicellulose and, finally, the fermentation of hexose and pentose sugars.
They involve the use of microorganisms, such as fungi (white, brown and soft rot
fungi), bacteria, or consortia of bacteria (Huzir et al. 2018; Amiri and Karimi 2018).

8.2 Biobased Energy Vectors from Food Waste

8.2.1 Bioethanol

Ethanol, as obtained from fermentation of renewable resources, has emerged for

decades as an alternative fuel in internal combustion engines due to its physical and
combustion properties; as well as its power generation capacity (Ilhak et al. 2019).
The octane number of ethanol is higher than that of gasoline, which allows the use of
ethanol in a spark ignition engine with a high compression ratio. On the other hand,
ethanol has a high volumetric efficiency due to its latent heat of vaporization value
(Ilhak et al. 2019). Moreover, this biofuel produces less carbon oxides on combus-
tion. However, ethanol as a fuel is not without problems. Its difficulty to ignite at low
temperatures makes cold starting difficult, which means that combustion is not
complete. In addition, it is corrosive to some of the engine parts, which should be
fabricated with materials featuring adequate physical resistance. Therefore, although
engines could work, in theory, with a blend of ethanol with gasoline up to 85:15, the
real practical limit seems to be 20% in engines without any engine mechanical
modification (Ilhak et al. 2019). Finally, ethanol is more expensive than
petroleum-based fuels, although its price is expected to decrease by about 8% in
the coming decades (OECD/FAO 2018).
Since the early 2000s, the production and use of biofuels has been prioritized in
the policies of various countries, however, the market for biofuels is expected to be
limited due to low investment in their research and development, with a projected
global expansion in the case of ethanol from 102.106 m3 in 2010 to 140.106 m3 in
2029 (OECD/FAO 2020).
8 Food Waste Biorefinery for Bioenergy and Value Added Products 191

The largest bioethanol producing country is the United States, with around 55%
of world production, followed by Brazil (30%), the European Union (5%), China,
Canada, India, Thailand and Argentina. The main expansion of this biofuel is
expected to take place in developing countries, with Brazil, India and Thailand,
together with China, being the most important (OECD/FAO 2018).
However, the feedstock currently used for its production is corn, sugarcane,
barley, wheat, sugar beet and sweet sorghum; agricultural products used for food,
whose use in obtaining bioethanol is framed within first generation biofuels (Cooper
et al. 2020). First generation biorefineries are based on raw materials usually
employed for food and feed, very rich in simple sugars (monosaccharides, disac-
charides) or edible polysaccharides (as starch). Therefore, as food production, in
term of mass, is far from the production of fuels need by Humanity (several billions
of tons annually), a high stress is created on food market, this being the main reason
for the search for other raw materials. A main advantage of first-generation ethanol
production is the process simplicity—very well-known due to its long-time use in
the food industry. Although the cost of biomass purchasing is high, up to 65% total
costs, lower technology and process-related costs in comparison to other raw
materials makes first-generation bioethanol production the first and most developed
option nowadays. As for the use of non-edible lignocellulose-based feedstock
(second-generation biorefineries), it involves 20–25% higher costs due to the tech-
nical complexity of required pretreatment and saccharification steps and the higher
complexity of the fermentation step, related to the antimicrobial activity present in
hydrolysates (Esmaeili et al., 2020). Thus, tax policies and monetary incentives are
needed for the transition from first to second generation biorefineries.
Fermentation varies depending on the raw material used, but most of its stages are
maintained with certain changes in process variables, such as pH (between 3.7 and
5.5) or temperature (between 30 and 40
C). Milling, fermentation and distillation
are performed in-series when using sugarcane and other sources of soluble sugars. It
is necessary to add gelatinization and hydrolysis stages (enzyme-based mashing-
saccharification) prior to fermentation when polysaccharide-rich feedstock is
employed (Ayodele et al. 2020). In both cases, up to 95% glucose or sucrose present
in the feedstock can be obtained. Fermentation is usually carried out with Saccha-
romyces cerevisiae, having genetically modified yeasts proven to enhance its pro-
ductivity. Finally, in a multistage distillation process, 95% ethanol is obtained,
which can be subsequently dehydrated to 99.99% using azeotropic distillation
(Ayodele et al. 2020) or membrane technology (Khalid et al. 2019). This technology
has been used in the United States, Brazil and the EU for more than two decades.
First-generation bioethanol production competes for farmland with food produc-
tion. This issue is at the centre of sustainability debates as large areas of arable land
with a high need for rainfall or irrigation are required (Ayodele et al. 2020). Thus,
most recent research focuses on obtaining the so-called second-generation
(2G) bioethanol. The raw materials used to produce 2G ethanol are classified into
three main groups: agricultural residues, forestry residues and energy crops. This
chapter puts the spotlight on agricultural and agrofood residues due to their abun-
dance, particularly sweet sorghum and sugar beet bagasse, and straw and corn
192 M. Ladero et al.

stubble. The chemical composition of these residues presents some variation in their
components: cellulose (between 33 and 48%), hemicellulose (between 18 and 27%)
and lignin (between 7 and 21%) (Ayodele et al. 2020; Jeevan Kumar et al. 2020).
Lignocellulose pretreatments are more complex than those applied to starch or
feedstock rich in free simple sugars (first-generation biorefineries). These pretreat-
ments aim at obtaining cellulose, hemicellulose and lignin fractions with adequate
purity to boost the development of 2G ethanol (Romaní et al. 2018; Cheah et al.
2020). They are critical to the economics of 2G ethanol.
The pretreatments studied are very varied and can be classified into different
categories: physical processes (milling, extrusion, freezing, microwave and ultra-
sound processes), chemical processes (treatment with acids, bases or ionic liquids,
organosolv, oxidation with sulfite, alkaline wet oxidation and ozone), physicochem-
ical processes (steam explosion, treatment with liquid hot water and ammonia fibre
explosion) and, finally, biological processes (digestion by fungi and bacteria) (Cheah
et al. 2020; Dhiman and Mukherjee 2020; Jeevan Kumar et al. 2020).
Hydrothermal treatments enclose the use of supercritical water as a quite
established operation, which requires temperatures higher than 374
C. This condi-
tion provokes both the denaturalization of the walls of plant cells, the degradation of
hemicellulose and the transformation of lignin into its monomers. The advantages of
this pretreatment are high energy conversion, low corrosion and no need for catalysts
(Ayodele et al. 2020). There are some problems related to the high temperature and
pressure values required, entailing the use of sophisticated equipment, special
materials and reducing gases. Hydrothermal treatment includes emerging technolo-
gies as the use of ionic liquids. Ionic liquids modify the lignocellulose biomass
structure in a way that requires subsequent stages of regeneration, filtration, washing
and distillation prior to hydrolysis and fermentation (Ayodele et al. 2020).
An effective pretreatment must provide large amounts of cellulose and hemicel-
lulose prior to hydrolysis to monomers (mainly glucose and xylose). In addition,
pretreatment causes the generation of inhibitory compounds for subsequent stages
(enzymatic hydrolysis, if applicable, and fermentation). The pretreatments that
generate highly inhibiting compounds are acid pretreatment, organosolv and steam
explosion (Cheah et al. 2020).
The following hydrolytic stage is an enzymatic process that can be combined with
the fermentation stage (Jeevan Kumar et al. 2020). The basic operation uses two
separate stages and is known as ‘Separate Hydrolysis and Fermentation’ (SHF). Its
main disadvantage is the inhibition of cellulolytic enzymes by the accumulation of
reducing sugars in the reaction medium during the enzymatic stage. Also, since two
reactors are needed, the investment in process equipment becomes more expensive
(Jeevan Kumar et al. 2020). To solve the problems posed in the SHF approach, the
‘Simultaneous Saccharification and Fermentation’ (SSF) process was developed, in
which the hydrolysis and fermentation stages are carried out jointly in a single
reactor. Obviously, this procedure is possible only with similar optima conditions
for both transformations (Jeevan Kumar et al. 2020). Another advancement is
‘Consolidated Bioprocessing’ (CBP), which consists of simultaneous enzyme pro-
duction, saccharification and fermentation by the same microorganism, decreasing
8 Food Waste Biorefinery for Bioenergy and Value Added Products 193

the need for exogenous enzymes and reducing operation costs (Jeevan Kumar et al.
2020). A last alternative is the partially consolidated bioprocess (PCBP), a combi-
nation of simultaneous pretreatment and saccharification and subsequent fermenta-
tion (Jeevan Kumar et al. 2020).
Pretreatment and saccharification stages can jointly result in a complex mixture of
hexoses (glucose, mannose and galactose) and pentoses (xylose and arabinose).
Therefore, microorganisms (as pure or mixed cultures) capable of transforming all
of them to ethanol whilst being highly ethanol tolerant are needed (Ayodele et al.
2020; Jeevan Kumar et al. 2020). S. cerevisiae is the most widely used microorgan-
ism in the production of 1G bioethanol from glucose. However, 2G bioethanol
production faces the described challenges, requiring other microorganisms in addi-
tion (Ayodele et al. 2020; Jeevan Kumar et al. 2020) or to obtain ad hoc cell factories
to produce bioethanol using different approaches such as metabolic engineering,
genetic engineering or adaptive evolution (Sakar et al. 2020; Sharma and Arora
2020).
One strategy is the use of ethanol-producing bacteria (EPB), such as Zymomonas
mobilis, capable of fermenting both types of sugars. Another strategy is based on the
use of mixed cultures, called co-fermentation. In this case, microorganisms capable
of fermenting pentoses, such as Candida shehatae, Kluyveromyces marxianus,
Pichia stipis and Pachysolen tannophilus, are used together with S. cerevisiae. In
the case of using co-fermentation, the SHF and SSF processes (SHF, SSF) are
referred to as ‘separate hydrolysis and co-fermentation’ (SHCF) and ‘simultaneous
hydrolysis and co-fermentation’ (SSCF) (Ayodele et al. 2020; Jeevan Kumar
et al. 2020). Finally, the combined production of 1G and 2G bioethanol can lead
to profitable processes (Ayodele et al. 2020).
As for starch-rich FW, although fermentation approaches are similar to those
addressed above, the enzymatic saccharification is achieved by amyloglucosidases,
α-amylases, fungal mixtures, including proteases for more complex food matrixes
still rich in carbohydrates (Saeed et al. 2018). In fact, the treatment of household FW
benefits from the use of complex mixing systems within the reactors and the use of
several enzymatic cocktails (Loizidou et al. 2017). Finally, ethanol fermentation is a
valuable pretreatment in the process of biogas (or methane) production by anaerobic
fermentation (Sun et al. 2020; Karimi and Karimi 2018).

8.2.2 Biobutanol

Butanol is a 4-carbon alcohol (C4H10O) receiving increasing attention as a substitute

to conventional transportation fuels. It can be produced from biomass
(as “biobutanol”) as well as fossil fuels (as “petrobutanol”), both having the same
chemical properties.
1-butanol (or n-butanol), iso-butanol and tert-butanol are valuable gasoline addi-
tives due to their physiochemical properties. Compared to ethanol, butanol can be
blended to any ratio with gasoline as well as diesel without additional infrastructure.
194 M. Ladero et al.

Table 8.1 Main processes to produce n-butanol and isobutanol

Product Processes Feedstock Conditions Performance Challenges
n-butanol Traditional Propylene T  100
C, P  Conversion Fewer energy
(Oxo + syngas 20 atm, cat ¼ Rh,  high requirements by
synthesis) Co Selectivity developing new
 medium catalysts
From Ethanol T  150
C, P ¼ Conversion Reduce environ-
ethanol 1 atm, cat homog.  40% mental pollution
¼ Ru,Ir Selectivity and
 90% non-reusability
problems of the
catalyst
ABE Waste C. acetobutylicum Productivity Evolve enhanced
biomass or E. coli ¼ 0.5–6.5 g/ microorganisms
(Lh) with higher toler-
ance to the sol-
vent
Develop continu-
ous fermentation
and new methods
of product
recovery
Isobutanol Traditional Methanol T  200
C, P  Yield  Reduce energy
(Guerbet + 80 atm, cat ¼ 75% consumption
reaction) n-propanol Ni/MeONa through new
catalysts
From Methanol T  180
C, P ¼ Conversion Reduce environ-
ethanol + Ethanol 1 atm, cat homog.  75% mental pollution
¼ Ru Selectivity and
 99% non-reusability
problems of the
catalyst
ABE Waste C. acetobutylicum Productivity Evolve enhanced
biomass or E. coli  0.2 g/ microorganisms
(Lh) with higher toler-
ance to the sol-
vent
Develop continu-
ous fermentation
and new methods
of product
recovery

It also shows easy transportation and less corrosion because of its low vapour
pressure, has a vaporization heat slightly higher than gasoline and its low solubility
in water reduces the potential of groundwater contamination (Pugazhendhi et al.
2019; Bankar et al. 2013). A summary of the different processes to produce
n-butanol and isobutanol is shown in Table 8.1.
Although bio-n-butanol had been used on a large scale and its biologically
fermentative production has a long history, the higher octane number of isobutanol
8 Food Waste Biorefinery for Bioenergy and Value Added Products 195

has attracted increasing attention in the fuel industry. However, biological isobutanol
is harder to separate and purify due to its low concentration in the fermentation broth
(Fu et al. 2020; Bankar et al. 2013).
In the petrochemical route, n-butanol can be produced using three industrial
processes: propylene oxo synthesis, conversion of n-butanol from ethanol and the
Reppe synthesis, oxo synthesis being the most common in industry. In this process,
propylene and syngas feed streams are converted with cobalt or rhodium catalysts
(Huzir et al. 2018; Jin et al. 2011).
n-Butanol serves as a precursor for producing paints and plastics and is also an
intermediate in the production of butyl acrylate, methacrylate and as a solvent in the
production of dyes, oils and waxes. The n-butanol market size is forecasted to reach
US$6.74 billion by 2025 with an estimated annual growth rate of 6.5% during
2020–2025. Major players in the n-Butanol market are Arkema (France), BASF
(Germany), Dow Chemical (US), Eastman Chemical (US) and Mitsubishi Chemical
(Japan) (The Express Wire 2020). However, butanol and its isomers present the
disadvantage of a quite low production compared with ethanol fermentation, which
is 10–30 times higher (Bankar et al. 2013; Chen et al. 2014).
On the other hand, the global isobutanol market is expected to reach US$1.18
billion by 2022. In 2014, synthetic isobutanol accounted for 58% of the total market,
while bio-based isobutanol is expected to grow 7.0% from 2015 to 2022. Some of
the main companies involved with biobutanol production are Butamax (US), Gevo
(US), BUTALCO (Switzerland), Green Biologics (UK) and Cathay (China) (Grand
View Research 2016).
Agricultural and food residues with a high content of cellulose and hemicellulose
and low lignin content are an ideal substrate for biobutanol production. This
production occurs in two different steps, mainly extracellular breakdown of poly-
meric carbohydrates of biomass and intracellular two-stage ABE fermentation using
C. acetobutylicum. In the first step, hydrolysis of the polymers is made by the
numerous enzymes secreted, while in the ABE fermentation acetone, butanol and
ethanol are produced in 3:6:1 ratio (Pugazhendhi et al. 2019; Bankar et al. 2013;
Chen et al. 2014).
Butanol toxicity due to its accumulation is the major problem during fermenta-
tion. Producing >7.4 g/L of butanol suppresses Clostridium growth and limits
butanol yield leading to an increase of the production and recovery costs. To solve
this issue, there is an ample toolbox in genetic engineering of the biosynthesis
pathways. Thus, researchers have developed solventogenic Clostridia tolerant
strains towards butanol production and have restricted their spore formation.
Besides, interest has also been shown in E. coli and S. cerevisiae, which are well
tractable and rapidly growing (Pugazhendhi et al. 2019; Fu et al. 2020; Bankar et al.
2013)
Batch bioprocessing is the most used method to conduct the fermentation,
although, in an economical comparative investigation, continuous fermentation
was found to be preferred for large-scale production. Among the techniques to
recover butanol from the fermentation broth, distillation is the most traditional and
widely applied method. However, due to water having a lower boiling point than
196 M. Ladero et al.

butanol, researchers have developed new recovery techniques to decrease the cost of
the separation, like adsorption, gas stripping and pervaporation (Pugazhendhi et al.
2019; Bankar et al. 2013).
Due to the problems associated with the ABE fermentation and the increasing
urgency to substitute petrochemical routes, ethanol upgrading to butanol has
received much attention in recent decades. There are two main pathways to achieve
this transformation, namely: the direct dimerization of two molecules of ethanol and
the multistep tandem synthetic route known as the Guerbet reaction. The later
includes the dehydrogenation of ethanol, followed by aldol condensation (under a
strong inorganic base like NaOEt), dehydration and final hydrogenation to produce
n-butanol or isobutanol (Wu et al. 2018; Kulkarni et al. 2018; Wingad et al. 2016).
To carry out this upgrading, recent progress in catalyst development has been made,
which involves homogeneous catalysts, such as iridium and ruthenium complexes;
and heterogeneous catalysts, including metal oxides and zeolites like MgO or Al2O3,
hydroxyapatite (HAP) and supported metal catalysts like Ni/Al2O3 have been
explored. These reactions are performed under relatively high temperatures and
pressures (Wu et al. 2018; Kulkarni et al. 2018; Wingad et al. 2016).

8.2.3 Biodiesel

Biodiesel is defined as the monoalkylesters of long chain fatty acids derived from
renewable liquid feedstocks for use in compression ignition engines. Biodiesel is a
worldwide recognized potential alternative for substituting petroleum-based diesel.
Biodiesel has a series of advantages compared with conventional diesel, including its
renewable origin, biodegradability, non-toxicity and the virtual nonexistence of
sulfur and aromatics compared to diesel. In addition, biodiesel is of geostrategic
value for countries as it provides support to local economies and reduces energy
dependency (Hajjari et al. 2017; Foroutan et al. 2020; Hamza et al. 2020).
The key aspects of biodiesel application are the choice of feedstock and the
design of a suitable production process. The main drawbacks for the implementation
of biodiesel are the production costs, associated to raw material costs, and their
effective supply as well as the development of efficient production processes to
satisfy the demands as fuel. The properties of biodiesel as fuel and the required
process production (including previous treatment and purification) development are
strongly correlated with the fatty acid content or degree of unsaturation of the
feedstock. Conventionally, biofuels including biodiesel are classified based on
their feedstock and production technologies into different generations. These gen-
erations include biofuels produced from edible oil seeds, non-food oil crops and
wastes, algae, and the genetically engineered oil crops. The debate between using
edible and non-edible oil feedstock for biodiesel is driven by feedstock availability
and cost, greenhouse gas emission, economic efficiency of using fertile lands as well
as fuel vs. food/feed competition. A suitable alternative for biodiesel relies on
non-edible vegetable oil; however, controversy arises regarding the use of soil
8 Food Waste Biorefinery for Bioenergy and Value Added Products 197

resources for the massively needed supply. In this context, waste-oriented oils/fats
have been proposed as the excellent options to produce biodiesel (Ramos et al. 2019)
with a major dual advantage: cost and environmental impact. Utilizing it as feed-
stock for biodiesel means a double contribution in making the environment safe and
cleaner. Hence, waste based-oil is considered an economically and socially viable
feedstock for low cost biodiesel production on a commercial scale (Hamza et al.
2020; Goh et al. 2020).
Biodiesel is synthetized by the process of esterification or transesterification
reactions of fatty acid or vegetable oil in the presence of monohydric alcohol,
catalyst, and defined conditions of temperature over a period of time.
Transesterification is the catalysed process of trading the alkoxy group of an ester
by an alcohol such as methanol and ethanol (acyl acceptor) to convert the triglyc-
erides of the oil to fatty acid alkyl esters and glycerol. In biodiesel production,
different alcohols can be used. Methanol and ethanol, and the reaction product
produced when methanol is used is called a fatty acid methyl ester mixture
(FAME), whereas if the alcohol is ethanol, the product obtained is a fatty acid
ethyl ester mixture (FAEE). The mild reaction conditions needed, the fast reaction
time and the easy phase separation combined with its low-cost and industrial
availability make methanol the most used alcohol in biodiesel production, although
it is not a renewable bioresource as ethanol (Ramos et al. 2019).
Besides the properties of the original feedstock, the type of catalysts used in the
transesterification reactions is another critical element affecting the biodiesel pro-
duction. They could be chemical compounds, such as acids and/or bases, and
enzymes, working in homogenous or heterogeneous phase. The overall process
involves three consecutive reversible reactions which produce intermediate mole-
cules of di- and monoglycerides. Reactions involved and catalysts have been
reviewed in detailed in recent literature (Ramos et al. 2019; Changmai et al. 2020;
Hamza et al. 2020). The following paragraphs cover the basics of reaction catalysis
in the interplay with feedstock properties.
Homogeneous base catalysis was vastly implemented due to its simplicity, mild
reaction conditions and high productivity and conversion. However, it is very much
limited by the free fatty acid content. The homogeneous acid-catalysed reaction is an
alternative since it can simultaneously catalyse esterification and transesterification.
However, it deals with a slower reaction rate and the energy and methanol demand is
higher. As a consequence, general drawbacks of the homogeneous catalysis are the
result of side reactions of saponification and hydrolysis; or the high capital cost and
energy required for the process, the high cost of separation and purification of
catalysts and glycerol as well as the need for neutralization and wastewater treatment
(Ramos et al. 2019).
To overcome the previous disadvantages, heterogeneous catalysed reactions have
been developed involving different solid materials. Heterogeneous catalysts facili-
tates separation and are less sensitive to high FFA content. As drawbacks, they are
costly and energy intensive as high temperatures and high alcohol:substrate ratios
are needed (Ramos et al. 2019).
198 M. Ladero et al.

Finally, enzyme catalysis based on lipases has been considered. Enzyme-

catalysed reactions offer simple biodiesel purification procedures with reduced
energy requirement due to the mild operating conditions. Additionally, a high
selectivity of transesterification can be achieved with regards to the feedstock,
offering a vast range of substrate because of the capability to esterify both
glyceride-linked and non-esterified fatty acids in one step, not resulting in side
reactions such as saponification. This shows the additional advantage of producing
high-grade glycerol as by-product whilst also being environmentally accepted.
However, the use of enzymatic catalysis is limited, mainly due to their higher cost
compared to chemical catalysts, slower reaction rates than that using alkaline
catalysts, and enzyme inhibition (Ferrero et al. 2016; Ramos et al. 2019; Ching-
Velasquez et al. 2020). The inactivation by the alcohols used as substrates (mainly
methanol) and glycerol produced in the reaction is another problem in this process.
Advances in biodiesel manufacturing techniques from natural oils and fats using
conventional and advanced technologies, to improve transesterification and biodie-
sel productivity have been recently reviewed (Kirubakaran and Arul Mozhi Selvan
2018; Ambat et al. 2018; Gebremariam and Marchetti 2018; Tabatabaei et al. 2019;
Goh et al. 2020).
FW-based oil has been acknowledged as a suitable renewable source for biodiesel
production. Waste oil can be waste edible oil (WEO) or animal fats derived. Each
year, large amounts of WEO are produced by the food industry, restaurants and
households around the world. It has been estimated that European countries alone
produce 100,000–700,000 tons of WEO per year with a potential cost reduction by
60–90% (Foroutan et al. 2020). Use of waste oil as feedstock is very much related to
the current regulatory frameworks for waste oil collection and recycling practices
(Goh et al. 2020). However, the application of waste oil has some limitations,
including their high free fatty acid and water content, which influences the final
ester yield in the base catalysed reactions. Hence, to improve the yield and efficiency
of biodiesel production, it is imperative to perform an oil characterization, especially
regarding the acid value (Kirubakaran and Arul Mozhi Selvan 2018; Ching-
Velasquez et al. 2020). Animal fats, which are the waste produced after meat
processing and cooking, are another source for biodiesel production. Animal waste
feedstock has economic, environmental, and food security advantages over edible
oils (Kirubakaran and Arul Mozhi Selvan 2018; Ambat et al. 2018; Ching-
Velasquez et al. 2020). However, animal waste fats with higher saturated fatty
acids and free fatty acids require complex production techniques (Kirubakaran and
Arul Mozhi Selvan 2018; Ambat et al. 2018; Ching-Velasquez et al. 2020).
Generally, waste-based oil must be submitted to some preliminary characterisa-
tion and processing before subjecting it to biodiesel production. First, the collected
waste oil must be freed from debris or other impurities through a simple filtration
technique. The water content can be removed through drying. Further pretreatment
stages may be needed to reduce the FFA value to<1%; otherwise, saponification will
occur, which will greatly affect the biodiesel yield when basic catalysts are used
(Kirubakaran and Arul Mozhi Selvan 2018; Hamza et al. 2020; Hamza et al. 2020).
Regarding the limitations during the reaction, although the methods vary in terms of
8 Food Waste Biorefinery for Bioenergy and Value Added Products 199

reaction mechanisms and conditions, the emphasis is to improve the blending of the
solvents to enhance the rate of reaction and produce higher biodiesel yields
(Kirubakaran and Arul Mozhi Selvan 2018; Hamza et al. 2020; Hamza et al. 2020).
Before commercialization, some purification procedures may be performed to
maintain quality standards for the final product to be considered biodiesel. Impurities
come from solids, unsaponifiable materials present in the feedstock itself, including
catalyst residues, water, glycerol and excess alcohol from the reaction, such as polar
compounds, dimers, mono and diacylglycerols and free fatty acids. There are several
methods for removing such impurities, such as membrane filtration, centrifugation,
and distillation as well as wet and dry washing (Fonseca et al. 2019; Ramos
et al. 2019).
Last trends of manufacturing biodiesel from waste oil focuses on developing new
heterogeneous catalysts, enabling technologies, reactor concepts (Gupta et al. 2020)
and reaction media to intensify the reactions and increase productivity (Bankovi-
ć-Ilić et al. 2017; Degfie et al. 2019; Ching-Velasquez et al. 2020). New heteroge-
neous chemical catalysts include waste egg shell and heterogeneous base catalysts or
carbon based nanocatalysts (Banković-Ilić et al. 2017). Immobilized lipases as
heterogeneous biocatalysts are under constant development to increase productivity
and decrease production costs (Cavalcante et al. 2021). Recent examples are the
application of lipases from Rhizomucor miehei (RML), lipase B from Candida
antarctica (CALB) and lipase from Thermomyces lanuginosus to overcome current
limits of enzyme catalysis to increase the biodiesel production yield and rate (Babaki
et al. 2017; Badoei-dalfard et al. 2019; Ching-Velasquez et al. 2020). Intensification
of the reaction is being assisted by enabling technologies as microwave assisted
intensification of biodiesel production from waste cooking oil using heterogeneous
base catalysts (Gupta and Rathod 2018) or ultrasound system for the enzymatic
transesterification of oils using combi-lipases as biocatalyst (Poppe et al. 2018).
Among new reaction media, the use of low-cost deep eutectic solvents (DESs) has
been investigated as a new reaction medium for enzymatic biodiesel production from
waste oils with reuse of the catalyst (Merza et al. 2018).

8.2.4 Biogas, Biohydrogen, Biohythane and Volatile

Fatty Acids

The fermentation of organic substrates, including FW, can lead to gaseous products
of interest mainly in the energy sector, but also as building blocks for many other
industries. Biogas is one of such products, which is defined as a mixture of methane,
CO2 and small amounts of other gases produced by anaerobic digestion (AD) of
organic matter. Commonly, the methane content is in the range from 45 to 75% v/v,
with most of the remainder being CO2. The composition varies with the feedstock
and the fermentation process used, which will inevitably affect the energy content of
biogas, whose lower heating value (LHV) varies from 16 to 28 MJ/m3. Globally, the
200 M. Ladero et al.

production of biogas has steadily increased over the last two decades from 0.28 EJ in
2000 to 1.33 EJ in 2017, with the EU contributing as much as half of the supply,
followed by Asia with about a third. However, as of 2017, this is only about 2% of
the overall bioenergy supply and has potential to represent a higher share in
estimated growing capacities that only in the EU could increase up to 20  109 m3
(World Bioenergy Association 2019).
Biomethane, also referred to as renewable natural gas, consists mainly of pure
methane, which can be obtained by purification and upgrading of biogas by remov-
ing CO2 and other gases or by gasification of solid biomass followed by methana-
tion. It has a LHV of 36 MJ/m3 and can use the distribution channels and be applied
in technologies that currently use natural gas. The biomethane market prospects also
show an outstanding potential increase in demand. The International Energy Asso-
ciation estimates growths up to about 45 Mtoe by 2030 and 75 Mtoe under a Stated
Policies Scenario, which could reach as much as 110 Mtoe by 2030 and above
200 Mtoe by 2040 under a Sustainable Development Scenario with developing
countries in Asia leading the way. This product sees a broad-based growth across
all sectors where natural gas is present (buildings, power and heat, industry), whilst
also tapping into markets like transport (International Energy Association 2021).
Hydrogen shows great advantages as fuel candidate, as it has a high energy yield
(142.4 kJ/g) and heating efficiency (about 2.75-fold with respect to hydrocarbons). It
is a clean fuel as only water is produced from combustion and can made use the
infrastructure associated with the rapid development of fuel cell technologies (Wang
and Yin 2018). Current worldwide hydrogen value chains currently indicate a
production of hydrogen from fossil-based natural gas and coal amounting to
196 Mtoe and 75 Mtoe, respectively. The production of hydrogen as by-product of
other processes is about 48 Mt H2, only 0.3 of which comes from renewables, which
points towards an opportunity of increasing the share. By 2050 transport, industrial
energy and particularly the use of hydrogen as industrial feedstock will have an
increased market share, such as in methanol and ammonia production (International
Energy Association 2021). As for biohydrogen in particular, it was calculated that
about 15.5 Mt could be produced using waste from crops like potatoes, wheat, corn,
sugarcane or barley by 2030 (Alavijeh et al. 2020).
In connection with methane and hydrogen, the term hythane was coined and
patented by the company Hydrogen Component Inc., which tested mixtures of these
gases in internal combustion engines. Mixtures containing 20% v/v H2 showed a
reduction of NOx emissions whilst maintaining energy efficiencies. Significantly,
these mixtures did not require changes in engines or infrastructures.
Finally, the term volatile fatty acids (VFA) typically refers to organic acids of
chain length from two to up to six carbons. VFAs have been used in many
applications, including energy related technologies, such as the generation of elec-
tricity in microbial fuel cells, in the synthesis of complex polymers or in textiles,
food and pharma products. In general, the market value of VFAs is related to the
length of the chain, which drives the production towards heavier products rather than
lighter, there is even a trend towards the generation of higher fatty acids (Esteban and
Ladero 2018).
8 Food Waste Biorefinery for Bioenergy and Value Added Products 201

Policies to promote the generation of all of these gaseous products from renew-
able substrates, particularly FW and other agricultural wastes show a particular
alignment with the UN SDGs, particularly SDG 7 (Affordable and clean energy)
and SDG 12 (Responsible consumption and production). The drive towards the
production of each of the mentioned products from organic substrates will greatly
depend on the fermentation technology used. The following epigraphs will cover the
particularities of their production with a focus on FW. Table 8.2 summarizes the
main results of selected references of processes to obtain the mentioned products.
Biogas is produced by single-stage AD of organic matter such as wastewater
sludge, manure, crop residues or, naturally, FW. AD is a process that consists of four
steps, namely: (1) hydrolysis, where lipids, proteins, carbohydrates and other large
polymers are broken down into smaller molecules by anaerobic bacterial consortia
including Cellulomonas clostridium, Bacillus thermomonospora, Ruminococcus
baceriodes among others; (2) acidogenesis, during which bacteria like Streptococ-
cus, Lactobacillus or Bacillus transform the monomers into short chain organic acids
and alcohols; (3) acetogenesis, where the former products are converted into H2,
CO2 and acetic acid by bacteria like Clostridium, Acetobacterium or
Syntrophomonas; and (4) methanogenesis, in which Methanolobus, Methanosarcina
or Methanococcus reduce the previous products to CH4.
The first AD plant treating FW dates from 1939 in the USA, although this type of
processes has attracted interest in Europe in the last decades, reaching a capacity of
150 Mton/year (Bolzonella et al. 2018). The quantity and quality of biogas, i.e., its
biomethane content, depend on the type of inoculum and operational factors like pH,
temperature, hydraulic retention time (HRT), solid retention time (SRT), stirring and
loading rate (Tabatabaei et al. 2020). Largely, the biogas produced is also influenced
by the composition of the feedstock. In the case of FW, this can be disadvantageous
as they could have low C/N ratios, high lipid concentrations, insufficient macro- and
micronutrients or there could be toxic compounds, all of which may trigger different
inhibition mechanisms that may unfavour the steps of AD to different degrees
(Tabatabaei et al. 2020).
Biogas production is a slow process owing to the long doubling time of the
microorganisms involved in these stages, being methanogenesis the rate-limiting
stage in the AD process leading to higher HRTs. In other cases, the hydrolysis stage
can be cumbersome if complex organic substrates are used, hence leading to toxic
byproducts and VFAs. To decrease HRTs and enhance biogas production, one
possible solution involves the upstream pretreatment of the feedstock to increase
its digestibility. In this sense, biological pretreatment using fungi, microbial consor-
tia or enzymes is beneficial as they are relatively simple, do not require additional
chemicals and the investment is typically low. On the other hand, such pretreatments
require long reaction times and may be hard to apply in some substrates. Further
lines of work on pretreatment feature techniques like microaeration and metabolic
engineering of microorganisms (Tabatabaei et al. 2020). Additionally, components
like fats or terpenes need to be removed to enhance the productivity as they show
inhibition effects and methane formation can be reduced. In the case of terpenes,
these compounds are known to have antimicrobial and antioxidant properties and are
 202

Table 8.2 Examples of biological processes to obtain biogas, biohydrogen, biohythane and VFA from FW sources
Food waste Type of AD Inoculum Pretreatment reactor Operating conditions Main results Reference
Fruit, vegeta- Two-stage Activated sludge Mechanical homogeniza- T ¼ 35
C YCH4 ¼ 460 mL/g Wang et al.
ble and (acidogenic and from wastewater tion HRT ¼ 10 days + 20 VS (2014)
kitchen waste methanogenic) treatment plant Pilot plant CSTR (2 and days CVFA ¼ 2582 mg/
4 m3) OLR ¼ 4.5 g VS/ (L day) L
Tomato Mainly Methanogenic sludge Lycopene extraction and T ¼ 55
C YCH4 ¼ 105 mL/g Allison and
pomace methanogenic from UC Davis enzymatic saccharifica- HRT ¼ 90 days VS Simmons
tion CSTR OLR ¼ 1 g VS/ (L day) (2017)
Kitchen waste Full four-stage oper- Activated sludge Lipid extraction T ¼ 55
C YCH4 ¼ 531 mL/g Algapani
ation codigested with from wastewater Shaken bottles (120 mL) HRT ¼ 30 days VS et al. (2017)
sludge treatment plant OLR ¼ 4.24 g VS/ CVFA ¼ 510 mg/g
(L day) VS
Household Two-stage Brown water from None for FW; preheating For H2: YCH4 ¼ 728 mL/g Paudel et al.
food waste (acidogenic and University toilets for sludge CSTR (10 and T ¼ 37
C; HRT ¼ 0.3 VS (2017)
methanogenic) 40 L) days; OLR ¼ 47.8 g VS/ YH2 ¼ 99.8 mL/g
(L day) VS
For CH4: CVFAb¼ 2200 mg/
T ¼ 37
C; HRT ¼ 20 L
days; OLR ¼ 37.2 g VS/
(L day)
M. Ladero et al.
8 Food Waste Biorefinery for Bioenergy and Value Added Products 203

also used as food colorant, so isolation of this type of compounds could be interest-
ing not only from the perspective of increasing yields of biogas, but also towards an
integral valorisation of FW (Allison and Simmons 2017).
Hydrogen from different biomass sources can be obtained through different
(thermo)chemical methods that include the gasification of hydrocarbons, steam or
aqueous phase reforming and the corresponding water-gas shift reactions. In addi-
tion, a number of biological processes using lignocellulosic and carbohydrate-rich
substrates like FW have attracted interest in the last few years, including direct and
indirect biophotolysis (Sampath et al. 2020). Both require phototrophic microorgan-
isms like cyanobacteria or microalgae like Chlorella or Chlamydomonas to capture
solar radiation and perform oxygenic photosynthesis, by means of which H2 can be
generated in a single or two-step process.
In dark fermentation, bacteria like Clostridium, Enterobacter, Cellulomonas or
Thermotoga can be used, in which hydrogenases are the key catalyst for the
generation of H2, which is produced without any oxygen or light inputs. Feedstock
like FW or other type of waste can be used, although it appears that lipid and protein-
rich substrates are detrimental to H2 production compared to others rich in carbo-
hydrates (Ntaikou et al. 2010).
In photofermentation, light-dependent purple sulfur and, especially, non-sulfur
bacteria like Rhodobacter sphaeroides or Rhodopseudomonas palustris are suitable
to decompose organic acids to H2 with nitrogenases and hydrogenases catalyzing the
process (Hitam and Jalil 2020).
Hybrid systems can increase biohydrogen productivity combining
non-photosynthetic and photosynthetic bacteria. The organic acid byproducts gen-
erated during dark fermentation by bacteria like Clostridium butyricum serve as
substrate for photoautotrophic microorganisms like Rhodopseudomonas faecalis in
the presence of light in the second stage (Azwar et al. 2014).
Apart from physical pretreatments that are also applicable to the production of
biogas to make the complex polymeric structure more accessible in FW or other
biomass, certain chemical pretreatments are meant to increase the yield to hydrogen.
For example, chloroform, BESA and O2 inhibit methanogenic species (Wang and
Yin 2018).
Owing to the importance of hydrogen production processes, a techno-economic
analysis of a dark fermentative hydrogen production process from FW in China
recently showed that it could be profitable operating with a supply of above 0.3 ton/
day of FW. The unit production cost of the product would be US$1.02 per cubic
meter with labour having the highest impact on the costs (Han et al. 2016).
In the past few years, the production of biohythane has attracted a good deal of
attention, for which the use of FW and sewage sludge have had prominent use.
Contrary to the sole production of biogas, the substrates are subjected to a two-stage
AD consisting first of a dark fermentative phase followed by a methanogenic step.
This operation allows the reduction of the overall required fermentation time and
reactor volumes, which makes it attractive from the operation point of view
(Bolzonella et al. 2018). The two main types of feedstock typically used are FW
and sewage sludge or codigestion of both. LCA research on biohythane shows that a
204 M. Ladero et al.

two-stage AD process produces a methane amount similar to the single-stage

configuration, although the concomitant production of H2 helps to reduce an addi-
tional 10% the overall CO2 equivalent emissions of the process (Franchetti 2013).
Acetic, propionic, (iso)butyric and (iso) valeric acid are commonly denoted as
VFAs. These are the main byproducts generated following AD of biomass as
intermediates of the different stages. Their production will vary depending on the
composition of the substrate used, especially if they are complex and have high
organic load rates, lipid content or low C/N ratios. Additionally, the operational
conditions used will determine to what extent the metabolic pathways in the acido-
and acetogenesis stages of AD will occur. In general, low solid to inoculum ratios
and low HRT lead to an increase in VFA production. Also, the quality of VFAs is
dependent on the temperature conditions as under mesophilic conditions acetic acid
is the main VFA produced during hydrolysis and acidogenesis; on the other hand
thermophilic operation favours the generation of butyrate (Zhou et al. 2018;
Srisowmeya et al. 2020).

8.3 Biobased Chemicals from Food Waste

8.3.1 5-Hydroxymethylfurfural and Furfural

Lignocellulosic biomass consists of cellulose (35–50%), hemicellulose (20–35%)

and lignin (10–25%) as major components. From the hydrolysis of the former two
fractions, hexoses like glucose and fructose and pentoses like xylose maybe
obtained, from whose dehydration 5-hydroxymethylfurfural (HMF) and furfural
can be obtained (Esteban et al. 2018). Both are very highly valued compounds that
act as building blocks for further chemicals given their chemical functionality.
From transformations of HMF, further furan derivatives like 2,5-diformylfuran,
2,5-furandicarboxylic acid or 5-hydroxymethyl-2-furan carboxylic acid may be
obtained with interest as pharmaceuticals, agrochemicals, flavors and fragrances or
dimethylfuran and methyl tetrahydrofuran, valuable fuel additives. As for furfural,
compounds like 2-methyl tetrahydrofuran and 2-methylfuran are derivatives that
have proven to be bioadditives that improve fuel performance. Their industrial
relevance has led to the first plants for HMF (between US$330,000 and US
$386,000 per tonne) production having been established in the last few years, such
as the ones by AVA Biochem or GF Biochemicals, the latter of which focuses on
levulinic acid using HMF as intermediate. As for furfural, with a market price of US
$2800–US$3300 per tonne, its market is estimated to grow from US$551 to about
US$700 million from 2019 to 2024 (Bhaumik and Dhepe 2015), which has led to an
increasing number of industrial efforts. Starting with Quaker Oats Company in the
1920s, many companies have patented technologies for furfural production, such as
Petro Chemie, Krupp or Stake Technologies (Esteban et al. 2020).
The source of sugars from which the dehydration takes place are (hemi)cellulosic
fractions from lignocellulosic biomass, including FW. With the generation of furans
8 Food Waste Biorefinery for Bioenergy and Value Added Products 205

Fig. 8.3 Concept for the production of furans from carbohydrate-rich FW using a biphasic liquid
system

in mind, ideally FW with a high fraction of carbohydrates would be most productive.

Unlike most of the other products covered in this chapter, HMF and furfural are
obtained through chemocatalytic processes as these compounds are inhibitors of
microorganisms. For the reaction to occur, hydrolysis to obtain individual sugars
happens first, followed by the dehydration in a polar phase, usually aqueous, by an
acid catalyst. FW like pasta, potatoes and bread have been used to obtain HMF,
furfural and other furans using green solvents like γ-valerolactone (GVL) as medium
(Lui et al. 2019; Yu et al. 2018).
However, in the continued presence of acid catalysts and water, HMF can
rehydrate to levulinic and formic acid and both HMF and furfural can polymerize
and form undesired humins. For this reason, many authors have followed a biphasic
approach, whereby nonpolar solvents extract the furans in situ as they are generated
and hence avoid the aforementioned reactions. By doing this, the furans can after-
wards be isolated and the organic solvent be recycled into the process. In addition, if
a molecular catalyst is used, it can remain in the aqueous phase and be reused.
Figure 8.3 depicts this concept.
Following the latter approach, some works have reported the production of HMF
using FW. For instance, melon rind waste has been used as carbon source to obtain
HMF under microwave irradiation using montmorillonite KSF as catalyst, with
which the hydrolysis to hexoses like glucose and fructose and their dehydration
was achieved. A biphasic H2O/THF was used to improve the performance of the
system by extracting HMF with respect to single phase operation, reaching a 6%
overall recovery from the original FW source (Lucas-Torres et al. 2016). Typically
water is used as the polar phase, although it has been found that choline chloride-
206 M. Ladero et al.

based (ChCl) deep eutectic solvents can help hydrolyse carbohydrate polymers. A
biphasic system with ChCl-H2O as polar phase and GVL as extracting phase was
reported for the production of HMF from carbohydrate-rich FW like bread or rice
waste, for which AlCl36H2O was used as catalyst. Yields up to 18.60% were
obtained with GVL greatly helping to increase the extraction efficiency, thereby
avoiding the degradation of HMF. Very importantly, the ChCl-H2O solvent showed
high recyclability (Ji et al. 2020).

8.3.2 Citric Acid

Citric acid (CA) is one of the most widely used organic acids: its production was
2 Mtons in 2020, with China being the world’s largest producer. The global market
could reach US$3.6 billion in 2025, growing more than 5.24% by then. Currently, it
is mainly consumed in the food, pharmaceutical and personal care industries as
acidulant, antioxidant, preservative, flavouring, buffer and astringent. Other appli-
cations include leather tanning, metal finishing and phosphate replacement in deter-
gents (Amato et al. 2020, Mores et al. 2021). Great research efforts have focused on
the production of citric acid through fermentation processes. Aspergillus niger is the
main producer due to its easy manipulation and its high selectivity/yield towards the
acid of interest in comparison to other microorganisms such as Yarrowia lypolytica
yeast, which also generates isocitric acid (Mores et al. 2021). A. niger can produce
CA from glucose, sucrose, maltose, galactose, mannose and fructose. This is a good
reason to employ impure raw materials with high carbohydrate content such as cane
molasses or corn starch.
In recent years, several studies have focused on FW valorisation to reduce costs
(Amato et al. 2020). Ozdal and Kurbanoglu (2019) produced 68.6 g/L citric acid
through the most widely used method in the industry, submerged fermentation. The
best results were observed with 0.15 g/L of KH2PO4 and 4 g/L of chicken feather
peptone (CFP). This nitrogen source was obtained from a complex pretreatment with
two consecutive hydrolysis with 6 N H2SO4 at 70
C, 24 h and 130
C, 4 h, followed
by the addition of different hydroxides, filtration and evaporation. The carbon source
was obtained by pretreatment of sugar beet molasses with H2SO4 1 N at 90
C,
15 min and its subsequent addition of 0.1 g/L of K4Fe(CN)6. Employing the same
type of operation, Liu et al. (2018) used waste cooking oil as a carbon source,
co-producing 12.6 g/L of citric acid and 4 g/L of erythritol at pH 6 and an osmotic
pressure of 0.75 osmol/L. Hou and Bao (2018) produced 136.3 g/L of citric acid by
simultaneous saccharification and fermentation (SSF) of corn stover, pretreated with
H2SO4 5% w/w, 175
C, 5 min and detoxified with Amorphoteca resinae ZN1 at
28
C, 4–7 days. Roukas and Kotzekidou (2020) obtained 351.5 g/kg dry pome-
granate peel by solid state fermentation, a promising method with low or no
pretreatment or complex equipment, although with a lower capacity to use nutrients
(Table 8.3).
8 Food Waste Biorefinery for Bioenergy and Value Added Products 207

Table 8.3 Citric acid production processes from FW

CA
production
Substrate Microorganism Type of operation and yield Reference
Sugar beet A. niger Batch, submerged, 30
C, pH 68.8 g/L Ozdal and
molasses MO-25 6, 200 rpm, 7 days, 4 g/L CFP. 0.48 g/g Kurbanoglu
with CFP 0.15 g/L KH2PO4 (2019)
Waste Y. lypolytica Batch, submerged, 28
C, pH 12.6 g/L Liu et al.
cooking oil M53 6, 200 rpm, 7 days, 0.75 osmol/L 0.48 g/g (2018)
Corn stover A. niger SIIM Batch, SSF. Reactor 1: 50
C, 136.3 g/L Hou and
M288 pH 4.8, 12–48 h. Reactor 2: 0.75 g/g Bao (2018)
1 vvm air
Pomegranate A. niger B60 Solid state, 25
C, pH 8, 8 days, 351.5 g/kg Roukas and
peels 3% methanol, moisture 75% 0.78 g/g Kotzekidou
(2020)

8.3.3 Lactic Acid

Lactic acid (LA) is a very versatile compound that arouses much interest, accounting
its production through fermentative processes for around 80–90%. The global
demand for LA is estimated to increase from 1220 kt in 2016 to 1960 kt in 2025
(López-Gómez et al. 2019). The chemical route generates racemic mixtures of this
acid, requiring expensive separation processes. However, through microbial fermen-
tation, D- or L-lactic isomers can be generated. LA is widely used as acidulant in the
food industry. Furthermore, it is used in the manufacture of topical ointments,
lotions, parenteral solutions, cleaning agents, solvents, humectants and in the gen-
eration of oxygenated chemicals such as esters. In addition, polylactic acid (PLA) is
a green, biodegradable and biocompatible polymer obtained by ring opening from
lactide, a cyclic lactic acid dimer. Table 8.4 presents some LA production routes
from FW.
Among LA-producing microorganisms, the bacteria of the genus Lactobacillus
should be highlighted, being the most abundant genera. In recent years, numerous
studies have been carried out based on the use of FW as a cheap source of nutrients
(López-Gómez et al. 2019; Rawoof et al. 2020). Ahmad et al. (2020) studied the
effect of different pH and total solids content on dark fermentation of FWs without
inoculation of external microorganisms. The authors obtained a maximum LA
concentration of 12.87 g/L after having carried out an enzymatic pretreatment with
a cellulase at 50
C, pH 5–5.5 for 72 h. In order to reduce production costs, Jiang
et al. (2019) obtained 79.1 g/L of L-lactic acid through a simultaneous saccharifica-
tion and fermentation (SSF) in fed batch from 100 g/L of corncob residues
supplemented with 15 g/L peanut meal, 30 g/L CaCO3, 10 U/g cellulose, 15 U/g
glucosidase and 0.3 g/L protease. Through a continuous operation, Peinemann et al.
(2019) achieved a yield of 0.86 g/g with the addition of glucoamylase. However,
Costa et al. (2021) did not need to add any enzyme in their process, reaching a yield
208 M. Ladero et al.

Table 8.4 Lactic acid production processes from FW

LA
production
Substrate Microorganism Type of operation and yield Reference
Food waste Indigenous Batch, mode dark, 50
C, pH 6, 10 12.87 g/L Ahmad
microbiota days 150 gTS/L, 10 days 0.09 g/g et al.
(2020)
Corncob Bacillus Fed-batch, SSF. 50
C, pH 79.1 g/L Jiang et al.
residue coagulans H-1 6, 150 rpm, 84 h, 15 g/L peanut 0.76 g/g (2019)
meal, 30 g/L CaCO3, 10 U/g cellu-
lose, 15 U/g glucosidase, 0.3 g/L
protease
Food waste Streptococcus Continuous (0.44/day), SSF, 35
C 74 g/L Peinemann
(university sp. pH 6. 160 rpm, 148 h, 1 mL/24 h 0.86 g/g et al.
canteen) glucoamylase (2019)
RCW and L. casei DSM Batch, 37
C, pH 7, 50 rpm, 65.4 g/L Costa et al.
PP 20011 48 h, RCW:PP 90:10 0.78 g/g (2021)

of 78.3% in a batch operation from ricotta cheese whey (RCW) and centrifuged pear
processing (PP) residue in a 90:10 ratio.

8.3.4 Succinic Acid

Succinic acid (SA) is widely used in the food and pharmaceutical industry as an
additive, while it is an intermediate of the production of polybutylene succinate,
polyesters, polyols, resins, coatings and pigments in the traditional chemical indus-
try. In the bioeconomy era, SA is used mainly in the generation of intermediate
chemicals such as tetrahydrofuran, hydroxysuccinimide or maleic acid, among
others. In addition, the potential application of this acid in the manufacture of
biodegradable polymers should be highlighted (Dai et al. 2020). In 2017, the
bio-derived SA market was estimated at US$175.7 million and a compound annual
growth rate of 20% was forecasted, reaching a value of US$900 million by 2026
(Li et al. 2020). Table 8.5 features some studies on SA production starting from FW.
Bacteria isolated from the rumen of ruminants, including Actinobacillus.
succinogenes, Anaerobiospirillum succiniciproducens, and Mannheimia
succiniciproducens are the most promising candidates that can naturally produce
SA as the main product during anaerobic fermentation. However, some processes
have also been developed using fungi like Saccharomyces cerevisiae, Yarrowia
lipolytica, Byssochlamys nivea and Paecilomyces varioti. To achieve sustainable
production of bio-based SA, expensive sources of carbon and nitrogen, mainly
glucose and yeast extract, can be replaced by FW (Dai et al. 2020; Li et al. 2020).
For this reason, after optimizing the hydrolysis conditions, Li et al. (2018) used fruit
and vegetable waste (FVW) pretreated with 2% glucoamylase, 1% cellulase, 2%
hemicellulase and 0.25% pectinase at 55
C, pH 5, 300 rpm for 48 h. The authors
8 Food Waste Biorefinery for Bioenergy and Value Added Products 209

Table 8.5 Succinic acid production studies starting from FW

SA
production
Substrate Microorganism Type of operation and yield Reference
FVW Y. lypolytica Fed batch, isFBB, 28
C, pH 140.6 g/L Li et al.
PSA02004 6, 600 rpm, 2 L/min air, 324 h 0.46 g/g (2018)
Sugarcane Y. lypolytica Batch, 28
C, pH 6, 600 rpm, 2 L/ 33.2 g/L Ong et al.
bagasse PSA02004 min air, 102 h 0.58 g/g (2019)
FVW Y. lypolytica Batch, isFBB. 28
C, no pH con- 32.6 g/L Li et al.
PSA02004 trol, 600 rpm 3 L/min air, 54 h 0.61 g/g (2019)
HSCBs A. succinogenes Batch. 37
C, no pH control, 24.1 g/L Ferone
DSM 22257 150 rpm, MgCO3 30 g/L, 400 h 1.1 g/g et al.
(2019)

conducted a fed-batch type fermentation in an in situ fibrous bed bioreactor (isFBB).

They employed Y. lypolytica as a biocatalyst to reach a SA yield of 0.46 g/g. From
these results, Li et al. (2019) used the same substrate and pretreatment but without
pH control for the first time, achieving a yield of 0.61 g/g. Ong et al. (2019) needed
two pretreatments to obtain a yield of 0.58 g/g through a free cell batch type
fermentation with the same strain. In order to effectively use sugarcane bagasse,
this substrate was submitted to an alkaline treatment with NaOH 0.5 M, 80
C, 2 h
and an enzymatic treatment with cellulase 40 FPU/g at 50
C, pH 5, 150 rpm, and
96 h. To valorise a by-product stream from the beverage industry, Ferone et al.
(2019) used high sugar content beverages (HSCBs) as feedstock for SA batch
production by A. succinogenes. After adding 30 g/L MgCO3 to activate the meta-
bolic pathway of succinic acid generation, they obtained 1.1 g/g from
not-supplemented ACE juice without pretreatment.

8.3.5 Fumaric and Itaconic Acids

Fumaric acid (FA) and itaconic acid (IA) are included by the United States Depart-
ment of Energy on the list of top 12 building blocks to be obtained biotechnolog-
ically (Zhang et al. 2013). Both are known to be produced best using filamentous
fungi.
FA ((E)-2-butanodioic acid) production was linked to the Rhizopus genus until
50s in the XX century, when the petrochemical production route was developed.
Presently, FA is widely used on food and drug industries with promising applica-
tions in the polymer industry, having a global production of around 346 ktons/year
(Martin-Dominguez et al. 2018). This demand is satisfied through a petrochemical
process consisting of maleic acid isomerization. Very intensive conditions and the
presence of maleic anhydride are managed in this process, entailing a great environ-
mental risk. In an attempt to have fewer intensive requirements, enzymatic routes are
currently under study, although their maturity is still limited (Martin-Dominguez
210 M. Ladero et al.

Table 8.6 Fumaric acid production processes from FW

FA
Substrate Microorganism Pretreatment yield Reference
Apple pom- Rhizopus oryzae Oven dried 52 g/ Das et al.
ace (AP) 1526 kgAP (2015a)
Cane Rhizopus oryzae Hydrolyzation and activated 34.2 g/ Deng and
bagasse ATCC 20344 carbon adsorption L Aita (2018)
Food Waste Rhizopus RH 7-13 Milling, heating 31.26 g/ Liu et al.
Separation of liquid fraction. L (2016)
Food Waste Rhizopus RH Pulverization and hot water 23.94 g/ Fan et al.
7-13-807 extraction L (2020)

et al. 2018). Table 8.6 summarizes some fumaric acid production processes using
FW as substrate.
In the development of a new fermentative process through the biorefinery con-
cept, several renewable raw materials related with food and agro-food industries
have been studied by several authors. In the last decade, many authors have tested
different waste types, achieving good production yields using “classical”
pre-treatments like drying and enzymatic hydrolysis, very consolidated on lignocel-
lulosic biomass revalorization (Deng and Aita 2018). Also, different kinds of
operation have been tested (Das et al. 2015b), such as Solid State Fermentation
(SSF), where excellent yields are reached having simpler pre-treatments and process
conditions. The direct use of FW has been studied, too, reaching very promising
results, mostly requiring easier handling pretreatments (Fan et al. 2020).
IA is employed overall as a very versatile monomer in the chemical industry,
particularly in fibres and polymers, also having many other applications in pharma
and health industries (Yang et al. 2020). It is known to be a much consolidated
bio-product obtained by a fermentative process carried out by Aspergillus terreus
using glucose or sucrose as the substrates (Huang et al. 2014b).
With a global production of about 407 kton/year, IA would involve a potential
market of US$567 million in 2022 (Ramakrishnan et al. 2020). Substrates are
responsible for its high production costs; thus, it is necessary to find an alternative
raw material to reduce prices (Huang et al. 2014b) and move this process into the
green industry cycle. Lignocellulosic biomass has been studied as potential raw
material for this process (Yang et al. 2020). As compiled in Table 8.7, very
promising production yields are reached using different FW.

8.3.6 Diols

Diols constitute a very versatile family of compounds for the chemical industry. This
term refers to compounds containing two alcohol groups that are attractive
biorefinery products mainly for their potential as monomers in bio-based polyesters
8 Food Waste Biorefinery for Bioenergy and Value Added Products 211

Table 8.7 Studies focused on itaconic acid production from FW

FA
Substrate Microorganism Pretreatment yield Reference
Corn starch Modified Enzymatic hydrolysis 80 g/L Huang et al.
Aspergillus (2014a)
terreus
Bamboo Aspergillus Alkaline treatment, steam 41.54 g/ Yang et al.
terreus explosion and enzymatic L (2020)
AtYSZ-38 hydrolysis
Watermelon Aspergillus Drying, grinding and micro- 21.52 g/ Ramakrishnan
(Citrullus japonicas wave assisted solvent extraction kgCLR et al. (2020)
lanatus) rind
(CLR)

Table 8.8 Studies for the production of diols from FW

Diol
and
Substrate Microorganism Pretreatment yield Reference
Cooking Microbial Raw glycerol from waste cooking- 1,3- Wang et al.
Oil consortium oil-based biodiesel production PDO (2019)
27.77 g/
L
Sugar Enterobacter Sugar cane molasses provided by 2,3- Psaki et al.
cane ludwigii industry BDO (2019)
50.6 g/
L
FW Bacillus Homogenization 2,3- OHair
licheniformis BDO et al.
YNP5-TSU 6.8 g/L (2021)
Cardoon Engineered Steam explosion and enzymatic 1,4- De Bari
Escherichia coli hydrolysis BDO et al.
0.46 g/ (2020)
gglucose

(Sato et al. 2020). Table 8.8 shows some examples of diols produced from FW
sources.
One of the most useful diols is 1,3-propanediol (1,3-PDO), whose main applica-
tion is being a monomer for polytrimethylene terephthalate production (Wang et al.
2019). 1,3-PDO fermentative production is usually through co-production with other
diols (Sato et al. 2020) or other bio-based molecules (Wang et al. 2019). Literature
on the production of 1,3-PDO from FW is scarce. However, the work by Wang
perfectly showcased the concept of biorefinery, using wasted cooking oil to obtain
biodiesel and raw glycerol as by-product, which can be ultimately used as substrate
for 1,3-PDO production (Wang et al. 2019).
2,3-butanediol (2,3-BDO) is a very promising diol with promising applications in
several processes such as polyurethane production (Psaki et al. 2019). Its
212 M. Ladero et al.

fermentative production from lignocellulosic biomass is well reported, using stan-

dard pretreatments (Psaki et al. 2019). However, there are studies where 2,3-BDO
can be produced from direct use of FW in non-sterile conditions (OHair et al. 2021),
largely reducing costs due to the pre-treatment.
Finally, 1,4-Butanediol (1,4-BDO), with a global production of 2 million tons/
year, is a great building block used in the automotive and fibre (lycra) industries
(De Bari et al. 2020). There are some studies confirming the viability of implanting
local biorefineries for 1,4-BDO production using different lignocellulosic materials
(De Bari et al. 2020).

8.4 Bioingredients and Biomaterials from Food Waste

8.4.1 Biolubricants

Used oils like waste cooking oils are valuable sources of lipids that, under suitable
chemical transformations, can lead to obtain valuable chemicals. Specifically, lubri-
cants are an interesting family of compounds essential for tribological science, for
they are put to use to reduce overheating and friction in a variety of engines,
machinery, turbines and gear. In this sense, biolubricants pose an interesting oppor-
tunity due to their superior performance in terms of health and biodegradability
considerations. Vegetable oil-based bio-lubricants are recognized to have enhanced
lubricity, high viscosity, good anti-wear property, high viscosity index, increased
equipment service life, high load carrying ability, low evaporation rate, low emission
of metal traces into the atmosphere. To function as lubricants, waste oil must be
biologically or chemical processed (Esteban and Ladero 2018; Khodadadi et al.
2020). Table 8.9 presents some examples of the production of biolubricants from
FW.
Reactions of biolubricants manufacturing involve hydrolysis, transesterification,
epoxidation and oxirane ring-opening reactions (Li and Wang 2015; McNutt and He
2016). Both chemical and biological catalysis are applied. For example, the fatty
acid ethyl esters mixture, a fish oil residue obtained after the extraction of omega-3
polyunsaturated fatty esters, has been converted into mixtures of mono-, di-, and
triesters of trimethylolpropane by transesterification with a chemical catalyst
(Angulo et al. 2018). Many recent examples of the application of biocatalytic
reactions for the preparation of biolubricants can be found. In some cases trans-
formations involved enzymatic hydrolysis and chemically catalysed esterification to
obtain a maximum yield of biolubricant in minimum time (Chowdhury et al. 2013).
Oil can be produced from confectionery and wheat milling side streams. In that case,
nutrient-rich fermentation media were produced by a two-step bioprocess involving
crude enzyme production by solid state fermentation followed by enzymatic hydro-
lysis of confectionery industry waste. The extracted microbial oils were enzymati-
cally hydrolysed and the free fatty acids were esterified by Lipomod 34-MDP in a
solvent-free system with trimethylolpropane (TMP) and neopentyl glycol (NPG).
8 Food Waste Biorefinery for Bioenergy and Value Added Products 213

Table 8.9 FW valorisation to biolubricants

Substrate Catalyst Process Results Reference
Fatty acid Sodium Transesterification of 84% conversion of Angulo
ethyl esters ethoxide mixtures of mono-, di-, ethyl esters), enriched et al.
mixture from a and triesters of in trimethylolpropane (2018)
fish oil residue trimethylolpropane triesters (96%
selectivity)
Microbial oils Lipase cata- Hydrolysis of oil and Conversion yields Papadaki
obtained from lyst. esterification in a were 88% and 82.7% et al.
confectionery Lipomod solvent-free system with depending on the (2018)
industry waste 34-MDP trimethylolpropane source of oil
(TMP) and neopentyl
glycol (NPG).
Soybean oil Immobilized Esterification with 90% conversion was Fernandes
lipase from neopentyl glycol (NPG) achieved after only et al.
Candida and trimethylolpropane 6h (2021)
rugosa (TMP) alcohols in a
solvent-free medium

The highest conversion yields were 88% and 82.7% for NPG esters of R. toruloides
and C. curvatus, respectively (Papadaki et al. 2018).
Enzymatic transformation can be used for the synthesis of biolubricants from
by-products of soybean oil processing using the lipase from Candida rugosa (CRL)
both in free and immobilized forms, to produce biolubricants via their enzymatic
esterification with neopentyl glycol (NPG) and trimethylolpropane (TMP) alcohols
in a solvent-free medium (Fernandes et al. 2021). Another example is the production
of a bio-lubricant, which was obtained through esterification of WCOs free fatty
acids with neopentyl glycol by using Thermomyces lanuginosus lipase
(TL) immobilized on Fe3O4-CA (citric acid modified magnetite nanoparticles)
catalyst. The immobilized lipase was used for the biolubricant synthesis from
WCOs in a solvent-free system (Sarno et al. 2019). Immobilized enzyme catalysts
have also been used in esters from branched alcohols and dicarboxylic linear acids
(Serrano-Arnaldos et al. 2021). For example, a green and efficient strategy for the
preparation of octylated branched biolubricant from waste cooking oil involved
hydrolysis and esterification with 2-ethylhexanol employing lipase (Novozym
435) and further epoxidation and activation by a low-cost nucleophilic reagent,
octanoic acid, to prepare octylated branched biolubricant using an recyclable ionic
liquid, [HMIm][PF6], as catalyst (Zhang et al. 2020).

8.4.2 Bacterial Cellulose

Bacterial cellulose (BC) is a homopolymer composed of units of β-D-glucopyranose

linked by β (1!4) O-glycosidic bonds, as well as plant cellulose (PC). Cellulose
nanofibers are arranged in a ribbon-like structure of 3–4 nm thick and 70–80 nm
214 M. Ladero et al.

wide cellulose polymorphism I. Some bacteria genera are BC producers, among all
microorganisms Komagataeibacter sp. (known before as Gluconacetobacter) is the
most significant BC producer. Nevertheless, even though BC and PC have the same
molecular composition, they differ in their chemical and physical properties. BC has
high purity due to the absence of hemicellulose and lignin, high mechanical strength,
elevated water holding capacity, biocompatibility and high crystallinity (approx.
90%) make it an interesting material for a wide range of applications.
The most widespread culture medium for BC production is the Hestrin-Schramm
medium (HSM). The main drawbacks of HSM are the cost of carbon and nitrogen
sources and the requirement of a buffer to avoid the decrease of pH caused by the
gluconic acid release (Hussain et al. 2019). Depending on the mixing of culture
media, a pellicle in the broth surface can be obtained (most common) if it is statically
incubated or sphere-like particles, if submitted to stirring conditions. For BC pro-
duction, general conditions are 28–30
C, pH from 4.0 to 8.0 and an incubation
period from 3 to 25 days or more, in static conditions (Hussain et al. 2019).
Regarding the BC purification process, it involves an alkaline treatment followed
by a washing step. From an industrial point of view, BC generation is hindered by
both the considerable economic cost of the culture media (approx. 30% of process
economics) and productivity. Nowadays, there are companies that produce BC
employing coconut water as raw material and use batch reactors (tanks and trays)
for the production. Nonetheless, this source is not available worldwide. In the last
years, a variety of FW has been tested, reaching among 2.1–10.8 g/L from 3 to
28 days, depending on the raw material and culture condition (Fan et al. 2016;
Abdelraof et al. 2019; Kuo et al. 2019; Salari et al. 2019). Furthermore, pretreatment
steps are usually employed when FW is used in order to breakdown their polymers,
thereby enhancing carbon intake by the microorganisms. This approach shows
interesting results if the production of BC with HSM is compared to the higher
yield of BC achieved with FW (Table 8.10). The second possible step is to isolate or
modify BC producers using all the molecular biology, genetic and microbiology
tools available nowadays.
The broad diversity of BC application is caused by its remarkable physicochem-
ical properties added to its classification as GRAS (Generally recognized as safe) by
the FDA (Food and drug administration) in 1992. In the medical field, it has been
applied for the development of wound dress, dentary prostheses, tissue scaffolds,
blood vessel replacements and biosensors. In the food industry, it has been used as
packaging material (biopackaging), and a food ingredient like fat substituent,
probiotics immobilizer and stabilizing agent (Andriani et al. 2020). In the textile
industry, composite manufacturing, 3D printing material and optoelectronics are
examples of the extensive utilization list of this biopolymer (Andriani et al. 2020;
Hussain et al. 2019; Zhong 2020).
8 Food Waste Biorefinery for Bioenergy and Value Added Products 215

Table 8.10 Production of bacterial cellulose from FLW

Production
(g/L)
Fermentation Productivity
Substrate Microorganism Pretreatment condition (g/L day) Reference
Potato Gluconacetobacter Nitric acid Static condi- FWM: 4.72/ Abdelraof
peels xylinus ATCC hydrolysis tion; 0.79 et al.
10245 28–35
C, pH HSM: 1.21/ (2019)
6.0 and 4–6 0.30
days
Orange Gluconacetobacter Enzymatic Static condi- FWM: 3.40/ Kuo et al.
peels xylinus BCRC hydrolysis tion; 0.43 (2019)
12334 28–30
C, pH HSM: 0.97/
4.8, 8 days 0.19
Citrus Komagataeibacter Enzymatic Static condi- FWM: 5.7/ Fan et al.
wastes xylinus CICC hydrolysis tion; pH 5.0, 0.71 (2016)
No.10529 28–32
C, HSM: 3.9/
21 days 0.48
Beet Gluconacetobacter Beet molasses: Static condi- FWM: 4.56/ Salari
molasses xylinus PTCC 1734 acid hydrolysis tion; pH 5.5, 0.32 et al.
and Cheese whey: 28
C and FWM: 3.55/ (2019)
cheese Enzymatic 14 days 0.25
whey hydrolysis HSM: 3.26/
0.23

8.4.3 Polyhydroxyalkanoates (PHA)

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters (bioplastics) which

constitute an alternative to fossil-derived plastics. They are classified depending on
the polymerization units size in short-chain-length PHAs (scl-PHAs, 3–5 carbon
atoms) and medium-chain-length PHAs (mcl-PHAs, from 6 to more than 14 carbon
atoms) (Sabapathy et al. 2020). PHAs production is widespread among Bacteria and
Archaea domains, whose synthesis takes place under unbalanced growth conditions
with an excess of carbon source and they are accumulated inside cells. The most
common PHAs are polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and
their copolymer polyhydroxybutyrate-co-polyhydroxyvalerate (PHBV) (Anjum
et al. 2016). The scl-PHAs have poor mechanical characteristics due to their
crystallinity, mcl-PHAs have more suitable ones. That is the reason for the require-
ment of mixtures of different PHAs and their modification to improve their qualities
as a material. Nevertheless, they show significant application potential, because they
are biodegradable, biocompatible and they produce low end-of-life environmental
problems. In consequence, PHA has wide applicability as biomedical material such
as surgical sutures, regeneration materials and prostheses (Grigore et al. 2019).
These are biopolymers with interesting perspectives. There are also some applica-
tions in biodegradable food packaging and active packaging as well as in agriculture
as a substituent of thermoplastics. Furthermore, they are used as composites and 3D
printing inks (Grigore et al. 2019).
216 M. Ladero et al.

Table 8.11 Valorisation of FW using PHA production processes

Yield (% g
CDW)
Fermentation Productivity
Substrate Microorganism Pretreatment condition (g/L day) Reference
Sunflower Burkholderia Non specified 37
C, pH 7.0, 60/1.5 Kourmentza
used thailandesis 120 h aeration et al. (2018)
cooking E264
oil
Organic Microorganism Acidogenic Non controlled 55/6.7–11.8 Valentino
fraction of consortium fermentation temperature et al. (2018)
the munic- pretreatment (10–30
C),
ipal solid pH 7.8–8.4,
waste 120 days
Apple Pseudomonas Dilution and 30
C, pH 7.0, 49/1.5 Pereira et al.
pulp waste chlororaphis centrifugation 72 h, aireation (2021)
subsp. pretreatment
aurantiaca
DSM 19603
Grape Cupriavidus Organic 30
C, pH 7.0, 63/4.3 Kovalcik
pomace necator H16 extraction, 29.5 h, et al. (2020)
CCM 3726 enzymatic aireation
hydrolysis and
freeze drying

At present, PHAs comprise 1.7% of the 2.11 million tons of the global bioplastic
market. PHA production takes place using pure microbial cultures and definite
fermentation broth, which includes carbon sources such as pure sucrose and glucose,
organic acids (from 4 to 10 carbon atoms), alcohols and oils (Anjum et al. 2016).
With regards to production at pilot-scale and large-scale processes, to date fed-batch
production shows the highest productivities, despite these continuous and semi-
continuous reactors have already been tested (Sabapathy et al. 2020). Table 8.11
compiles a few examples of PHA production from FW.
Fermentation process parameters which are crucial in PHAs synthesis are the use
of elevated carbon to nitrogen ratio, control of pH and feedstock composition, all
together affect the production yield and polymer composition (Anjum et al. 2016).
However, the price of raw materials is one of the main disadvantages of current
PHAs production methodologies comprising up to 40–50% of the final production
cost. FW is an alternative substrate for cost-reduction in PHA production. For PHAs
synthesis, carbohydrate-rich or fatty acids-rich residues are adequate for producer
microorganisms. Moreover, pretreatments are often needed to enhance substrate
access and reduce the possible toxicity via detoxification steps. In that sense FW,
for instance, cheese whey (rich in lactose and proteins), cereal residues (e.g. wheat
straw and rice straw), sugar industry by-products (e.g. molasses), fat-rich residues
(e.g. frying oil and seeds with high oil level) are used to substitute expensive raw
materials. Final PHAs production yields obtained with this feedstock exceed 50%
8 Food Waste Biorefinery for Bioenergy and Value Added Products 217

PHA content per dry cell weight, and can reach more than 80% in some cases
(Table 8.11).
Another critical drawback in PHAs manufacturing is the accumulation inside the
microorganism as mentioned above, generating complex downstream processes
which involves cell lysis to extract PHAs. Purification costs can account around
30% of the process economy. These drawbacks result in a high price of PHAs around
5–6 EUR/kg, a disadvantage if compared to 0.8–1.5 EUR/kg for most common
petroleum-related plastics (El-Malek et al. 2020; Khatami et al. 2021; Sirohi et al.
2020).

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 Part IV
Downstream Processing for Resource
Recovery
Chapter 9
Valorisation of Anaerobic Digestate:
Towards Value-Added Products

F. Guilayn , J. Jimenez, F. Monlau, and C. Vaneeckhaute

Abstract Anaerobic digestion (AD) is considered an important brick of the circular

economy allowing to treat and stabilize organic residues, produce renewable energy
and promote the return to soil of organic matter and mineral nutrients. Digestate has
been historically seen as a biogas by-product with a fertilizing value while
representing a significant cost sector to AD plant operators. A paradigm shift is
now necessary as other renewable energy sources with lowering production costs
tend to disrupt an AD economic model greatly relying on subsidized biogas
valorisation either via electricity or purification to biomethane. This chapter is
focused on digestate post-treatments allowing to generate value-added products
while closing and enhancing the loop between major agricultural inputs and outputs,
the latter being indirectly present in digestates. Several options exist to generate
mineral fertilizers, soil amendments, organo-mineral fertilizers, biostimulants, bio-
control products, energy and beyond from digestate. Consumer behaviour and
regulatory framework evolution are necessary for increasing the demand and
enabling a more sustainable agriculture based on biosourced upcycled materials. A
major global milestone has been recently achieved as the EU has introduced a new
fertilizer regulation (CE 2019/1009) that will tend to boost the commercialisation of
digestate-based products within the world’s largest common market.

Keywords Biogas · Anaerobic digestion · Circular economy · Biowaste · Organic

waste

F. Guilayn (*)
SUEZ, CIRSEE, Le Pecq, France
e-mail: [email protected]
J. Jimenez
LBE, Univ. Montpellier, INRAE, Narbonne, France
F. Monlau
APESA, Pôle Valorisation, Cap Ecologia, Lescar, France
C. Vaneeckhaute
BioEngine, Chemical Engineering Department, Université Laval, Québec, QC, Canada

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 227
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_9
228 F. Guilayn et al.

Abbreviations

ABP Animal by-products

AD Anaerobic digestion
APL Aqueous pyrolysis liquid
Bt Bacillus thuringiensis
CMC Component material categories of EU regulation CE 2019/1009
CSTR Continuous stirred-tank reactor
DM Dry matter
EU European Union
LF Digestate liquid fraction after phase separation
MBT Mechanical biological treatment
OFMSW Organic fraction of municipal solid waste (here defined as obtained from
mixed-collection after MBT)
OM Organic matter
PAH Polycyclic aromatic hydrocarbons
PCB Polychlorinated biphenyls
PFC Product function categories of EU regulation CE 2019/1009
PFR Plug flow reactor
R&D Research and development
SF Digestate solid fraction after phase separation
SHF Separate hydrolysis and fermentation
SSF Simultaneous saccharification and fermentation
TAN Total ammoniacal nitrogen
VFA Volatile fatty acids
WWTP Wastewater treatment plant

9.1 Introduction

Improving soil quality and fertility by recycling organic waste products has become
a crucial approach to substitute mineral chemicals and to participate in climate
change mitigation by promoting carbon storage in soils. (Alburquerque et al.
2012; Gissén et al. 2014; Minasny et al. 2017). In this context anaerobic digestion
(AD) should play a crucial through the valorisation of digestate, the digested output.
This process is being successfully implemented in Europe as shown by the 18,202
plants recorded in 2018 by the European Biogas Association (EBA 2019).
AD consists of several biological reactions where the organic carbon is converted
to its most oxidised state (CO2) and its most reduced form (CH4). The methane is an
energy vector for directly producing electricity, heat and power or after purification
and injection in the natural gas grid. In an energetic and climatic crisis context, AD is
considered as an alternative for organic waste valorisation.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 229

Numerous positive aspects of AD as an organic waste treatment can be listed:

(1) organic waste stabilisation (Möller et al. 2010; Kothari et al. 2014), (2) carbon
and nutrients reuse promoting the conservation of natural resources (Vaneeckhaute
et al. 2017), (3) a cost-effective (Kothari et al., 2014), energy-positive and relatively
simple process applicable to a wide range of substrates (Appels et al. 2008; Capson-
Tojo et al. 2016), and (4) the production of value-added products (biogas and
digestate) from high moisture organic waste inputs (Guilayn et al. 2020b).
A paradigm shift is necessary for AD as other renewable energy sources with
lowering production costs tend to disrupt an AD economic model greatly relying on
biogas valorisation via electricity (usually via combined heat and power) or purifi-
cation to biomethane. For instance, according to Brémond et al. (2021), solar and
onshore wind had an expected leveled cost of electricity of US$5–6/MWh by 2020
while biogas cost reduction only allowed to reach a leveled cost of US$8–9/MWh.
In this context, digestate management is one of the key challenges for allowing AD
to be a full-sustainable and economic-efficient solution.
Indeed, AD has been mostly considered as an organic waste valorisation for
biogas production. In the last decade, interest has been grown for the digestate which
tends to be the main final product in terms of mass. The digestate consists of a
valuable mix of microbial biomass, minerals and undigested inorganic and organic
material (Guilayn et al. 2020b).
Digestates have been successfully used as an alternative fertilizer and can be more
effective than using NPK fertilizer in certain cases (Barłóg et al. 2020), reducing
agricultural costs while improving soil quality. Indeed, AD is known to conserve P
and mineralize N into ammoniacal nitrogen during organic matter biodegradation
leading to enhanced P and N recovery (later discussed) (Mazzini et al. 2020).
Direct landspreading of raw digestate and/or its mechanical separation fractions is
currently the most common digestate management strategy. However, adding to the
economic pressure to boost AD economic model (as discussed above), the direct
agricultural use of digestates as organic fertilizers is limited by several factors such
as transportation costs, organic matter quality, nutrient availability and environmen-
tal impacts. Indeed, agricultural use of digestate is strictly controlled by environ-
mental authorities. In some cases, they can be associated with environmental risks as
ammonia volatilisation, pathogens, organic micropollutants and heavy metal con-
tent, nutrients lixiviation, among other (Nkoa 2014). Furthermore, the biochemical
composition of digestate is related to the feedstock nature of AD, the process
conditions and the post-treatments (Alburquerque et al. 2012; Guilayn et al.
2019a; Barłóg et al. 2020; Akhiar et al. 2021). There is no one digestate but several
types of digestates. This variability could be an advantage by using feedstocks and
AD conditions as actuators for dedicated organic fertilizer production being in
adequation with the crop and soil needs. In this frame, digestate post-treatments
are necessary, especially for public environmental services providers operating
regional (centralized) and urban AD facilities treating waste (Guilayn et al.
2020b). However, current options for digestate post-treatments are limited, often
costly and with low creation of value.
230 F. Guilayn et al.

The use and development of digestate upgrading technologies is crucial and have
to take into account several objectives including (1) removing undesired digestate
characteristics (e.g. through chemical precipitation, nitrification/denitrification,
impurity removal), (2) reaching disposal requirements, (3) reducing logistics costs
by concentrating the valuable components (e.g. liquid/solid separation technologies
more or less advanced, drying, evapo-concentration); (4) producing high market-
value products (e.g. N-stripping, struvite crystallisation, biostimulants, etc.)
(5) ensuring homogenous quality over time; and (6) increasing market acceptance
and (7) creating new markets (Guilayn et al. 2020b).
In this chapter, an overview of digestates valorisation pathways is provided from
full-scale to the most promising approaches under development. First, the effects of
AD and the direct agronomical valorisation of digestate are discussed. Then, the
most relevant value-added products that can be obtained from digestates, according
to the author’s view, are presented and discussed. Finally, insights on the regulatory
framework in the EU and in North America are presented.

9.2 Anaerobic Digestion Effects on Digestate Properties

As AD will produce a series of systematic effects on its feedstock, several trends can
be generalized regarding the quality of digestates. A simplified overview of AD effet
on input is provided in Fig. 9.1.

Fig. 9.1 A simplified summary of most notable effects of AD. Areas are proportional to mass and
illustrate the case of a feedstock containing about 30% total solids
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 231

9.2.1 Mass Reduction

Due majorly to the conversion of OM to biogas and to a lesser extent due to the
consumption of water for hydrolysis, digestate mass and volume are always reduced
compared to the AD reactor input. As most of this mass loss is the biodegradation of
organic particles, the greater the dry matter (DM) content of the input, the more this
effect will be noticeable. For instance, full-scale Dry-AD can result in up to 10–25%
mass reduction (as 1-input/output) (Bolzonella et al. 2006; Schievano et al. 2011;
Banks et al. 2011). However, it is important to consider the overall process line,
notably considering AD pre-treatment. As an example, in the case of biowaste
wet-AD, the process line might rely on heavy dilution for depackaging and/or
hygienisation steps which can increase by two- to threefold the wet volume of AD
input compared to the plant feedstock (data from field expertise).

9.2.2 Organic Matter Stability

About 50 to over 90 % of input OM is converted to biogas during AD for typical

inputs. Usually, less biodegradable material such as fibrous feedstock (notably from
manure, lignified green waste and crop residues) and secondary/biological sludge
(i.e. waste activated sludge) will present a conversion rate in the lower range and the
opposite for non-fibrous and highly biodegradable feedstock (e.g. food waste and
primary sludge). The resulting digestates contain microbial biomass and concen-
trates the more stable fractions of OM. Beyond that, some authors defend the
presence of humification-like processes occurring in AD (microbiological processes
resulting in the complexification of the organic matrix) (Brunetti et al. 2012). From
the perspective of EU fertilizer regulation (CE 2019/1009), most of the digestates
should be conforming to the stability thresholds of 25 mmol O2/kg OM/h or 250 mL
biogas/gOM (Guilayn et al. 2017).

9.2.3 Nitrogen and Phosphorus

The mineralisation of input OM includes the biodegradation of proteins and amino

acids, thus the conversion of organic nitrogen into total ammoniacal nitrogen (TAN,
present as NH3 and NH4+). Total nitrogen of digestates is usually composed of
50–90% of TAN (Guilayn et al. 2019a), which is readily accessible to plants,
meaning that the digestate fertilizing value is greatly enhanced compared to inputs.
However, coupled to a neutral to alkaline pH, NH3-related issues (e.g. odor and
toxicity) are increased and must thus be considered for plant design (e.g. air treat-
ment and covered digestate storage) and land spreading (e.g. injecting techniques
must be preferred).
232 F. Guilayn et al.

Concerning phosphorus (P), during AD, P is theoretically conserved in the

digestate. Organic P forms will tend to be mineralized but the solubility and
bioacessibility of resulting minerals can be highly variable. Due to the overall
mass loss and conservation of P, a concentration increase can be expected. However,
some authors indicate low P losses (less than 10% for Schievano et al. 2011) while
others observe losses of up to 36% (Marcato et al. 2008). The reason is probably the
retention and accumulation of phosphorus in solid form in the reactor following
precipitation (Möller and Müller 2012). The AD effect on OM, nitrogen and
phosphorus will be further discussed in the next sections of this chapter.

9.2.4 Innocuity

No intrinsic AD mechanism is notable for decreasing AD feedstock innocuity other

than increasing ammonia concentration as discussed above. Indeed, the digestate
will inherit eventual hazardous characteristics from the feedstock. When hazardous
components are not anaerobically biodegradable, a slight concentration reduction
(due to loss in the reactor) or a slight concentration increase (due to digestate volume
reduction) will be observed (notably heavy metals, inert impurities and some organic
micropollutants). On the contrary, several innocuity parameters can be intrinsically
improved by AD such as pathogens (c.f. below) and odors (Hjorth et al. 2009).
Additionally, AD often enhances waste management practices as any biomethane
potential loss is a revenue loss for operators (even animal wellbeing) as shown by
Couturier et al. (2019) in a study with 45 French farmers operating AD plants. It is
also to be noted that a low quality feedstock might jeopardize digestate quality
whose disposal costs are often higher than 80 €/t in Europe.
Regarding pathogens, AD temperature (in the case of thermophilic AD), micro-
bial community acclimation (thus greater competitiveness for substrate) and VFA
inhibition can be related as factors promoting partial up to virtually total pathogen
kill/inactivation during AD (Jiang et al. 2020). This effect is though highly variable
depending on retention time and pathogen family. In certain countries, thermophilic
AD is recognized as a hygienising process. However, in the EU, AD alone is not
recognized as a standard hygienisation process by the animal by-product (ABP)
regulation (CE 1069/1009).
As discussed above, due to mass loss, trace metals can be slightly concentrated.
However, its bioaccessibility is often reported to be reduced (Marcato et al. 2009).
Regarding organic micropollutants, their decomposition is difficultly generalized,
being very compound-dependent (Stasinakis 2012).
Globally, it is widely accepted that AD greatly enhances the fertilizing value of
digestates compared to AD inputs. Post-treatment technologies may further increase
digestate fertilizing value, as well as enable other market destinations. For many of
these advanced technologies, AD can be also seen as a key pre-treatment step (next
sections).
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 233

9.3 Digestate Typology

Typical AD configurations (i.e. combination of feedstock and process line) arise

from a set of local/regional factors including regulation and policies (e.g. for renew-
able energy, waste (water) collection and treatment, land spreading), economy and
market (e.g. types of waste and existing technology providers).
As with any product from an engineered process, the composition of digestates is
a result of feedstock composition, process design, operational conditions and the
overall resulting process performance. It should thus be the case that the typical AD
configurations (input/process) should be linked to typical digestate quality (i.e.,
resulting in a digestate typology). From an EU point-of-view, the following AD
configurations can be highlighted, which had been recently linked to a digestate
fertilizing-value typology based on digestate quality database (Guilayn et al. 2019a).

9.3.1 Agricultural Digesters

In the EU, agricultural digesters are the most common, notably in Germany, due to
historical support policies. According to the European Biogas Association, there
were nearly 18,000 biogas plants in Europe in 2017 (landfills included), about 50%
of them being agricultural digesters in Germany. Depending on member state’s
national policy, typical inputs may vary. For instance, in France, agricultural AD
will typically co-digest manure/slurry, crop residues and/or catch crops. In Germany,
the legislation historically allowed AD of energy crops as the main crop cultivated
specifically for AD. These digesters are usually mesophilic continuous stirred-tank
reactors (CSTR) but agricultural dry-AD is also reported, especially for fibrous
manure. In Fig. 9.2, they can be related to digestate types 1 and 6. Higher dry matter
(>5% in Wet-AD, relating to interquartile ranges), volatile solids (>70% DM) and
C/N ratio (5–20) would be related to the more fibrous characteristic of agricultural
inputs. Agricultural digestates tend to be easier to commercialize from a regulatory
point-of-view. This is the most dominant digestate type as, in Europe, most biogas
installations are based on dedicated energy crops (Brémond et al. 2021). This trend
tends to continue but probably shift to rotational crops (catch crops) to avoid food
production competition while providing side benefits to the main crop (Brémond
et al. 2021).

9.3.2 Wastewater Treatment Plants

Wastewater treatment plant (WWTP) digesters are also numerous. Given the low
concentration of sewage sludge, they are usually large mesophilic CSTR. As the
sludge is conventionally digested prior to dewatering, these AD plants are usually on
234 F. Guilayn et al.

Fig. 9.2 Digestate typology based on a clustering analysis on a database of 91 samples (extracted
from Guilayn et al. 2019a)

site. WWTP digesters typically receive a combination of primary (more biodegrad-

able) and secondary (biological) sludge (less biodegradable) and AD plants with free
capacity might boost biogas production by accepting external organic waste when
allowed. In Europe, co-digestion with source-separated organic waste tends to be
limited/forbidden. In Fig. 9.2, they can be related to type 2. It is the cluster with the
lower DM range (typically <5%) and high phosphorus (>1% DM) and low potas-
sium (<2.5%) contents characteristics of sewage sludge. In Europe, in the case of
land spreading, sewage sludge digestates are usually spread under a waste status
and/or national standards. Some countries count with end-of-waste standards for
composted sludge (e.g. NF U 44-095 in France). They were not included as inputs
for the latest EU regulation on fertilizers (CE 2019/1009), except recently for struvite
recovery and ashes after incineration.

9.3.3 Centralized/Regional Digesters

Centralized digesters, also referred to as “regional” or “territorial”, are large-scale

biogas facilities (>1–2 MW and >50 kt/year), usually mesophilic CSTR treating a
great mixture of organic waste streams from a large area, such as food processing
waste, municipal/industrial sludge, household waste, green waste, agricultural
waste, among other. Most of these installations are wet-AD because typical
pre-treatment for depackaging and hygienisation relies on heavy dilution. Given
the very diversified types of inputs and mixtures, they are more difficultly clustered
but could be associated mainly to type 3 and to some type 2 and 5 digestates in
Fig. 9.2.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 235

9.3.4 Municipal Solid Waste Digesters

The organic fraction of municipal solid waste (OFMSW) is usually treated in large-
scale municipal biogas facilities. OFMSW is obtained after a mechanical biological
treatment (MBT) of mixed-collection urban waste. MBT of such a stream usually
produces a fibrous cellulosic biodegradable material with a structured bulk capacity
without needing a structuring co-substrate. MBT is thus often followed by a Dry-AD
thermophilic digester.
Another emerging configuration is Dry-AD of biowaste (source-separated) when
it contains a significant contribution of green waste serving as structuring material. It
is important to mention that some authors will also refer to source-separated
biowaste from municipal solid waste collection as OFMSW. In Fig. 9.2, they
compose most of type 3 and 5 digestates presenting low nutrient content that can
be associated to the typical cellulosic material obtained from MBT. In Europe, on the
one hand, the restrictions and end-of-waste scenarios described for sludge digestate
apply similarly to MBT digestate from mixed-collection waste. On the other hand,
source-separated biowaste digestate is clearly trending to reach more easily “end-of-
waste” status. For instance, biowaste (source-separated) was included in the latest
EU regulation on fertilizers (CE 2019/1009)
To conclude, even if AD configurations can be somehow generalized, the pro-
cess/digestate typology is also a result from a limited technical expertise imposing
design and operational rules that are being constantly challenged and optimized by
research. This process might take a significant time to reach the market, thus the
typology can be expected to be constantly evolving.

9.4 AD Operational Strategies for Enhancing Digestate

Value: The Case of Residual Organic Matter

As organic matter is the major digestate component (other than moisture), valorizing
digestate organic matter is one of the greatest levers for creating value. In the future,
a more defined economic value to stabilized carbon is expected through carbon
credits. Additionally, an increasing economic value tends to be associated for the
beneficial effect of organic matter in agriculture, especially as most soils are being
rapidly depleted in OM. In this scenario, optimizing AD for considering not only
biogas yield but digestate OM quality and quantity can be crucial. A change of
paradigm will be needed. Adapting the process line to enhance final digestate quality
while lowering biogas production has already been done to achieve better economic
results in one of the largest urban AD facilities in Europe (internal information
from SUEZ).
First of all, feedstock quality plays a major role in the proportion of OM degraded
during the anaerobic digestion process and during land spreading of the digestate
(Guilayn et al. 2019a, 2020b). As previously said, fibers-like feedstock enhance OM
236 F. Guilayn et al.

in digestate because lignin-like compounds are not biodegraded (i.e., manure, crop
residues, straw), whereas biowaste-like feedstock decrease OM in digestate because
the OM content is more biodegradable (i.e., OFMSW and biowastes).
All bibliographic sources agree that a fraction of the digestate’s OM remains
biodegradable once it is incorporated into the soil, making post-treatment interesting
by composting to achieve a more stabilized OM. This fraction of biodegradable OM
converted into methane and carbon dioxide is dependent on the efficiency of AD
performance in particular on parameters such as temperature, organic loading rate
and hydraulic residence time. On average, 2/3 of the biodegradable OM introduced
into the digester is transformed into biogas (Alburquerque et al. 2012).
In addition to feedstock and process parameters, the technology itself seems to
have an impact on the soil amending value of digestates: those resulting from
dry-AD seem to contain more OM consisting mainly of carbon chains very difficult
to degrade by the microorganisms. AD would not decompose the humic potential of
OM because the microorganisms do not break down lignin, complex lipids, steroids
and precursor molecules for humus formation (Jimenez et al. 2017). As previously
stated, some authors indicate the presence of humification-like processes occurring
in AD (Brunetti et al. 2012), but humification is a controversial topic for over a
century in soil science. However, a stabilized OM is obtained in the digestate due to
the transformation of labile fractions of OM in biogas and the formation of more
stable compounds (increased degree of aromaticity, accumulation of longer aliphatic
chains) according to Möller (2015).
To conclude, the digestates contain a fraction of residual biodegradable OM and a
proportion of stabilized pre-humic matter, which depends on the conditions of the
digestion and feedstocks but also on the post-treatment applied to the digestate.
These parameters can be used as actuators to control the quality of the digestate.

9.5 Digestate Landspreading: Practices and Limitations

9.5.1 Agronomic Digestate Value

The most classical valorisation of digestates and by-products is the agronomic use in
crop soils. Indeed, as previously mentioned, because of the stabilized OM and the
mineralized nutrients, digestates are considered as organic fertilizers if they are
properly managed. Three types of fertilisation can be obtained using organic resi-
dues in crop soils: (1) chemical fertilizing (available nutrients NPK used at short/
mid- term to feed the plants), (2) biological fertilizing (labile OM able to feed the soil
microorganisms at short term) and (3) physical fertilizing (amendment potential of
stabilized OM to improve the soil structure). Several different types of digestates
have been demonstrated to be effective replacements for mineral fertilizers while
reducing environmental impacts (Walsh et al. 2012; Koszel and Lorencowicz 2015;
Panuccio et al. 2019; Verdi et al. 2019). Nevertheless, regulatory constraints, good
management and spreading practices are necessary for controlling environmental
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 237

and health risks as it can be expected for any chemical fertilizers, raw manure and
sludge (Nkoa 2014).
Considering chemical fertilisation, mineral elements increase chemical fertility
by feeding the plants’ nutritional needs. Nitrogen fertilizing is the main target when
chemical fertilizer is considered. During AD, ammonia reacts with water in the
anaerobic environment to form ammonium found in the digestate. After land spread-
ing, nitrification by soil bacteria transforms ammonium into nitrate, which can be
assimilated by the plant, within a period of a few days to a few weeks. Losses in the
form of nitrous oxide or nitrogen oxide may occur during this process. However, the
total nitrogen stock remains constant compared to the undigested inputs, but the final
concentrations are higher, due to the loss of a quantity of carbon in the form of
biogas during the AD process (Tambone et al. 2009).
When the digestate is incorporated to the soil, part of the carbon is used as an
energy source for the soil microflora and nitrogen is used as a nutrient. A phenom-
enon of reorganization of mineral nitrogen is often observed: as soon as the digestate
is added to the soil, the microbial activity of the soil is reinforced with the production
of new cells because its incorporation provides a source of carbon energy. This
requires a proportional amount of nitrogen, which is part of the composition of many
essential molecules in cells. If the supplied OM does not contain enough of N to meet
this demand, microorganisms will take (and therefore immobilize) N soil solution to
be able to grow (Reibel and Leclerc 2018).
For raw and liquid digestate, with a lower C to N ratio compared to solid
digestate, studies report that the rate of net nitrogen mineralisation is usually positive
(Cavalli et al. 2016; Jimenez et al. 2020a). On the other hand, for the solid digestate,
net immobilisation of nitrogen, even in the medium term can occur, depending on
the digestate typology (Jimenez et al. 2020a). Indeed, the authors showed that the C
to N ratio is negatively correlated with soil mineralized N and N recovered in plant
tissues. It seems to be a good indicator for the mineralisation potential of organic N
and for apparent recovery fraction of N by plants as shown by Jimenez et al. (2020a).
As previously discussed, the nitrogen fertilizing value of digestate depends on:
(1) the mineral nitrogen content, which depends on the feedstocks and post-
treatments. More than half of the organic nitrogen contained in the raw digested
inputs is found as ammonium quickly mobilized by plants, conferring a strong
fertilizing potential. (2) The rate of mineralisation of the organic fraction of nitrogen
in digestates. The mineralisation kinetics depends on the type of feedstock and the
environmental conditions (temperatures and mainly soil moisture). However, the
mineralisation of organic nitrogen appears to play a minor role in the fertilizing
value. Little organic nitrogen remains in the soil after a growing season, thus
reducing the long-term effect of so-called residual nitrogen as well as long-term
nitrogen leaching (Reibel and Leclerc 2018). (3) The phenomenon of ammonia
volatilisation, which can in some cases reach 100% mineral nitrogen. This phenom-
enon is also a function of the type of feedstock, post-treatment, storage, spreading
and pedoclimatic conditions (Reibel and Leclerc 2018). The spreading conditions
have a strong influence on the apparent recovery fraction of N by crops: a liquid
digestate applied in surface can lose 35–100% of its ammonia (Nyord et al. 2008;
238 F. Guilayn et al.

INRA-CNRS-IRSTEA 2014). Climatic conditions on the day of intake and follow-

ing the day of intake also seem to be a strong factor explaining the variability
observed in certain field trials, because this parameter greatly influences the ammo-
nia volatilisation.
Concerning the N fertilizing potential, post-treatments highly impacts the imme-
diate availability of nitrogen and the digestates can be classified as follows, in terms
of the fertilizing value equivalent coefficient to short term (one crop year): Liquid
digestates> Raw digestates> Solid digestates> Digestates composted ~ Dried
digestate (Reibel and Leclerc 2018).
Concerning P fertilisation, as previously discussed, P is theoretically conserved in
the digestate but low to significant losses can be observed due to accumulation in the
reactor (Marcato et al. 2008; Schievano et al. 2011; Möller and Müller 2012).
Similar to nitrogen, organic P can be converted to inorganic P, but unlike nitrogen,
resulting phosphorus is mainly found in a solid form in the raw digestate. It is mainly
converted to ortho-phosphates that can be present as salts (such as calcium phos-
phate and struvite) and/or adsorbed to particles. For example, Zhang et al. (2012)
observed on samples of nine digestates of agricultural origin, an average of 70% of
phosphorus in solid form, against 30% in interstitial fluid. According to Reibel
(2018), the fertilizing value in P of the digestate depends first on the phosphorus
content, varying according to the feedstocks with total P contents extremely variable
(from 4 to more than 20 g P/kg DM). The highest levels are observed for digestates
from wastewater treatment (WWTP) sludge, with up to 21 g P/kg DM. Digestates
from pig slurry AD and animal by-products have also high levels of P but slightly
lower. In contrast, the lowest contents are associated with digestates from the
bio-waste and green waste AD (INRA-CNRS-IRSTEA 2014). However, the total
P content is not a sufficient indicator of the P fertilizing value of a digestate. P
speciation on availability and the soil physicochemical conditions govern the
apparent P recovery by plants. Indeed, Jimenez et al. (2020a) showed that P
speciation and post-treatments impact the P chemical availability as well as the P
recovery by plants in a calcareous soil where P is the lowest available for plants
(i.e. high pH value and carbonates levels).
Digestate post-treatments have also an impact on P speciation and P fertilizing
value. According to Reibel (2018) a classification can be made (%DM): P liquid
digestate from screw press> raw digestate> P digestate dried> P digestate solid
fraction separation by centrifuge> P digestate solid fraction separation by screw
press> composted digestate.
Indeed, the liquid phase which contains orthophosphates retains a fertilizing
value in phosphorus greater than the solid phase of digestate or compost, with
composting breaking down the concentration of P in the digestate. This classification
makes it possible to estimate the phosphorus concentration in the digestate. How-
ever, in terms of raw matter, a different order is obtained, the products richest in DM
allow providing more P (in% raw matter): P dried digestate> P composted
digestate> P digestate fraction solid separation by centrifuge> P digestate solid
fraction separation by screw press> digestate raw> liquid digestate.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 239

Moreover, contrary to the fate of N, physicochemical reactions govern the P fate

in soil. According to Ahmad et al. (2018), more than 80% of the applied P is easily
immobilized in soil by adsorption/precipitation reactions or conversion into organic
form. P can thus become partially/poorly unavailable for the plants.
Considering biological and physical fertilisation through OM, digestates can
provide stable OM to compensate soil humus mineralisation as soil improvers or
soil amendments. OM in soil is crucial to feed the microbiological activity, provide
bulk capacity which improves soil structure, increases cation exchange, pH buffering
and water retention capacities, adsorbs nutrients, among many other effects. In
practice, the complete distinction of mineral fertilizers and soil improvers is not
always pertinent. Some materials as dried digestates are both rich in OM and
nutrients, allowing acting both as organic soil improvers and “slow-release” fertil-
izers (Guilayn et al. 2020b). Moreover, mineral fertilizers can be used to enrich and
balance nutrient content on organic soil improvers such as composts, providing
readily available nutrients along with stable OM. In the literature the amending
potential of digestates varies from 10 to 354 kg C/t of raw digestate with the
following classification: Composted digestate> Solid digestate> Raw digestate>
Liquid digestate (Reibel 2018).

9.5.2 Limitations of Digestates Use in Agronomy

AD enhances undoubtedly the N availability for plants growth, but this process can
also enhance the potential loss of ammonia by volatilisation during storage (mini-
mized if the storage is covered) and spreading compared to initial materials (Reibel
2018). This increase can be partially offset by the better infiltration capacity of liquid
digestates in the soil and by covering actions during storage as well as appropriate
practices during spreading such as rapid burial, dry climatic conditions and cool
temperatures, non-bare soil, etc. Spreading recommendations are found more suit-
able for slurries and is not observed for solids, composted and dried digestates.
Greenhouse gases can also be produced during storage and spreading. However,
some studies showed that AD decrease CH4 and N2O emissions compared with
feedstock spreading (Holly et al. 2017; Reibel 2018).
Another limitation of some types of digestates is the metallic trace elements
content. AD tends to increase trace mineral concentrations via degradation of the
organic matrix with a clearer tendency for sewage sludge and OFMSW. For the
digested sewage sludge, speciation of metallic trace elements seems to be evolving
towards more stable forms and less available during digestion (Reibel 2018).
Concerning organic micropollutants, AD modifies the concentrations of organic
contaminants. For example, the anaerobic digestion of urban sludge has demon-
strated a potential for degradation of PAH, nonylphenol and PCB (Reibel 2018).
According to Guilayn et al. (2020b), digestate farmland spreading is the mainly
digestate management strategy. However, for the non-agricultural digestate pro-
viders, several legal and technical bottlenecks are found as follows: (1) digestate
240 F. Guilayn et al.

spreading regulation tend to be stricter for digestates form non-agricultural AD

feedstocks. For example, in the EU fertilisers regulation, digestate is an authorized
material but the text excludes some urban feedstocks as sewage sludge, OFMSW
and other mixed-stream substrates (CE 2019/1009). Consequently, those digestates
follow the waste regulations, requiring high administrative cost procedures for
spreading and homologation. (2) The agricultural needs are seasonal while the
production of digestates is continuous on a yearly basis. This aspect leads to storing
the digestate for a long time or transport to distant regions (King et al. 2013; Gong
et al. 2013). (3) The time variability of the digestate composition can occur and is
related to the feedstocks variability and performance of AD stability (Zirkler et al.
2014). This aspect can induce quality control issues for spreading and marketing
purposes. (4) The quantity of the produced digestate can exceed the capacity of the
local available soils for receiving nutrients (Vaneeckhaute et al. 2013; Nkoa 2014)
leading to an increase in transport costs. Dahlin et al. (2015) reported that the
digestate transport distance is become twice higher in the last years (i.e. 150 km).
Moreover, the number of large and centralized facilities had grown because of their
higher economic feasibility. However, most of these plants exceed the local nutrient
spreading capacity.
Despite the digestate value, its agricultural use as direct spreading is not enough
to overcome the digestate challenge, mainly in the cases of large-scale and/or
non-agricultural digesters. Adding to that, even for agricultural AD, increasing or
creating value for digestate is one of the major levers to balance decreasing sub-
ventions on biogas valorisation. New ways of digestate valorisation through
upgrading technologies are essential.

9.6 Digestate Phase Separation: A Key Step for Digestate

Spreading and Post-treatment

Digestate phase separation (also referred to as dewatering in the wastewater treat-

ment sector) is a key step for enhancing digestate management practices and
enabling digestate post-treatment advanced technologies. The most applied technol-
ogies are screw press and decanting centrifuge (sometimes combined in this
sequence) but vibrating screens and filter presses are also commonly related
(Guilayn et al. 2019b; Akhiar et al. 2021). These technologies greatly differ in
performance which is a combination of equipment performance and digestate type
driving technology choice. As a rule-of-thumb, fibrous agricultural digestates tend to
be separated by screw presses, which are poor performing compared to centrifuges
(Guilayn et al. 2019b).
The first point to be highlighted is the benefit of (partially) separating moisture
from solids. The reduced moisture of the solid fraction (SF) implies a reduced
transportation cost thus greater distances and surfaces for spreading. Adding to
that, other operational and agricultural benefits are related. The SF is better stackable
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 241

whereas the LF is better pumpable. The SF might contribute to the soil bulking
capacity, whereas the LF has better infiltration in soil. Regarding post-treatment
(later detailed), the SF can be post-treated by technologies needing a low-moisture
content (notably composting and drying) whereas the LF through technologies
needing low-solids and/or presenting clogging issues (notably nitrogen stripping,
struvite recovery, (vacuum-)evaporation and membrane filtration).
Adding to this “physical” effect, digestate phase separation enhances nutrient and
organic matter management in agriculture. The SF concentrates 30–90% of the OM
into 5–50% of the initial mass (Guilayn et al. 2019b). It can thus be better used as a
soil amendment (adding to the bulking capacity effect), composted or as constituting
growth medium material replacing peat (Nesse et al. 2019). The LF concentrates
most of the ammoniacal nitrogen and potassium, while phosphorus tends to follow
the organic matter and tends thus to be concentrated in the SF (Guilayn et al.
2019a, b). This nutrient fractioning effect can be used as a strategy for improving
nutrient management (e.g. different crops or seasons) and increasing the spreading
surface of the fractions by reducing the concentration of the limiting nutrient (usually
N or P).

9.7 Value Added Products from Liquid Digestates

Given the benefits described above, digestate post-treatment technologies are clas-
sified in this chapter by their applicability concerning digestate rheological state. To
the best of the author’s knowledge, this process classification has not yet been linked
to an actual rheological objective parameter, but the DM content (i.e., total solids) is
a usual practical indicator. The techniques described below for liquid digestates are
usually applied to LF or wet-AD digestates with less than 5–8% DM. After phase
separation, adding to the enhanced physical aspects described in the previous
section, it is important to mention that most of the “liquid-state” technologies
described below will benefit from an enhanced nutrient content (e.g., N-stripping,
microalgae, membrane filtration and evaporation).

9.7.1 Nutrient Fractionation

Nutrient fractionation is considered a mechanism for extracting target nutrients from

the liquid digestate stream into different product fractions. This can notably be
accomplished using various membrane filtration steps. As such, following the initial
phase separation, the liquid fraction could, for example, be further treated through
nanofiltration followed by reversed osmosis. In this case, a mineral nitrogen-
phosphorus rich fertilizer and nitrogen-potassium rich fertilizer can be produced in
sequence, as already implemented at full scale by the company NEREUS (France).
The produced water following reversed osmosis typically meets reuse standards. A
242 F. Guilayn et al.

full-scale system called the Biorek Process has been installed by BIOSCAN (Den-
mark). Besides reversed osmosis, microfiltration and ultrafiltration units can also be
combined with ammonia stripping (Vaneeckhaute et al. 2017). Operating tempera-
tures for membrane filtration typically range between 10 and 40  C. The pH is
typically between 6 and 8. The cost of membrane filtration for manure and digestate
treatment was evaluated through a large-scale pilot project conducted at eight
installations in the Netherlands. The estimated cost ranged between 9 and 13 €/t of
manure or digestate (de Hoop et al. 2011). Potential revenues for the reversed
osmosis concentrates were estimated at an average of 6.1 €/t. An important technical
issue when using membrane filtration is clogging and fouling of the membrane,
which can result in significant costs for the use of cleaning products. In order to
mitigate this problem, Vaneeckhaute et al. (2012, 2019) evaluated the performance
of vibratory membrane filtration (VSEP technology) for digestate purification at
demonstration scale. Although the chemical use was significantly reduced, the
energy requirements of this technology are not negligible. The potential pollution
of the recovered concentrate flows, for example with heavy metals, also remains an
important point of attention.

9.7.2 Concentration and Granulation

As previously discussed, digestate present a pool of valuable compounds notably

stabilized organic matter and nutrients. Liquid digestates and LF, however, present a
large content of moisture (usually over 90%) thus its valorisation is restricted to
landspreading over small distances (about <50 km). Spreading limits can be easily
attained, imposing higher transportation costs that would drive the investment on
digestate post-treatment.
According to the study of Fuchs and Drosg (2013), digestate evaporation is
economically feasible from about a reference transportation distance (no post-
treatment) of 80 km. Digestate (vacuum-)evaporation techniques are fully developed
and proposed by several companies. Global costs including operation expenditure
and capital amortisation are in the range of 15–30 €/t of digestate (Guilayn et al.
2020b, annex material). They can be used as a treatment for simultaneous nitrogen
stripping (due to temperature) and recovery as ammonium sulphate as described in
the next section. Due to clogging issues, suspended solids must be low and input
digestates are often required to present less than 1–3% dry matter. Anyway, scaling
issues due to the soluble mineral content have also been observed (industrial
expertise from SUEZ). Output concentrates is a dark liquid “syrup” presenting up
to 20–25% DM. Adding to that, it might be economically feasible to large-scale
facilities to further include a full drying/granulating step after evaporation such as
the ecoDry process proposed by SwissCombi (Switzerland). In the EU, both strat-
egies could be interesting for achieving end-of-waste status through the regulation
CE 2019/1009 as discussed by Guilayn et al. (2019a, b), but authorized post-
treatments are not yet clear.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 243

9.7.3 Nutrient Recovery as Pure and Reformulated Products

Several technologies have been developed in the last decades to extract pure and
formulated end products from liquid fractions of digestate. Based on a review
executed by Vaneeckhaute et al. (2017), the current most implemented technologies
at full-scale include struvite precipitation and crystallisation, as well as ammonia
stripping and scrubbing.
Struvite precipitation allows to recover soluble phosphorus (ortho-P) from waste-
water streams at high phosphorus loads (usually >80 kg P/day) (Vaneeckhaute et al.
2018). It typically requires the addition of a magnesium source (typically MgO, Mg
(OH)2 or MgCl2) and caustic soda (NaOH) to increase the pH (8.3–10) and induce
precipitation of MgNH4PO4
6H2O (struvite). Alternatively, the pH can be increased
through stripping of carbon dioxide, without the addition of NaOH. The
precipitation-crystallisation reaction usually takes place in a fluidized bed reactor
or a completely stirred tank reactor. Important parameters to control include pH,
temperature, mixing intensity, and the presence of impurities such as calcium.
Struvite can be reused as a valuable slow-release fertilizer product containing the
macronutrients N, P and Mg that support plant growth, equivalent to 12% P (28%
P2O5). Capital costs for producing struvite have been estimated at 0.05 €/day.
However, the overall benefits for the treatment plant can reach 0.52 €/day
(Vaneeckhaute et al. 2017). The latter is related to chemical savings (conventional
Fe/Al-salts), savings from reduced sludge handling and disposal, as well as savings
related to the reduced cleaning of uncontrolled struvite deposits. The market value
for struvite is variable, with numbers of 45 €/t in Belgium to 250 €/t in Japan
(Vaneeckhaute et al. 2017).
Nitrogen stripping and scrubbing allows to recover nitrogen from a variety of
wastewater streams at high ammonia (NH3) nitrogen concentrations (usually
>500 mg/L) (Vaneeckhaute et al. 2018). It involves the physical transfer of ammo-
nia from the aqueous phase to the gas phase at increased pH. The ammonia is then
absorbed in sulphuric acid by means of an air scrubber to produce liquid ammonium
sulphate. Ammonia stripping typically takes place in a packed bed tower or a
submerged aeration system. Key advantages of the first is its easy operation and
low capital cost, while key advantages of the latter are the absence of packing
material (no risk of fouling) and its ability to increase the pH without chemical
addition (through CO2 stripping). Acidic air scrubbing usually takes place in a
packed bed tower. Sulphuric acid and air are introduced into the tower in counter-
current. Important parameters to control are pH (typically 10–11) and temperature
(typically 60–70  C). The ammonium sulphate concentration in the recovered liquid
product ranges from 25 to 40% by weight. It can be valorised as a fertilizer product
rich in the macronutrients nitrogen and sulphur. Capital costs for stripping are
estimated between 500,000 euros and 1.58 million euros for a packed bed tower,
whereas this amounts to 3.5–15 M€ for a submerged aeration system. The opera-
tional costs range between 1.4 and 2.5 M€/year (Vaneeckhaute et al. 2017). The
market value of the recovered ammonium sulphate solution ranges between 80 and
244 F. Guilayn et al.

120 €/t of fresh weight. As such, under optimal process conditions, nitrogen strip-
ping and scrubbing can be viable.

9.7.4 Microalgae and Cyanobacteria Cultivation

The cultivation of microalgae and cyanobacteria has attracted a lot of research

attention these last decades as new valorisation routes for liquid digestate
valorisation (Franchino et al. 2013; Xia and Murphy 2016; Zuliani et al. 2016).
Liquid digestate is mainly composed of soluble compounds, water and nutrients
(N and K sources majority) that make it an ideal culture support for microalgae or
cyanobacteria (Nkoa 2014; Akhiar et al. 2017, 2021; Guilayn et al. 2019b).
Microalgae can be done in open systems (i.e. raceway) or in closed systems called
“photobioreactors” (Kirnev et al. 2020). Nonetheless the chemical properties of
digestate and especially its high turbidity and high content of ammoniacal nitrogen
can limit the microalgae growth (Xia and Murphy 2016). Due to their chemical
properties, liquid digestate from urban wastewater or agro-industrial wastes are more
appropriate than agricultural ones (Xia and Murphy 2016; Zuliani et al. 2016).
In order to overcome these chemical barriers (turbidity, ammoniacal nitrogen),
several treatment options can be implemented such as dilution with fresh water or
others agro-effluents with less turbidity (Franchino et al. 2013; Zuliani et al. 2016;
Markou et al. 2021). For instance, Franchino et al. (2013) have tested three
microalgae strains (Neochloris oleoabundans, Chlorella vulgaris and Scenedesmus
obliquus) on an agro-zootechnical digestate at different dilution ratios (1:10; 1:15;
1:20: 1:25). Specific growth rate of 0.26, 0.23, 0.49/day were observed for, respec-
tively, Neochloris oleoabundans, Scenedesmus obliquus and Chlorella vulgaris by
applying a dilution rate of 1:20 (Franchino et al. 2013). Similarly, Jimenez et al.
(2020a, b) have investigated at different dilutions (1:20, 1:30, 1:50) the
Monoraphidium sp. on agricultural liquid digestate in laboratory trials. Optimum
dilution factor was 1:50, with a specific growth rate of 0.13/day and a complete
nitrogen removal in 25 days. Markou et al. (2021) have also investigated Spirulina
cultivation in artificial seawater at 2.5%, 5%, 10% or 15% (v/v) of digestate in a
fed-batch mode. The highest production was noticed at 5% (1300 mg/L dry
biomass) but further increases in digestate concentrations lowered biomass accumu-
lation (1020 1150 mg/L dry biomass). Other investigations have investigated
alternative pre-treatment methods to avoid dilution including: activated carbon
treatment (Marazzi et al. 2017), flocculation-biological contact oxidation (Zhou
et al. 2019), aerobic treatment (Wang et al. 2019) and struvite precipitation (Jiang
et al. 2018).
The cultivated microalgae can be further used in different applications such as
biofuels such as biodiesel (Sialve et al. 2009), bioethanol and biohydrogen (Harun
et al. 2010; Yun et al. 2012). The microalgae can be also sent back to the biogas plant
for methane production (Sialve et al. 2009; Mussgnug et al. 2010). Other alternatives
can be animal feed (Madeira et al. 2017), fertilizers and biostimulants (Hidalgo
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 245

2015; Ronga et al. 2019a). Microalgae can also be used for the upgrading of biogas
and several studies have been done in the last decades (Tongprawhan et al. 2014;
Rodero Raya et al. 2019). Finally, when considering the valuation chains it is
important to consider the contamination risks (i.e. pathogens, organic and inorganic
contaminants) and biomass safety concerns (Markou et al. 2018) and for this purpose
human-consumption applications are often not considered. Up to date, some R&D
projects have been implemented in Europe coupling liquid digestate with microalgae
cultivation. Among these projects, the AlgaeBiogas project (https://algaebiogas.eu/)
has investigated the use of liquid digestate for microalgae cultivation.

9.7.5 Biological Nutrient Recovery Strategies

Liquid digestate and LF can be used more indirectly for the recovery of value-added
products by providing nutrients and other compounds (such as phytohormones) for
living organisms. This is the case for microalgae recovery (presented previously),
but also for a large range of emerging technologies. It can be highlighted (not
exhaustive): (1) Nutrient and phytohormone source for hydroponics (Antón et al.
2017; Ronga et al. 2019b; Guilayn et al. 2020a), (2) Macrophytes recovery. A full-
scale demonstration plant of duckweed recovery was recently constructed in Europe
(Pascual 2016) and (3) Liquid culture medium. For instance, liquid digestates have
been successfully used as a culture medium to produce bioplastics (Passanha et al.
2013) and ethanol (Ujor et al. 2020).

9.8 Value Added-Products from Solid Digestates

As introduced during the “liquid-state” valorisation section, the DM content is a

usual practical indicator for the feasibility of the technologies described in this
section. The techniques described below for solid digestates are usually applied in
SF or dry-AD digestates with more than 20–25% DM. Adding to the enhancement of
physical properties described for “solid-state” technologies (Sect. 9.6), most “solid-
state” technologies will benefit from a greater OM content of SF after phase
separation, such as composting, post-fermentation and thermal conversion.

9.8.1 Composting

Composting process consists in an aerobic biological decomposition of biodegrad-

able organic matter into CO2 and microorganisms. Digestate composting is a
commonly used post-treatment in agricultural, urban, and industrial AD plants.
Compost of digestate has a good amendment potential compared with raw digestate
246 F. Guilayn et al.

and solid or liquid phases (Reibel and Leclerc 2018). Due to the more favourable
thermodynamics and aerobic conditions, a higher variability of microorganisms
(i.e. bacteria, actinomycetes, fungi) can be found both effects allowing a higher
OM decomposition than AD (Guilayn et al. 2020b).
Industrial composting can be controlled by several parameters as the retention
time (from 3 weeks up to 1 or 2 months), the optimal C to N ratio between 20 and
40, the temperature, the moisture (> 60%), the bulking agent and aeration rate and
method (Epstein 2011). Composting is an exothermic process. Temperatures reach
more than 70 C within the piles inducing an authorized hygienisation process of
digestates in several countries. Temperature, exposure time and the number of
turnings are usual regulatory parameters varying from country to country. For
example, in Germany, temperatures of 55, 60 and 65  C are requested for a period
of respectively 14, 6 and 3 days (Amlinger and Blytt 2013). Largely due to the self-
heat reaction, water loss occurs during composting and decreases transportation
costs. Levasseur et al. (2017) reported a mass loss from 30 to 50%. As physical
impurities and trace metals are conserved during composting, depending on the mass
loss rate and the bulking agent used, these compounds can be either concentrated or
diluted (compared to digestate). Besides, bioaccessibility and solubility of heavy
metals can be reduced due to their strong bonds to compost organic matter (Smith
2009).
Composting is usually performed on the solid phase of digestates. Besides, some
digestates need bulking agents because of (1) the low part of biodegradable OM
allowing temperature increase, (2) the physical structure of digestates which do not
allow sufficient aeration and (3) the C to N ratio of digestates which are lower the
optimal values (Tremier et al. 2014; Zeng et al. 2014, 2015). Common bulking
agents are green waste, wood chips, sawdust and compost grinding wastes (Epstein
2011). A recent and new technique for liquid wastes is based on the spreading and
the constant turning of the liquid into a saturated support bulking material
(Chiumenti 2015; Levasseur et al. 2017).
Composting can convert ammonia to nitrates through nitrification. However,
depending on the process operation, ammonia can be largely volatilized, denitrified
(under anoxic conditions) and lost as N2 or even N2O (a strong greenhouse gas).
Zeng et al. (2012) observed 2–43% of total N loss as ammonia and up to 76%
including nitrification-denitrification. More recently, the addition of biochar has
been used as co-substrate to avoid NH3 volatilisation due to adsorption and support
for nitrifying bacteria (Wu et al. 2017). Researches indicate that industrial
composting can be optimized to produce minimal amounts of NH3 and N2O (<1%
input N) (Chiumenti 2015), but without a diffcult-to-achieve complete nitrification,
an important N2 emissions still represent a relevant loss in value.
Despite all the advantages of the composting process, composting is not neces-
sarily a low-cost technology. Moreover, the compost producer usually receives a
price near to zero or even pays an intermediary distributor. According to Dahlin et al.
(2015) and (Guilayn et al. 2020b), composts price goes from under 0 to 7 €/t, way
below other value-added products that can be recovered from digestates.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 247

Regarding composting costs, the long retention time and the bulking agent
volume induce an installation four times or more larger than the surface needed
for AD. Furthermore, providing aeration and the subsequent air treatment for a high
surface can be extremely costly. The use of forced aeration and/or mechanical
turning is needed. Low-cost passive aeration based only on convection (heat moving
up) is usually not allowed for producing certified composts despite its attested
effectiveness. Adding to that, composting leachate treatment can be challenging
(Roy et al. 2018).

9.8.2 Digestate Drying

Solid digestates and solid fractions can be dried through thermal and solar drying
equipment such as those conventionally applied to wastewater sludge for several
decades. Drying equipment present a high capital investment (typically over 500 k
€), being mostly restricted to large facilities. Commonly related equipment are belt,
rotary and disk dryers. Belt driers seem more suited as rotary and disk dryers can be
damaged by digestates containing large particles. Fire risk due to self-heating and
ignition risk of explosive atmosphere must be considered during design and opera-
tion of digestate drying and storage facilities (Guilayn et al. 2020b).
From a fertilisation perspective, thermal drying induces ammoniacal nitrogen loss
through volatilisation as NH3. It can be though recovered as ammonium salt/acidic
solution through air collection and treatment (Vaneeckhaute et al. 2017). However,
in practice, many operators can be reluctant to operate air treatment equipment
(i.e. acid washing towers) in less acidic levels (e.g. pH 5–6), as any emission risk
might represent air pollution, neighbourhood complains and penalties. In such cases,
the final product can be too acidic for farmers (pH 2–3) and represent a disposal cost
superior to 40 €/m3 in Europe (SUEZ expertise). In the opposite, digestate can be
acidified prior to thermal drying to avoid nitrogen loss (Pantelopoulos et al. 2016).
By removing most of digestate moisture, thermal drying can be an effective
option for enabling large-scale facilities to export digestate to distant areas, while
maintaining product stability over time. Transportation costs can be required to be
further reduced by pelletisation, as it increases product bulk weight from 100 to over
600 kg/m3 (Dahlin et al. 2015).

9.8.3 Growth Media

Digestate can be used as a growth medium for plant seedlings, partially/totally

substituting conventional non-renewable materials such as peat. Zhang et al.
(2013) successfully used solid digestates as a growth medium for tomato, while
reporting a growth biostimulant effect. The solid portion as well as a solid state
digestate may be used as growth medium for insects and invertebrates. Some
248 F. Guilayn et al.

experiences have already been carried out by using either the liquid or the solid
fraction of the digestate as obtained from conventionally applied separation tech-
niques (Cesaro 2021). O’Brien et al. (2019) successfully tested solid digestates
derived from dairy manure and food waste in the cultivation of the fungal species
Pleurotus ostreatus.
Waste-based composts have been extensively studied as growth media in hydro-
ponic cultivation, while the use of the digestate is more recent. The potential of the
anaerobic digestate has been also explored as growth medium in hydroponic systems
(Cesaro 2021). These can be considered as an engineered plant cultivation method,
which uses soil-less growth medium and a nutrient solution. Ronga et al. (2019b)
evaluated the cultivation of baby leaf lettuce in hydroponic systems, using both the
solid and the liquid fraction of digestate as alternative growth medium and nutrient
solution, respectively.
Cerda et al. (2019) have proposed a solid-state fermentation (aerobic) technology
based on solid food waste digestate as substrate/support medium. They have
observed interesting yields for the production of Bacillus thuringiensis (Bt), the
major biological pesticide in the organic farming market. The final product can be
used as a Bt-enriched compost-like product, or, preferentially, an extraction step
should be added to produce a further value-added extract for foliar application.

9.8.4 Thermal Conversion

These last decades, a high interest has been brought among the scientific communi-
ties to valorise the solid fraction of digestates through thermo-chemical processes
(mainly hydrothermal, gasification and pyrolysis) (Freda et al. 2019; Miliotti et al.
2020; Cesaro 2021). Most of the studies were performed on digestate originating
from OFMSW, agricultural or WWTP sludges. Among them a specific attention has
been paid on the pyrolysis process (Torri and Fabbri 2014; Neumann et al. 2015;
Monlau et al. 2015a; Tayibi et al. 2021). Pyrolysis is defined as the thermal
decomposition of the organic matrix under non-oxidizing or very low-oxidizing
stoichiometric atmospheres, and occurs in the temperature range of 250–1200 C
(Bridgwater 2012; Biswas et al. 2017). During pyrolysis, organic matter such as
lignin, proteins, cellulose, and hemicelluloses are thermally broken down and
rearranged forming three major products (Fig. 9.3): (1) biochar (carbonaceous
solid fraction), (2) bio-oil (mainly composed of 15–30% w/w liquid of wide variety
of organic components and 85–70% of aqueous pyrolysis liquid (APL)) (Fabbri and
Torri 2016), and (3) syngas composed of non-condensable gases (i.e. CO, CO2, CH4
and H2) (Tayibi et al. 2020). Depending on the operating conditions
(i.e. temperature, heating rate, residence time) the pyrolysis process can be divided
into three main subclasses: slow, fast and flash pyrolysis (Laird et al. 2009).
Syngas can represent an interesting supplementary energy source that can be
further converted into heat or heat/electricity alone or mixed with biogas in boilers,
engines but also used for methanol production (Gollakota et al. 2016; Giuliano et al.
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 249

Fig. 9.3 The role of biochar in improving the anaerobic digestion chain

2020). Syngas has also attracted recently the attention as hydrogen source in
biological methanation processes (Schwede et al. 2017; Grimalt-Alemany et al.
2018). In parallel, bio-oil is generally separated into an organic and aqueous
phase, the first one can be used as a blend with existing transportation fossil fuels
or directly used as transportation fuels (Gollakota et al. 2016). On the contrary, the
aqueous phase can be used as new feedstock in the AD process in co-digestion with
other organic wastes (Torri and Fabbri 2014), but specific attention should be paid to
the furans and phenolic compounds present that can inhibit the microbial commu-
nities of the AD process (Barakat et al. 2012; Monlau et al. 2014).
Pyrolysis has been applied on solid digestate from OFMSW (Giuliano et al.
2020), agricultural wastes (Feng and Lin 2017; Monlau et al. 2015a; Tayibi et al.
2020), biowastes (Opatokun et al. 2017; Giwa et al. 2019) and wastewater sludges
(González-Arias et al. 2020). In most cases, slow pyrolysis was applied with
250 F. Guilayn et al.

temperatures ranging from 350 to 750  C (Neumann et al. 2015; Ghysels et al. 2020)
with most of the studies operating at 500  C (Monlau et al. 2015a; Ghysels et al.
2020; Tayibi et al. 2021). Tayibi et al. (2020) have investigated the pyrolysis
(500  C, 10  C/min, 1 h) of solid digestate treating sludges and agricultural bio-
masses and reported biochar, bio-oil and syngas yields of 37%wt, 34%wt, 29%wt
respectively. Similarly, Karaeva et al. (2021) reported biochar, bio-oil and syngas
yields of 41%wt, 31%wt, 28%wt, respectively, after pyrolysis at 550  C of solid
digestate from agricultural wastes. After pyrolysis of solid digestate, syngas energy
of 12.9 MJ/Nm3 (Tayibi et al. 2021), 9.5–15.7 MJ/Nm3 (Monlau et al. 2015a),
13.1 MJ/Nm3 (Neumann et al. 2015) were reported. The organic phase of bio-oil
generally exhibited energy yields of 23.5 MJ/kg (Tayibi et al. 2021), 28.4 MJ/kg
(Monlau et al. 2015a), 33.9 MJ/kg (Neumann et al. 2015), 24.9–32 MJ/kg (Ghysels
et al. 2020).
Biochar can be used for several applications especially for improving the stability
of composting and AD (Torri and Fabbri 2014; Fagbohungbe et al. 2017; Xiao et al.
2017), in agronomic applications alone or in combination with fertilizers like liquid
digestate (Laird et al. 2010; Semida et al. 2019; Guilayn et al. 2021; Tayibi et al.
2021), or for gas treatment like biogas purification and upgrading. A lot of synergies
have been identified in the recent years by associating AD and pyrolysis and most of
them are using biochar as a central element as shown in Fig. 9.3 (Fabbri and Torri
2016; Fagbohungbe et al. 2017; Luz et al. 2018). Up to date, even if such processes
are mature and applied at industrial scale, there is no real example that has been
tested at industrial scale for such dual symbiotic approach.

9.8.5 Bioethanol Production

As previously mentioned, the solid digestate from the AD process especially coming
from agricultural biogas plants represents a promising perspective for the production
of biofuels such as bioethanol (Logan and Visvanathan 2019). Indeed, during the
AD process of agricultural residues such as crops and manures, part of the organic
matter is not degraded and among them a significant part of cellulose that can be
used for bioethanol production in a biorefinery concept (Ruile et al. 2015; Santi et al.
2015). Another approach for bioethanol fermentation commonly consists of two
steps: enzymatic hydrolysis and fermentation, which can be realized separately or
simultaneously. Two strategies can be generally applied: enzymatic hydrolysis
separately from fermentation, as separate hydrolysis and fermentation (SHF), or
simultaneously, as simultaneous saccharification and fermentation (SSF). In general,
the advantage of SHF is the ability to perform each step (i.e. enzymatic hydrolysis
and fermentation) under optimal temperature conditions (40–50  C for enzymatic
hydrolysis and 30  C for fermentation), whereas SSF offers the opportunities to limit
contamination and reduce the size of reactors (Cotana et al. 2015; Sukhang et al.
2020).
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 251

Recent studies have focussed on the bioethanol fermentation from solid digestate
and obtained a low ethanol yield (Monlau et al. 2015b; Sheets et al. 2015; Sambusiti
et al. 2016). Indeed, the solid digestate represents a highly recalcitrant biomass
especially due to its high lignin and crystalline cellulose content that can limit its
further biodegradation by enzymes during the saccharification process (Santi et al.
2015; Sambusiti et al. 2016). For these purposes, pretreatment technologies can be
applied such as mechanical, thermal, thermo-chemical, physical, biological or a
combination of them (Cotana et al. 2015; Sambusiti et al. 2016; Carrere et al.
2016). Until now, the pretreatments that have been reported in the literature to
enhance bioethanol fermentation from solid digestate are comprised of thermo-
acid (Teater et al. 2011; Stoumpou et al. 2020), thermo-alkaline (Yue et al. 2011;
Stoumpou et al. 2020), ozone (Wang et al. 2016) or mechanical (Sambusiti et al.
2016) treatment.
For instance, Yue et al. (2011) investigated the impact of the nature of digestates
(from CSTR and a plug flow reactor (PFR)) on bioethanol production. Ethanol
production of 105 g/kg dry digestate was noticed for the digestate from the CSTR
and 85 g/kg dry digestate for the digestate from the PFR. Similarly, Stoumpou et al.
(2020) have investigated acid and alkaline pretreatment on solid digestate (from
anaerobic digestion of wheat straw) to improve bioethanol production. Acid
pretreatment led to low bioethanol production probably due to the production of
inhibitors. In parallel, ethanol fermentation presented yields up to 65% from alkaline
pretreated digestate and all the available glucose was consumed, implying that no
inhibitory factors were present (Stoumpou et al. 2020).
From an industrial point of view, it is important that pretreatment technologies of
solid digestate take into account the ease of implementation, their cost and the
potential production of inhibitors (i.e. furans, polyphenols) (Barakat et al. 2012;
Jönsson and Martín 2016; Sukhang et al. 2020). Up to date, to our knowledge, there
is no industrial plant producing second-generation bioethanol from solid digestate.
In general, second-generation bioethanol is struggling to find industrial profitability
even if some units are already operational or under construction, most of them in
Europe and the United States. Although the first industrial-scale units appeared in the
United States (DuPont, Abengoa, Ineosbio, KiOR and Poet), some initiatives are
also existing in Europe such as the demonstrative Futurol platform (180,000 L/year)
in France and the Beta Renewables (approx. 80,000 m3/year) plant in Italy
(Crescentino).

9.8.6 Nutrient Recovery from Incineration Ashes

Nutrients can be extracted from the remaining ashes after combustion of biodegrad-
able wastes. Since phosphorus and potassium are non-volatile, these macronutrients
are concentrated in the ashes (Schoumans et al. 2010). Nevertheless, these ashes can
also be rich in heavy metals, including for example cupper (Cu), cadmium (Cd) and
zinc (Zn). The available process to extract phosphorus from ashes are generally
252 F. Guilayn et al.

classified as thermochemical and wet-chemical technologies. An example of the first

class is the process developed by Outotec (Finland), where alkaline reagents are
added to the ashes at a temperature of 1000  C, thereby gasifying the heavy metals
(Hermann and Schaaf 2018). Phosphorus is bound and recovered as CaHPO4, which
can be sold as mineral fertilizer. An example of the second class is the process
developed by the company EcoPhos (Belgium), where hydrogen chloride (HCl) is
added to the ashes to chemically extract the phosphorus (Bolland 1996; Takhim et al.
2018). Phosphorus recovery efficiencies higher than 90% have been achieved as
such (Saerens 2017; Schoumans et al. 2010; Takhim et al. 2018). Nevertheless,
currently, few full-scale installations exist for digestate treatment due to the rela-
tively high costs for combustion (up to 10 euros per ton of fresh weight) and the need
for a thorough flue gas cleaning system (Vaneeckhaute et al. 2017).

9.9 Regulatory Framework in EU and North America

Quality management of digestate and its (by-/co-)products involves a wide range of

permits and quality standards to ensure its safety and effectiveness. Strict local
regulation usually applies for spreading these products under a waste status in law.
End-of-waste schemes may vary regionally, and they tend to impose both product
quality/innocuity parameters, process design/operation criteria and good practices.
Such regional/international differences are a strong barrier for developing true
markets around digestate (by-/co-)products.
The EU has recently enforced a new Fertiliser Regulation allowing a homogenous
definition of quality and process criteria applicable to several types of materials
including digestates (CE 2019/1009). More broadly, this regulation represents a
huge international milestone as it promotes the trade of biosourced fertilisers
obtained from organic waste streams within the largest common market in the
world. It establishes 11 Component Material Categories (CMCs) to produce differ-
ent CE-labeled fertilising products. Concerning digestates, two CMC are related:
CMC 4 for fresh crop digestate and CMC 5—Digestate other than fresh crop
digestate. Different CMCs can be used to constitute a wide range of so-called
Product Function Categories (PFC), as detailed in the Annex I to the EU Regulation.
Several PFCs can be related to products coming from digestates (PFC 1 Fertiliser,
PFC 3, Soil Improver, PFC 4, growing medium, PFC 6 Plant Biostimulant). More
recently, phosphate salts (such as struvite), ashes from incineration and biochar were
included in the regulation as three new CMC. These categories represent even
further a new milestone as phosphate salts and incineration ashes coming from
WWTP sludge will be accepted. In the future, more CMC can be included as new
recycled products become scientifically proven to be effective and safe. It could be
the case, for example, for ammonium sulphate from acid scrubbing, recently added
to a French standard (NF U42-001-1 A1)
The most generic regulation currently in place in North America includes the US
Environmental Protection Agency (EPA) Classes of biosolids, which applies to
9 Valorisation of Anaerobic Digestate: Towards Value-Added Products 253

products derived from sewage sludge. The EPA issued a 40 CFR Rule (Part 503) that
categorizes biosolids as Class A, Class A EQ (Exceptional Quality) or Class B,
depending on the level of pathogens and the ability of the material to meet or exceed
Vector Attraction Reduction (VAR) requirements. Hereby, for Class A biosolids,
pathogens must be reduced to virtually non-detectable levels and the material must
also comply with strict standards regarding metals, odors and VAR. Class A EQ is
used to describe a biosolids product that not only meets, but exceeds, all Class A
pathogen reduction, metals and VAR requirements. Finally, Class B biosolids
contain higher levels of detectable pathogens than Class A biosolids and may require
a permit from the EPA with conditions on land application, crop harvesting and
public access. Additional state, provincial or local regulations may apply and are
currently under development for digestate and digestate-derived products.

9.10 Perspective

AD is a fully industrial, trending and politically encouraged technology. It is

expected to play a crucial role for achieving a circular economy while fighting
climate change. Digestate is traditionally seen as a by-product of AD as a renewable
energy technology that produces biogas or biomethane. However, it is usually the
major AD product in terms of mass. It represents nowadays a major cost centre for
most of industrial operators while presenting valuable compounds that can be
concentrated, fractionated and/or chemically/biologically transformed to generate
value-added products described in this chapter.
Today, full-scale post-treatment processes such as composting, drying and N/P-
recovery generate negative to moderate net value, but can effectively enable and
enhance digestate return to soil. Increasing demand for these products could increase
their price in the future, while emerging technologies such as microalgae and thermal
conversion could start true biorefineries from digestates, presenting significant
potential for creating value.
Adding to technological bottlenecks, all these options need a trusted end-of-waste
regulatory status that can be different interdependently according to AD feedstock
category, local regulation and public/consumer acceptance. Fortunately, this situa-
tion is evolving fast along with the urgency to reshape the economy. The EU has
provided an example and a global milestone with the new and still evolving
regulation on fertilizers (CE 2019/1009). It allows the commercialization in the
world’s largest common market of several product categories that can be derived
directly or after post-treatment of several types of digestate and digestate
by-products.
254 F. Guilayn et al.

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Chapter 10
Biochar Produced from Organic Waste
Digestate and Its Potential Utilization
for Soil Remediation: An Overview

Suchanya Wongrod, Gilles Guibaud, Stephane Simon, Piet N. L. Lens,

David Huguenot, Yoan Pechaud, and Eric D. van Hullebusch

Abstract Contamination of metal(loid)s and organic pollutants in soils has caused

detrimental effects to the environment, so that there is a need to develop appropriate
treatment approaches to resolve the soil pollution. In recent years, biochar produced
from biowaste material has been widely used as a potential adsorbent in remediating

S. Wongrod (*)
Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-
la-Vallée, France
Université de Limoges, PEIRENE, Équipe Développement d0 indicateurs ou prévision de la
qualité des eaux, URA IRSTEA, Limoges, France
IHE Delft Institute for Water Education, Delft, The Netherlands
Environmental Technology Program, School of Energy, Environment and Materials, King
Mongkut’s University of Technology Thonburi, Bangkok, Thailand
Environmental and Energy Management for Community and Circular Economy (EEC&C)
Research Group, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand
e-mail: [email protected]
G. Guibaud · S. Simon
Université de Limoges, PEIRENE, Équipe Développement d0 indicateurs ou prévision de la
qualité des eaux, URA IRSTEA, Limoges, France
P. N. L. Lens
IHE Delft Institute for Water Education, Delft, The Netherlands
D. Huguenot · Y. Pechaud
Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-
la-Vallée, France
E. D. van Hullebusch (*)
Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508), UPEM, Marne-
la-Vallée, France
IHE Delft Institute for Water Education, Delft, The Netherlands
Université de Paris, Institut de Physique Du Globe de Paris, CNRS, UMR 7154, Paris, France
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 263
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_10
264 S. Wongrod et al.

organic and inorganic pollutants in soils. This review aims to overview the produc-
tion technologies and characterization of biochars derived from sewage sludge
digestate (SSD) and the organic fraction of municipal solid waste digestate
(OFMSWD) and their applications for soil treatment. Critical discussions on the
sorption mechanism, important factors influencing contaminant retention in soils,
bioavailability of trace elements with biochar addition, and potential effects of
biochar application to soils are summarized. The interaction mechanisms involved
between the SSD and OFMSWD-derived biochars and contaminants, the main
factors influencing the biochars’ sorption efficiency and possible effects to the
environment are discussed in this review.

Keywords Biochar · Sewage sludge · Municipal solid waste · Heavy metal

removal · Organic pollutants removal · Soil amendment

10.1 Introduction

The increment of food supply for human beings, rapid expansion of industrial
production and intensive agricultural activities are raising concerns. There are
many negative impacts on the environment, e.g. discharges of metal laden waste-
waters into rivers, spills of petroleum hydrocarbons to soils, transportation of
veterinary pharmaceuticals from animal manures into surface runoff and soil leach-
ate on farmland. Thus, water and soil ecosystems are prone to biomagnification and
bioaccumulation of toxic chemicals into living organisms through the food chain.
Soil contamination requires risk assessment studies and remediation to increase
environmental safety by reducing the mobility and toxicity of pollutants.
Several treatment technologies have been sought to minimize pollution (Beesley
and Marmiroli 2011), e.g. excavation, solidification and stabilization, soil washing,
phytoremediation and bioremediation (El Sawwaf and Nazir 2012; Iturbe and López
2015). However, these conventional technologies are often expensive for in situ
treatment and may lead to nutrient losses in soils (Beesley et al. 2011). Supplemen-
tation of biochar to soil is an alternative to promote environmental sustainability by
converting organic waste by-products, for instance, the solid digestate from biogas
plants, into a value-added product, i.e. biochar (Meng et al. 2013; Tan et al. 2015).
Solid digestate is an organic by-product generated by wastewater treatment
plants. Their treatment before a final disposal is required to decrease volume and
reduce unpleasant odors. Moreover, these solid digestates are considered as hazard-
ous waste due to the remaining pathogens that can possibly be transferred to the soil
(Al Seadi and Lukehurst 2012). Therefore, sludge management with environmental-
friendly and cost-effective technologies are required. Conventional technologies to
dispose sludge such as landfilling or direct use in agriculture may be restricted,
respectively, due to limited landfill site availability and possible transportation of
pollutants to the farmland (Devi and Saroha 2016). Therefore, the valorization of
digested sludge arises as an interesting approach to treat solid digestate and conse-
quently produce biochar.
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 265

Biochar is a black solid char derived from thermal conversion technologies

(e.g. pyrolysis, hydrothermal carbonization, and gasification) of biowaste materials
in a limiting oxygen environment (Inyang et al. 2016). Pyrolysis produces biochar as
a main product, and bio-oils and syngas as by-products. There are extensive studies
on the use of biochars derived from agricultural residues (e.g. rice straw, pine chips,
and bamboo) and animal manures (e.g. swine solid, dairy manure, and poultry litter)
for soil and wastewater treatment (Ahmad et al. 2014; Ok et al. 2015; Sohi et al.
2010; Tan et al. 2015). Only a few studies focused, however, on the use of biochar
produced from organic waste digestate. The solid digestates such as sewage sludge
digestate (SSD) and the organic fraction of the municipal solid waste digestate
(OFMSWD) are considered as alternative materials for biochar production
(Neumann et al. 2014; Pituello et al. 2014; Wongrod et al. 2018a, b).
Solid digestate-derived biochars have been increasingly used for soil remediation
due to its improved properties after pyrolysis, for instance, more available oxygen-
containing functional groups (e.g. carboxyl or hydroxyl), and surface properties
(e.g. surface charge and hydrophobicity). For example, Zielińska et al. (2015)
reported the increase of specific surface area (2–4 times), macro- and micronutrient
content, and aromaticity in sewage sludge biochar, compared to its origin feedstock,
particularly at higher pyrolysis temperature (500–700  C). The biochar also provide
several benefits to the soil, e.g. immobilize toxic pollutants, increase nutrient reten-
tion to enhance crop yields, and provide longer carbon sequestration in soils
(Lehmann and Joseph 2009).
Biochars are applied in four main areas: carbon sequestration, soil fertility
enhancement, bioenergy production and contaminant remediation. This chapter
describes recent findings on the characterization and application of biochars derived
from SSD and OFMSWD for remediating organic and inorganic contaminants in
soils. The interaction mechanisms involved between biochars and contaminants, the
main factors influencing the biochars’ sorption efficiency and possible effects to the
environment are discussed in detail.

10.2 Biochar Production from Organic Waste Digestates

10.2.1 Biochar Production Technologies

Figure 10.1 shows various thermal conversion processes to produce biochar includ-
ing slow pyrolysis, fast pyrolysis, gasification, and hydrothermal carbonization
(HTC). Prior to pyrolysis or gasification, drying of the digestate to reduce the
moisture content to less than 10% is required, while no dehydration is needed for
HTC. Under slow pyrolysis, biochar is produced at low heating rates (10–30  C/min)
and long residence times (5 min–12 h) at temperatures ranging from 300 to 650  C
(Kambo and Dutta 2015). In general, higher temperatures result in lower biochar
yields due to partial degradation of lignin and cellulose (Kambo and Dutta 2015).
Table 10.1 presents the production conditions and yields of bioproducts of the
different thermal conversion technologies. The biochar produced under slow
266 S. Wongrod et al.

Fig. 10.1 Thermal conversion processes for biochar production. Adapted from Kambo and Dutta
(2015); Novotny et al. (2015); Sohi et al. (2010)

Table 10.1 Production conditions and yields of solid, liquid and gas products from thermal
conversion processes
Production conditions (operating Gas Liquid Solid
temperature, heating rate, residence (syngas) (bio-oil) (biochar)
Mode time) (%) (%) (%)
Fast pyrolysis 400–900  C, 100–1000  C/s, s–min ~13 ~75 ~12
Slow pyrolysis 250–700  C, 10–30  C/min, 15 min– ~35 ~30 ~35
2h
Hydrothermal 180–260  C, 5–10  C/min, 15 min–2 h ~5 ~25 ~70
carbonization
(HTC)
Gasification 800–950  C, 50–100  C/s, 10–20 s ~85 ~5 ~10
Adapted from Belcher and Masek (2013); Duku et al. (2011); Iwasaki et al. (2014); Kambo and
Dutta (2015); Sohi et al. (2010)

pyrolysis provides high biochar yields (25–35%) and oxygen-contained functional

groups such as hydroxyl and carboxyl on the biochar surface (Kambo and Dutta
2015), influencing the sorption capacity of the biochar. Fast pyrolysis is a process in
which biofuel (bio-oil) is produced as a main product, and biochar and syngas are
generated as by-products. Under fast pyrolysis, the process performs at 400–700  C
with a very high heating rate (~1000  C/s) and short residence time (< min) (Mohan
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 267

et al. 2014). High bio-oil (75%) and low biochar (10–15%) yields are often achieved
in fast pyrolysis (Table 10.1). In order to obtain homogenous biochar, pretreatment
of the feedstock, e.g. sieving to a small particle size (<2 mm) and drying (65–80  C)
to reduce the moisture to less than 10% are required (Pituello et al. 2014). Due to
lower biochar yields and higher operation costs from fast pyrolysis, the slow
pyrolysis is a more favorable option to produce biochar.
Gasification is a combination of thermal and chemical processes which is mainly
for fuel-gas production (Novotny et al. 2015). The fuel gases are mainly composed
of hydrogen (H2) and carbon monoxide (CO) that can be further used as renewable
energy sources for internal engine and power supply in industries (Novotny et al.
2015). Nevertheless, due to very high operating temperatures (800–950  C) during
gasification, the obtained biochars are relatively small, thus less desirable for biochar
production (Table 10.1). In addition, biochar produced from a hydrothermal carbon-
ization process shows low stability, i.e. high O/C ratio, whereas biochar produced
under slow pyrolysis acts as an effective sorbent due to its physicochemical
properties.
Biochar yields vary mainly depending on the operating temperature, which
strongly influences the biochar properties (Singh et al. 2010). For instance, at
increasing pyrolysis temperature (from 300 to 700  C) of SSD, a higher surface
area (from 11 to 26 m2/g) and ash content (66–87%) but lower biochar yields (from
83 to 65%) and O/C ratio (from 0.56 to 0.05; implied higher biochar stability) were
obtained (Yuan et al. 2015). In addition, the origin of feedstock also plays a role in
the biochar properties. Agrafioti et al. (2014) reported that rice husk contains lower
volatile matter (%) than sewage sludge and organic solid waste, which resulted in its
lower biochar yield.

10.2.2 Organic Waste Digestates and Their Derived Biochars

The solid sludge by-product (solid digestate) obtained from anaerobic digestion can
be used as substrate for biochar production using pyrolysis technology (Fig. 10.2).
By coupling anaerobic digestion with the pyrolysis process, more energy recovery
and sustainability of the digestion plant were found since the bioenergy could be
used to dry the solid digestate prior to pyrolysis (Monlau et al. 2015). This chapter
focuses on two types of solid digestates: sewage sludge digestate (SSD) and organic
fraction of the municipal solid waste digestate (OFMSWD).

10.2.2.1 Sewage Sludge Digestate

Sewage sludge digestate (SSD) is the organic residue remaining after the anaerobic
treatment of wastewater and waste activated sludge. The management of SSD is
currently a big concern because of the large quantities of solid digestate generated at
the wastewater treatment sites. The valorization of the solid digestate is an
268 S. Wongrod et al.

Fig. 10.2 Biochar production from organic waste digestate and its applications in soil
bioremediation

alternative to reduce the volume and eliminate organic pollutants in the digestate.
Waqas et al. (2014) reported a significant reduction of the polycyclic aromatic
hydrocarbons (PAHs) concentration after the conversion of sewage sludge into
sewage sludge-derived biochar.
Table 10.2 represents the composition of biochar derived from sewage sludge.
From Table 10.2, the SSD is rich in Cu, Cr, Pb and Zn (Pituello et al. 2014) as well as
Fe and P (Yuan et al. 2015) due to the addition of FeCl3 for phosphate precipitation
during the wastewater treatment. Moreover, the SSD biochar is rich in minerals
compared to biochars produced from biomass residues. This may result from the
diversity and complexity of sewage sludge (Zielińska et al. 2015). Sewage sludge
(59%) and its derived biochar (72%) have a high ash content, corresponding to the
dominant mineral fractions (such as SiO2, CaSO42H2O and CaCO3) contained in
the sludge (Zielińska et al. 2015). In addition, the SSD may contain undigested
organic compounds (such as carbohydrates, lipids and proteins) preliminarily from
the wastewater, inorganic elements from soils and synthetic materials from the
substrates (Devi and Saroha 2016).

10.2.2.2 Organic Fraction of the Municipal Solid Waste Digestate

The organic fraction of municipal solid waste digestate (OFMSWD) is a by-product

after anaerobic biological treatment of municipal solid waste. Municipal solid waste
in Europe often comprises of a large organic fraction (30–40%) with high moisture
content and biodegradability (Cesaro et al. 2019). This is due to the large content of
organic waste, food waste and leftovers from residences, restaurants, factories and
markets (Peng and Pivato 2017). The specific location and collecting time play a
Table 10.2 Properties of biochars derived from sewage sludge digestate
10

Experimental P
Process for biochar conditions Yield Ash C H O N S (% or Surface area
production Tpa, RTb, HRc (%) pH (%) (%) (%) (%) (%) (%) g/kg) (m2/g) Reference
HTC 225, 16, NAd 50 NA NA 38 NA NA NA NA 6% NA Huang et al.
(2018)
Slow pyrolysis 250, 4, 3.3 69 NA NA 36 NA NA NA NA 4% NA Huang et al.
(2018)
Slow pyrolysis 250, 1, 16–19 NA 6.9 48 28 NA NA 3 0.8 17 g/kg 0.8 Pituello et al.
(2014)
Slow pyrolysis 300, NA, 10 72 5.3 52 32 2 8 3 4 NA NA Hossain et al.
(2011)
Slow pyrolysis 300, 0.5, 17 62 6.0 NA 39 4 NA 7 NA NA 4 Agrafioti et al.
(2013)
Slow pyrolysis 400, NA, 10 63 4.9 63 20 1 4 2 4 NA NA Hossain et al.
(2011)
Slow pyrolysis 450, 1, 16–19 NA 7.2 67 22 NA NA 2 0.6 21 g/kg 7 Pituello et al.
(2014)
Slow pyrolysis 450, 4, 3.3 47 NA NA 29 NA NA NA NA 7% NA Huang et al.
(2018)
Slow pyrolysis 500, 25 54 7.1 73 18 0.7 4 2 3 5% 31 Zielińska et al.
(2015)
Biochar Produced from Organic Waste Digestate and Its Potential. . .

Fast pyrolysis 500, NA 28 9.0 65 19 1 8 4 0.2 NA NA Arazo et al.

(2017)
Slow pyrolysis 600, 25 51 11.0 77 18 0.4 1 2 4 5% 24 Zielińska et al.
(2015)
Slow pyrolysis 700, 25 48 12.2 79 18 0.2 0.7 1 4 6% 54 Zielińska et al.
(2015)
Slow pyrolysis 700, NA, 10 52 12.0 72 20 0.5 NA 1 6 NA NA Hossain et al.
(2011)
269

(continued)
Table 10.2 (continued)
270

Experimental P
Process for biochar conditions Yield Ash C H O N S (% or Surface area
production Tpa, RTb, HRc (%) pH (%) (%) (%) (%) (%) (%) g/kg) (m2/g) Reference
Gasification 850, 0.57, NA 36 NA 74 21 0.5 NA 1 NA NA NA Freda et al.
(2018)
Fast pyrolysis 900, 0.033, NA 13 NA 52 33 NA 27 NA 1 3% 27 Deng et al.
(2017)
a
Tp refers to pyrolysis temperature ( C)
b
RT refers to residence time (h)
c
HR refers to heating rate ( C/min)
d
NA refers to not available
S. Wongrod et al.
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 271

significant role in the variability of the OFMSWD (Alibardi and Cossu 2015). In
contrast, Davidsson et al. (2007) showed minor effects in chemical composition of
the OFMSWD collected from different origins.
Table 10.3 shows the chemical and physical composition of biochar produced
from different OFMSWD at various production temperatures. The OFMSWD is
usually rich in Cu and Zn (Pituello et al. 2014), which further concentrate after the
anaerobic digestion of the organic waste (Tampio et al. 2016). Direct use of this
OFMSWD sludge in the field may be restricted by the presence of odors, mainly
volatile organic compounds (VOCs), and potential soil contamination by leachate
(Cesaro et al. 2019). Hence, a digestate treatment to stabilize the OFMSWD via
thermal processes (e.g. pyrolysis and gasification) (Garlapalli et al. 2016) is often
performed to eliminate the organic compounds, reduce the sludge quantities and
obtain valuable bioproducts (i.e. biochar, bio-oil and syngas).

10.3 Characterization of Organic Waste Digestate-Derived

Biochars

10.3.1 Physicochemical Properties

Biochar derived from different bio-sources and pyrolyzed under several conditions
shows a wide range in chemical compositions, e.g. carbon, hydrogen, nitrogen, ash,
and metal content. The nature of the feedstock and the pyrolysis temperature are the
two most important parameters that strongly influence the chemical properties of
biochar. Low temperature biochars (300–400  C) contain several oxygen functional
groups that can interact with inorganic or polar organic chemicals through electro-
static forces and precipitation, whilst biochars produced at high temperature
(500–900  C) show high hydrophobicity which is likely to bind with organic
contaminants (Ahmad et al. 2014).
The main components of organic materials are water, hydrocarbons (HCs), and
tars (condensed polyaromatic compounds). Antal et al. (2003) demonstrated that C,
H and O contained in biochar tends to vaporize into H2O, H2, CO and CO2 with
increasing temperature. Typically, C and N are volatilized during pyrolysis, and
higher C:N ratios in biochar than in feedstocks are observed (Karaj et al. 2011;
Nartey and Zhao 2014; Yuan et al. 2015). In general, the volatilization of N and K
occurs at low temperatures, while P, S, Ca and Mg tend to mobilize at higher
temperature, and metal elements like Fe and Mn are likely to remain in the biochars
(Rodriguez 2010).
Biochar produced from SSD and OFMSWD have higher ash contents than
biochar obtained from wood and crop residues (de la Rosa et al. 2014; Pituello
et al. 2014). High ash contents in biochars can result from large mineral fractions
contained in the feedstock. de la Rosa et al. (2014) demonstrated that 69.5% of ash
was found in sewage sludge-derived biochar, whilst biochar made from wood
 272

Table 10.3 Properties of biochars derived from the organic fraction of municipal solid waste digestate
Experimental P
Process for biochar conditions Yield Ash C H O N S (% or Surface area
production Tpa, RTb, HRc (%) pH (%) (%) (%) (%) (%) (%) g/kg) (m2/g) Reference
Slow pyrolysis 250, 1, 16–19 NAd 7.2 37 33 NA NA 4 1 17 g/kg 0.7 Pituello et al.
(2014)
Slow pyrolysis (mixed 300, 2, NA 92 7.8 12 40 5 53 0.3 NA 0.08 g/kg NA Rehrah et al.
material) (2015)
Slow pyrolysis 350, 1, 16–19 NA 7.6 44 34 NA NA 4 0.9 20 g/kg 5 Pituello et al.
(2014)
Slow pyrolysis 400, 0.33, NA NA 8.0 6 48 12 31 1 0.1 NA 20 Jin et al. (2014)
Slow pyrolysis 450, 1, 16–19 NA 7.4 55 29 NA NA 3 0.8 24 g/kg 27 Pituello et al.
(2014)
Slow pyrolysis 500, 0.33, NA NA 8.5 9 59 9 20 1 0 NA 29 Jin et al. (2014)
Slow pyrolysis 550, 1, 16–19 NA 7.1 61 26 NA NA 2 0.6 26 g/kg 77 Pituello et al.
(2014)
Slow pyrolysis 600, 0.33, NA NA 9.0 6 70 8 13 1 0.1 NA 29 Jin et al. (2014)
a 
Tp refers to pyrolysis temperature ( C)
b
RT refers to residence time (h)
c
HR refers to heating rate ( C/min)
d
NA refers to not available
S. Wongrod et al.
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 273

contained an approximately 6 times (10.6%) lower ash content. The main composi-
tion of ash is alkaline salts of Na, K, Ca and Mg (de la Rosa et al. 2014). Many
studies showed that the percentages of ash in biochars increased along with higher
temperature, this may result from the higher organic matter volatilization with
increasing temperatures (Cantrell et al. 2012; Tsai et al. 2012; Zielińska et al.
2015). Additionally, the presence of Ca, Fe and Al oxides on the biochar solid
matrix can be attributed to the precipitation of target pollutants on the biochar
surface. The studies of Agrafioti et al. (2014) showed that biochars produced from
sewage sludge and the organic fraction of municipal solid waste contained much
higher mineral fractions (i.e. CaO, Fe2O3 and Al2O3) than biochar produced from
rice husk (<1.3% w/w). CaO was found in both SS (17% w/w) and OFMSW (50%
w/w) biochars, whereas Fe2O3 and Al2O3 were mainly found in the SS biochar (13%
and 5% w/w, respectively), but were negligible in the OFMSW biochar (< 1.5 % w/
w) (Agrafioti et al. 2014).
The physical characteristics of biochar, such as BET (Brunauer, Emmett and
Teller) specific surface area (SBET) and the crystallographic structure, also play a role
in adsorption of contaminants as well as retention of water and nutrients. Rodriguez
(2010) stated that biochars have a relatively large SBET and high porosity, which
vary depending on the type of organic material and the production conditions.
Typically, micropores enhance the sorption capacity of contaminants while
mesopores are mostly related to adsorption between solid and liquid phases, whereas
macropores are significant for the bulk soil structure and aeration in soils. However,
a higher SBET may not always corresponds to a better sorption for metal elements.
Agrafioti et al. (2014) showed that biochar produced from organic digestates have a
higher sorption efficiency for As(V) and Cr(III) than rice husk-derived biochar even
they have a much lower SBET (10–30 times) than the latter. This suggests that the
sorption mechanism of trace elements onto these biochars is predominated by
electrostatic interactions (i.e. anionic and cationic trace elements attraction) rather
than physical adsorption. Nevertheless, a high SBET and pore volume can enhance a
faster mass transfer between contaminants and biochar pores (Agrafioti et al. 2014).

10.3.2 Modification of Biochars

Biochars are recently applied as media for retention of organic and inorganic
contaminants in soils. However, a low sorption capacity has been found in certain
conventional biochars and thus a modification post-treatment to improve the sorp-
tion efficiency for target pollutants becomes necessary. Several pretreatment
methods aiming to improve the surface charges of biochar can be applied, such as
treatments with acidic or basic chemicals, steam activation, magnetization, and
surfactant modifications (Rajapaksha et al. 2016). The selection of the treatment
method for biochar depends on the type of contaminants such as hydrocarbons
compounds, metal(loid) elements, anionic and cationic ions and the hydrophobicity
and polarity of the biochar (Rajapaksha et al. 2016).
274 S. Wongrod et al.

Activation of biochar with steam was found to have higher nutrient retention
times available for plants than the raw biochar and activated biochar showed almost
doubled positive effects on the soil than non-activated biochar (Borchard et al.
2012). Regarding the difficulty in removing powdered biochars from aqueous
solution after the treatment, magnetic modification of biochar is an interesting
technique. Devi and Saroha (2014) found that zero valent iron (ZVI) impregnated
biochar composites, synthesized by FeSO47H2O as a chemical reagent and NaBH4
as a reducing agent, provided simultaneous adsorption and dechlorination of
pentachlorophenol.
Chemical modifications of biochars can be carried out by treatment with acidic
chemicals such as phosphoric acid (H3PO4), sulfuric acid (H2SO4) and nitric acid
(HNO3), an oxidizing agent such alkaline chemicals such as sodium hydroxide
(NaOH) and potassium hydroxide (KOH) (Rajapaksha et al. 2016). H3PO4 is one
of the common reagents used to modify chemical structures of adsorbents (Kang
et al. 2011). Treating with H3PO4 helps to break down lignocellulose, aliphatic and
aromatic biomass and further forming phosphate and polyphosphate to avoid con-
traction during the development of pores (Yang et al. 2011). H2O2, as an oxidant, has
been used to modify biochar and induce more oxygen-containing functional groups,
particularly carboxylic groups, on its surface (Gao et al. 2012). In addition, KOH
was found to induce higher SBET and more available functional groups on the
biochar surface (Jin et al. 2014). Moreover, biochar treated with urea had more
available N functional groups and surface basicity (Stavropoulos et al. 2008).
Regarding the improved properties of biochars after modification, the sorption
capacity of the modified biochars towards both inorganic and organic pollutants
have been investigated. The Pb sorption capacity was enhanced from 0.88 mg/g (raw
biochar) to 22.82 mg/g on H2O2-modified biochar, in accordance with more carbox-
ylic groups available on the biochar surface (Gao et al. 2012). Modification of
municipal solid waste-derived biochar with KOH was found to enhance the sorption
efficiency of As(V) 1.3 times, in agreement with its higher SBET and more available
surface functional groups (Jin et al. 2014). Rajapaksha et al. (2014) stated that
removal of organic pollutants could be improved by the π-π electron donor interac-
tions of biochar with the aromatic rings of organic pollutants. Biochar treated with
urea was also found to have higher uptake rates of phenol due to better π-π dispersion
interactions (Stavropoulos et al. 2008).

10.4 Applications

Biochar derived from organic waste digestate is currently considered as an effective

amendment for fertilization in agriculture. According to the high carbon content in
biochar, it acts as a good conditioner for soils to enhance their physical, biological
and chemical properties (Ahmad et al. 2014). Biochar can retain up to 18% of water
in soils, provide a nutrient sink for plants and a habitat for living organisms, thus
improving soil structure and enhancing crop yields (Verheijen et al. 2010).
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 275

Apart from application of biochar as a soil amendment, it also plays a role as an

active medium for pollution sinks in soils. Soil contaminants can be classified into
two main groups: organic and inorganic. The most common organic pollutants are
PAHs, petroleum hydrocarbons, pesticides, industrial organic solvents, pharmaceu-
ticals (e.g. carbamazepine and diclofenac), and antibiotics (e.g. sulfamethoxazole)
(Jung et al. 2013; Qiu et al. 2009; Williams et al. 2015; Xu et al. 2011; Zheng et al.
2013). The main sources of organic pollutants in soils are inappropriate anthropo-
genic activities such as mineral extraction, waste disposal, and spreading of fertilizer
on soils (Ahmad et al. 2014; Wang et al. 2016; Zhang et al. 2015; Zheng et al. 2013).
Potential toxic metals such as Pb, Zn, Cu, Cd and Cr, and metalloids like As and
Se are well-known inorganic pollutants which are non-biodegradable and persistent
in the environment. Toxicity mainly involves the dissolved and bioavailable fraction
of the metals, since they can be taken up by organisms (Florence et al. 1992). The
release of dissolved metals to the environment can result in their transfer,
bioaccumulation (i.e. accumulated in the tissue of organisms) and biomagnification
(i.e. increased concentration in animals higher in the food chain) (Wuana and
Okieimen 2011). Generally, the bioaccumulation of metals in ecosystems results
from anthropogenic sources such as direct use of digested organic sludge as fertilizer
on soils, use of pesticides in agriculture, discharge of high-metal contaminated
wastewater into rivers as well as irrigation using industrial or metal mining waste-
water (Tan et al. 2015; Zhang et al. 2013).
Because of the large specific surface area, high porosity and the presence of metal
oxides on sludge-derived biochar, metal ions can be sorbed or precipitated onto the
biochar, leading to lower concentrations of pollutants in soils and water runoff. Most
organic pollutants are hydrophobic and are, therefore, preferentially retained by
biochar. In addition, non-carbonized fractions on the surface of sludge-based biochar
contain oxygen functional groups such as –OH, –COOH and C¼O, which can
effectively enhance the sorption ability for the PAHs (Waqas et al. 2014; Zielińska
and Oleszczuk 2016).

10.4.1 Sorption Mechanisms

Figure 10.3 shows the main mechanisms of sorption for organic and inorganic
pollutants onto the biochar surface. The main mechanisms between biochar and
organic pollutants are electrostatic forces, active binding of oxygen functional
groups on biochar pores with polar organic compounds and bonding of non-polar
organic compounds on hydrophobic sites of biochar (Fig. 10.3). In general, organic
waste-derived biochar produced at high pyrolysis temperatures possesses a higher
sorption ability for hydrocarbon compounds due to their large SBET, high porosity
and high hydrophobicity on the biochar surface.
Sorption mechanisms between biochar and metal elements include ion exchange
between these elements and Na, Ca or K present on the biochar surface, electrostatic
attractions with the available surface functional groups (e.g. carboxyl and hydroxyl)
276 S. Wongrod et al.

Fig. 10.3 Interactions of biochar with positively and negatively charged pollutants

and co-precipitation of metals with oxide groups of biochar and humic substances
(Ahmad et al. 2014; Inyang et al. 2016; Zhang et al. 2013).

10.4.2 Environmental Factors Influencing Contaminant

Retention in Soils

Zhang et al. (2013) stated that degradation of organic pollutants is significantly

influenced by hydrolysis reactions, which can be enhanced by the initial solution pH,
contact time, temperature and initial pollutant concentration. In addition, the pres-
ence of mineral fractions and metal oxides on sludge-based biochar surfaces also
affects the sorption efficiency of organic pollutants.
The initial solution pH plays an important role in the sorption interactions
between biochar and pollutants. The adsorption capacity of methylene blue on
municipal sewage sludge-derived biochar was enhanced when the pH was increased
from 3 to 8 (Fan et al. 2016). This indicates that alkaline conditions (at high pH
conditions) favored the sorption of methyl blue on the biochar via electrostatic
interactions. Similarly, Oh and Seo (2016) reported significant increases of the
sorption efficiency of biosolid-derived biochar for halogenated phenols
(e.g. triclosan) or pharmaceuticals (e.g. ibuprofen) when the pH was adjusted from
4 to 7. In addition, they found that the sorption of halogenated phenols is clearly
related to the chemical and physical properties of the biochar (i.e. carbon content and
specific surface area) and properties of the halogenated phenols (i.e. hydrophobicity)
(Oh and Seo 2016).
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 277

The adsorption and partitioning of organic pollutants in soils are also affected by
the clay minerals and the organic carbon content present in the native soil (Ahmad
et al. 2014; Zhang et al. 2015). Zielińska and Oleszczuk (2016) found that the
presence of dissolved organic carbon (DOC) and clay minerals in the soil play
also a role in reducing the sorption ability of phenanthrene and pyrene by sewage
sludge-derived biochar. Zhang et al. (2015) investigated that with the same biochar
content, a low organic carbon content in the soil exhibited a 10 times higher
adsorption coefficient (Kf) than soil with a high organic carbon content, and
98 times higher compared to the control soil. This shows that biochar enhances
more the sorption ability of soils with a low organic carbon content.
Table 10.4 shows the sorption characteristics of organic waste digestate-derived
biochar for organic and inorganic contaminants. With different composition in the
feedstock, biochar can behave differently in the sorption interaction (Table 10.4).
The mobility of Cu is significantly affected by the organic carbon content in biochar
(Beesley et al. 2010). Yang et al. (2016) studied the effect of coexisting Al on P
sorption in acidic polluted soils upon the addition of biochar made from sewage
sludge. Al was shown to reduce the on the biochar via pH buffering against biochar
alkalinity (Yang et al. 2016). They also reported the enhancement of Pb-K
co-precipitation at lower pH and coexisting Al, whilst the co-precipitation of Pb-P
on sewage sludge biochar was the dominant Pb sorption mechanism at higher pH
(Yang et al. 2016).

10.4.3 Effect of Production Temperature on Biochar

Properties

By varying the production temperatures, unequal effects on physicochemical prop-

erties of organic sludge-based biochar have been observed (Pituello et al. 2014).
Generally, the pH value increases along higher pyrolysis temperatures due to the
volatilization of some acidic functional groups during the pyrolysis (Laghari et al.
2016). The pH of digested organic waste biochar ranges from 7 to 11 in most cases.
Pituello et al. (2014) found almost neutral pH on both municipal organic waste and
sewage sludge digestate-derived biochars at production temperatures between
250 and 550  C, whereas Zielińska et al. (2015) reported a higher pH of around
11–12 on sewage sludge biochar at pyrolysis temperatures from 600 to 700  C
(Table 10.2). In addition, biochars produced at higher temperatures possess higher
specific surface areas, while biochar yields decrease along higher operation temper-
atures (Tables 10.2 and 10.3). Kim et al. (2013) reported large increases in pH and
BET surface area (SBET) at pyrolysis temperatures from 400 to 600  C, leading to an
enhancement of cadmium (Cd) sorption onto biochar. However, opposite results
have been observed by Ding et al. (2014) as the sorption of lead (Pb) was reduced
from 21 to 6.1 mg/g when the pyrolysis temperature increased from 250 to 600  C.
 278

Table 10.4 Sorption characteristics for organic and inorganic contaminants on biochar produced from organic waste digestate
Isotherm/
CRa, kinetic Adsorption
Biochar feedstock Pollutant pH contact time models capacity Main mechanisms Reference
Municipal solid As(V) 6.0 5–400 mg/ Langmuir/ 24.49 mg/g Surface complexation with O-contained Jin et al.
waste digestate L, 30 h PSOb functional groups (e.g. carboxylic and phe- (2014)
(500  C) nolic); physical sorption via surface area and
Municipal solid As(V) 6.0 5–400 mg/ Langmuir/ 30.98 mg/ pore volume of biochar
waste digestate + L, 30 h PSO g
KOH
(500  C)
Municipal organic As(V) 6.0–7.7 90 μg/L, Freundlich/ 3.54 μg/g Adsorption of As(V) with mineral fractions Agrafioti
waste digestate 72 h PSO of biochar (i.e. CaO, Fe2O3 and Al2O3); et al.
(300  C) Cr(III) 7.0–9.5 170 μg/L, Freundlich/ 42.37 μg/g electrostatic interactions between negative (2014)
72 h PSO surface charge of biochar and Cr(III) cations
Sewage sludge As(V) 6.0–7.7 90 μg/L, Freundlich/ 4.25 μg/g
(300  C) 72 h PSO
Cr(III) 7.0–9.5 170 μg/L, Freundlich/ 30.12 μg/g
72 h PSO
Waste-art-paper Pb 5.0 20–400 mg/ Langmuir/ 51.2 mg/g Precipitation of Pb with carbonate to form Xu et al.
digestate (300  C) L, 24 h PSO PbCO3 and Pb2(OH)2CO3 on biochar (2017)
surfaces
Municipal sewage Methylene blue 2.0–11.0 50–150 mg/ Langmuir/ 12.58 mg/g Endothermic adsorption; electrostatic inter- Fan et al.
sludge + tea waste L, 24 h PSO action; ion exchange; surface complexation (2016)
(300  C)
Paper mill sludge Tricaine 7.0–7.5 10–500 mg/ Freundlich/ 87.4 mg/g The p–p interactions between aromatic Ferreira
(800  C) methanesulfonate L, 500 min PSO structure of tetracycline and graphite-like et al.
sheets of biochar (2017)
a
CR refers to concentration range
b
PSO refers to pseudo-second-order
S. Wongrod et al.
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 279

This can be attributed to the loss of oxygen-containing functional groups at higher

pyrolysis temperatures.
During pyrolysis, the organic materials degrade to volatile matter, resulting in
mass reduction and shrinkage when converted into biochars (Rodriguez 2010). In
addition, lower volatilization rates of the C content than the O, N and H contents
were observed at higher operating temperatures, leading to a decrease in the H/C,
O/C and (O+N)/C ratios. The H/C ratio represents the degree of carbonization of
biochars as H is primarily associated with organic substances within biomass: the
lower H/C ratio results in a higher carbonized biochar (Tan et al. 2015). The lower
O/C ratio of biochar demonstrates the higher aromaticity and hydrophobicity on its
surface, whereas the reduction of biochar polarity is affected by a low (O+N)/C ratio
due to a decrease of polar functional groups at higher pyrolysis temperatures (Chen
and Zhou 2008; Chun et al. 2004).
The structural modifications of organic sludge-based biochars start from 350 to
550  C due to the decomposition of macromolecules (i.e. proteins and carbohy-
drates), resulting in the disappearance of amines and carboxyl groups and the
formation of amorphous carbon (Pituello et al. 2014). Moreover, Pituello et al.
(2014) stated that C is rearranged into more complex structures of amorphous and
aromatic rings, leading to a higher biochar stability than its raw feedstock. In
addition, the aromatic C with a variety of functional groups predominates in biochar
produced at 350  C (Pituello et al. 2014), which may induce sorption interactions
between the biochar and target contaminants. Zielińska and Oleszczuk (2016)
reported higher retention of phenanthrene and pyrene in soil amended with sewage
sludge-derived biochar produced at higher pyrolysis temperatures (i.e. from 500 to
700  C). On the other hand, Oh and Seo (2016) found that the adsorption efficiency
of halogenated phenols or pharmaceuticals was not influenced by the pyrolysis
temperature of the biochar.

10.4.4 Remediation of Organic and Inorganic Contaminants

in Soils

10.4.4.1 Remediation of Organic Contaminants

In recent studies, biochar produced from digested organic sludge has been recog-
nized as a potential tool for the sorption of organic pollutants in soils (Khan et al.
2013; Waqas et al. 2014; Zielińska and Oleszczuk 2015). The presence of micro-
pores and mesopores on the biochar surface as well as aromatic structures and
functional groups help to retain toxic contaminants like PAHs, atrazine and
sulfamethazine in soils (Cao et al. 2011; Rajapaksha et al. 2014; Vithanage et al.
2014). Soil amended with biochar reduced the dissolved concentrations of phenan-
threne and pyrene compared to soils without biochar addition (Zielińska and
Oleszczuk 2016). Addition of 5% w/w sewage sludge-derived biochar to soil
enhanced the sorption capacity of phenanthrene from 8.3 to 20.3% (Zielińska and
280 S. Wongrod et al.

Oleszczuk 2016). Also, the sorption of pyrene in soil amended with sewage sludge-
derived biochar was increased from 14.5 to 31.7% with respect to control soil
(Zielińska and Oleszczuk 2016). Mukherjee et al. (2016) investigated the potential
adsorption-desorption of pesticides (i.e. bentazone, boscalid and pyrimethanil) using
digestates from a biogas plant (i.e. maize silage, manures and urine) in a loamy sand
soil. The partition coefficient (Koc) values of the pesticide boscalid were approxi-
mately 5 times higher in soil mixed with 30% digestate and 5% biochar compared to
the control soil.
Application of sewage sludge and its derived biochar to soils significantly
decreased the available PAHs concentration (Waqas et al. 2014). Also, supplemen-
tation of sludge and sludge-based biochar decreased the bioaccumulation of PAHs
into Cucumis sativa L. by 44–57% with biochar amendment and 20–36% with
sewage sludge amendment (Waqas et al. 2014). These results show that biochar
promotes a higher efficiency in the immobilization of PAHs than the raw sewage
sludge digestate during soil remediation. However, a higher uptake rate of
terbuthylazine in biochar amended in low organic carbon soil was found compared
to soil with a high organic carbon content. This suggests that the presence of
dissolved organic carbon in soil may block the biochar pores, resulting in lower
accessibility of pesticides to biochar (Ahmad et al. 2014).

10.4.4.2 Remediation of Inorganic Contaminants

In situ treatment of metals by addition of alkaline chemicals (i.e. lime) or natural

organic waste materials as soil amendments has been noticed to stabilize metal
elements in soils (Gadepalle et al. 2007; Kumpiene 2010). Biochar can be used as
a tool to immobilize metals in contaminated soils through several interactions as
illustrated in Fig. 10.3. Extensive studies on biochar applications as carbonaceous
materials for the stabilization of inorganic pollutants in soil have been performed
(Ahmad et al. 2014; Mohan et al. 2014; Zhang et al. 2013).
Table 10.5 shows the application of organic waste digestate-derived biochar for
organic and inorganic contaminant remediation. From Table 10.5, Méndez et al.
(2012) showed that the application of sewage sludge-derived biochar increases the
environmental safety of soils. Lower metal leaching of Cu, Ni and Zn from biochar-
soil amendment than soil mixed with raw sewage sludge digestate occurred after
200 days of incubation (Méndez et al. 2012). In addition, sewage sludge-derived
biochar has also been reported to increase soil respiration with respect to the control
soil (Méndez et al. 2012). Conversely, Khan et al. (2013) showed that sewage
sludge-derived biochars facilitate adsorption of As, Cr and Pb in soils. However,
Cu, Zn and Cd tended to mobilize due to higher availability of these elements in
biochars (Table 10.3). Thus, the supplement of sludge-based biochar to soils should
be verified before field applications.
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 281

Table 10.5 Application of organic waste digestate-derived biochar for organic and inorganic
contaminant remediation
Pyrolysis
Inorganic Biochar temperature
contaminant feedstock ( C) Effect Reference
As, Cd, Cr, Co, SSD 500–550 Immobilization of As, Cr, Co, Ni Khan et al.
Cu, Ni, Pb and and Pb (rise in pH) but mobiliza- (2013)
Zn tion of Cu, Zn, and Cd in soils
Cu, Cd, Ni, Zn SSD 500 Biochar increased soil respiration Méndez
and Pb compared to control soil but lower et al.
rate than soil with SSD. Lower (2012)
leaching of Cu, Ni and Zn in soil-
biochar amendment than in soil
amended with SSD
Cu, Pb, Zn, Cd SSD 450 Biochar enhanced soil fertility and Liu et al.
and Cr crop productions. Reduction of (2014)
metals availability for plant which
decrease the toxicity in contami-
nated soil
As, Cd, Cu, Pb SSD 550 Soil + SSD biochar: significantly Waqas
and Zn decreases of As, Cu and Pb con- et al.
centrations (2014)
Soil + SSD amendment: increases
in metal concentrations
Phenanthrene SSD 500 and 700 Application of 5% w/w biochar Zielińska
(PHE) and enhanced 2.4- and 2.2-times and
pyrene (PYR) higher sorption capacity for PHE Oleszczuk
and PYR, respectively in soils. (2016)
The sorption increased with
biochar produced at higher tem-
perature. DOC and clay minerals
present in the soils reduced the
efficiency of PHE and PYR sorp-
tion by biochar
Polycyclic aro- SSD 550 PAH concentrations in biochar Waqas
matic hydrocar- significantly decreased after et al.
bons (PAHs) pyrolysis. Biochar addition to soil (2014)
reduced availability and
bioaccumulation of PAHs into
Cucumis sativa L.
Halogenated OFMSWD 400 The sorption efficiency of biochar Oh and
phenols effectively increased at pH 4 or Seo (2016)
7. Surface area and carbon content
of biochar and hydrophobicity of
halogenated phenols mainly affect
the phenol sorption
Pharmaceuticals OFMSWD 400 pH significantly affected the Oh and
(i.e. ibuprofen) sorption capacities of biochar for Seo (2016)
ibuprofen due to the point of zero
charge (PZC) of sorbents and
deprotonation of the
pharmaceutical
282 S. Wongrod et al.

10.4.5 Bioavailability of Trace Elements with Biochar

Addition

The mobility and fate of metals in contaminated soil amended with biochar have
been widely studied (Beesley et al. 2014; Houben et al. 2013; Kargar et al. 2015;
Nartey and Zhao 2014). The effects on metal bioavailability in soils vary depending
on the nature of the biochar feedstock and type of metal (Zhang et al. 2013).
Addition of organic wastes as soil amendments to contaminated soils can increase
the bioavailability of metal(loid)s (Nartey and Zhao 2014). Tampio et al. (2016)
reported that plants had a higher metal contents and lower N bioavailability upon
amendment of a soil with a mixture of vegetable waste digestate and activated-
sludge. However, addition of biogas digestate from food waste and the organic
fraction of municipal solid waste to the soil showed positive outcomes as they
provided more bioavailable nutrients in the soil (Tampio et al. 2016).
Biochar produced from municipal wastewater treatment sludge contains mineral
oxides and a carbonized organic fraction which serves as a good sorbent for metals
(Zhang et al. 2016). The precipitation of metal (Pb and Zn) phosphate and more
available sorption sites were observed after aging of biochar for 30 days, ensuring
durability of metal immobilization in sludge-derived biochar during soil amendment
(Zhang et al. 2016). Campos and De la Rosa (2020) reported that Cu and Pb were
effectively immobilized in soils amended with biochar, which affirmed the promis-
ing potential of biochar to recover land polluted with heavy metals. It is important to
understand that one type of biochar cannot be representatively used as sorbent to
remediate all types of metals. Hence, the use of solid digestate-derived biochar in
soils should be verified for each particular biochar type to ensure the environmental
safety for field application.

10.5 Potential Effects of the Application of Sludge-Derived

Biochars to Soils

10.5.1 Stability and Durability Effect of Biochars in Soils

It is important to understand the stability of biochar in a soil environment to quantify

its persistence in soils and to be able to manage its fate in the environment in a long
term, e.g. for soil nutrient supplementation and enhancement of the water holding
capacity. Biochar can be degraded by biotic (e.g. microbial incorporation or oxida-
tion in carbon respiration) and abiotic (e.g. chemical oxidation or solubilization)
processes (Kuppusamy et al. 2016). The degradation of digestate derived biochar is
influenced by the available functional groups on the biochar surface and the aroma-
ticity of the adsorbent (Kuppusamy et al. 2016).
The molar ratio of oxygen to carbon (O:C) indicates the stability of biochar in
which black carbon is present (Spokas 2010). The stability of biochar increases with
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 283

lower O:C ratio and biochar with an O:C ratio lower than 0.2 shows a half-life of
more than a thousand year, whilst a reduction of the biochar half-life to less than a
hundred year has been observed with an O:C ratio higher than 0.6 (Kuppusamy et al.
2016). Also, the pyrolysis temperature and feedstock source are two main parame-
ters affecting the O and C content of a biochar (Spokas 2010). The O:C ratio is likely
to provide a strong indicator of biochar stability in soil rather than solely the
operation conditions or feedstock type (Spokas 2010). In addition, the biochar
persistence in soil can be predicted from the fraction of aromatic rings and
non-aromatic compounds present in the biochar (Singh et al. 2012). Hernandez-
Soriano et al. (2016) observed that addition of biochar to loamy soil slowed down the
soil respiration rate compared to soils amended with raw organic residues during
237 days of incubation. These results showed that the carbon metabolism in soil
amended with biochar enhanced the carbon stability in the soil (Hernandez-Soriano
et al. 2016). After 4 years of soil-biochar incubation in a field experiment, a
significant increase of the C:N ratio of the microbial biomass was found in
biochar-amended soil (Zhang et al. 2014). Additionally, they suggested that biochar
can reduce the variability of the environmental conditions for microorganisms and
thus decrease the temporary fluctuations in C and N dynamics.
Apart from the biochar applications as soil amendments, the use of biochar as
active sorbent to retain contaminants in soils in a long term has also been reported.
Liu et al. (2019) found that after 240 days of sorption equilibration of lincomycin
(an antibiotic) by biochar, about 75% of sorbed lincomycin on the biochars could not
be extracted anymore with acetonitrile/methanol extractant. This suggests that
biochar has a great potential to retain antibiotics in a long term. Moreover, Stefaniuk
et al. (2017) found a significant decrease in the PAHs concentrations after a long-
term incubation of sewage sludge and/or biochar in the soil (1.5 years), which were
reduced by 19% upon the addition of sewage sludge and by 35% with the addition
(5.0%) of sewage sludge and biochar. He et al. (2019) found that a soil incubation
with sewage sludge derived biochar can immobilize Pb and Cu in a long term
(2 years).

10.5.2 Biological Assessment

Table 10.6 shows possible effects of sludge-derived biochar addition to soils.

The possible effects of soil biota upon addition of sludge-derived biochar to con-
taminated soils can be determined in batch sorption tests and soil incubations. In
general, the biological assessment is performed to illustrate the biochar efficiency to
reduce the contaminant levels using plants, earthworms or bacteria as test organisms
because these are primary producers and consumers in the food chain and can
transfer pollutants contained in fatty tissues to predators or humans.
Generally, contaminated land sites are poorly fertilized soils which have little or
no cover of the vegetation on the top soil (Beesley et al. 2011). Application of
biochar on these polluted sites can reduce the concentration of pollutants in the soil
284 S. Wongrod et al.

Table 10.6 Possible effects of the application of sludge-derived biochar to soils

Effect
Material Dosage/soil type Positive Adverse Reference
Sewage sludge 1:0.3 w/w (21 kg Increased numbers of Increased bio- Malińska
(SS) and its SS + 6 kg woodchips) cocoons by 213% after availability of et al.
derived + 8% BSS / SS and 4 weeks, and earth- Cr to E. fetida (2017)
biochar sew- BSS worms by 11 times
age sludge before composting and
(BSS) 5 times after
composting. Reduced
bioavailability of Cd
and Zn to E. fetida
Wastewater 0.907 kg/m2 / Agri- Increased cherry Not reported Hossain
sludge biochar cultural soils tomatoes yield by 64% et al.
(chromosol) and increased P and N (2010)
available in soils
Metal bioavailable in
biochar below the
Australian limit level
for food
Sewage sludge 1.36 kg/m2 (7.5 t/ Increased maize Not reported Gwenzi
(SS) and its ha SS + 0.68 kg/m2 growth and total bio- et al.
derived BSS) / Tropical clay mass by 49% and (2016)
biochar (BSS) soil 329%, respectively.
Decreased Pb, Cu and
Zn concentrations in
maize by 22% com-
pared to SS treatment
alone
Sewage sludge 3% w/w / Tropical Increased fungi to Unchanged Paz-
biochar, soils (Acrisol and bacteria ratio in soils ratio of fungi Ferreiro
Miscanthus Ferralsol) planted with amended with sewage to bacteria in et al.
and pine wood proso millet (Panicum sludge biochar soils treated (2015)
biochar miliaceum L.) with biochar

pore water (Beesley et al. 2011). Biochar added in agricultural soils can provide
positive impacts on plant growth such as maize (Agegnehu et al. 2016; Gwenzi et al.
2016) due to the liming effect and a longer nutrient retention time in the soils (Sohi
et al. 2010). Conversely, adverse effects on plant growth were found by van Zwieten
et al. (2010) since biochar-soil amendment induced reduction of biomass production
by wheat and radish.
Earthworms serve as ecosystem engineers as they play important roles in soils,
i.e. they accelerate organic matter degradation, increase nutrient availability to plants
and nutrient recycling in ecological systems (Beesley et al. 2011). Therefore,
earthworms are considered as vital soil animals for which a reduction of contami-
nants becomes significant to avoid an over-uptake (Sizmur et al. 2015). From
Table 10.6, addition of sewage sludge-derived biochar in a mixture of sewage sludge
and wood chips integrated with vermicomposting showed 11- and 5-times higher
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 285

amounts of juvenile earthworms, respectively, before and after composting

(Malińska et al. 2017). Also, the concentrations of Cd and Zn bioavailable to
E. fetida was reduced with biochar application in the pre-composting of sewage
sludge (Malińska et al. 2017). However, increased Cr concentrations were observed
during biochar-vermicompost treatment (Malińska et al. 2017).

10.5.3 Agronomic Benefits of Organic Waste

Digestate-Derived Biochars

Soil quality deterioration is a worldwide problem and has been particularly observed
in agricultural areas. Addition of organic amendments to soils may be beneficial to
restore the soil quality. Addition of labile organic materials to soil can provide
bioavailable nutrients for crops and soil microbiota like microbes and fungi. How-
ever, a low nitrogen content in the organic substrates can limit the plant growth (Sohi
et al. 2010). Supplementation of alkaline biochar to acidic soils can enhance crop
yields through increasing soil pH and reducing acidity constraints which inhibit the
plant productivity (Ahmad et al. 2014). The efficiency of biochar amendment
depends on the biochar buffering capacity that varies with the nature of feedstock
and the pyrolytic temperature (Ahmad et al. 2014; Tables 10.2 and 10.3).
The use of biochar, compost and their combination in a tropical agricultural soil
has been studied: significant increases in total N, available P, nitrate nitrogen and
ammonium nitrogen in biochar-compost-soil systems compared to the control were
found (Agegnehu et al. 2016). Besides, biochar mixed with compost increased crop
biomass by 10–29% and helped to reduce N2O emissions compared to other
treatments (Agegnehu et al. 2016). Gwenzi et al. (2016) reported that with the
application of sewage sludge mixed with its derived biochar (15 or 7.5 t/ha of
each organic amendment) on tropical clay soil showed a 47% enhancement of
maize growth and was comparable to inorganic fertilizer (49%). Additionally,
adding biochar and sewage sludge in the tropical clay soil reduced Pb, Cu and Zn
uptake by the maize plants by 22% with respect to the presence of sludge alone
during the treatment (Gwenzi et al. 2016).
Supplementation of 12% biochar significantly reduced total nitrogen losses and
GHGs emissions, i.e. CH4, N2O and NH3, respectively by 92.85, 95.14 and 58.03%
during sewage sludge composting (Awasthi et al. 2016). However, low dosage
(2–6%) of biochar added to compost released more CH4 and N2O than the control
treatment (Awasthi et al. 2016). In addition, the humic (42%) and fulvic (28%) acid
concentrations increased during biochar-compost treatment with 12% biochar addi-
tion (Awasthi et al. 2016). van Zwieten et al. (2010) applied paper mill waste sludge-
derived biochar in two different agricultural soils, i.e. a ferrosol (high agricultural
potential) and calcarosol (low-moderate agricultural potential) in a greenhouse
study. The results showed high N uptake rates by wheat crops in biochar-ferrosol
amended soil and 250% increases in biomass production of soybean and radish
286 S. Wongrod et al.

compared to the control (van Zwieten et al. 2010). However, the calcarosol showed
varied results, i.e. increases in soybean biomass but decreases of wheat and radish
biomass (van Zwieten et al. 2010).

10.5.4 Possible Adverse Effects

Biochar application to soil can alter microbial activities since the degradation of soil
organic carbon provides energy for microbes, with addition of hardly degradable
carbon (i.e. biochar) to the soil. Thus, possible adverse effects on the microbial
community in soil can occur, generally depending on the types of organic residues as
feedstock, biochar dosage and time interval of its application (Sohi et al. 2010).
Weyers and Spokas (2011) reported a reduction of earthworm biomass when organic
sludge-derived biochars were added to soils.
It is also important to consider priming effects when biochar is applied to soil.
The priming effect is a phenomenon occurring when higher rates of soil organic
carbon decomposition take place corresponding to more organic materials
supplemented to the soil. This enhances the activity of the microbial population
due to the release of energy during the decomposition of organic substrates, which
accelerates the mineralization of soil organic carbon (Fontaine et al. 2003). Positive
priming effects were observed by faster decomposition rates of soil carbon when
amended with biochar, resulting in microbial activity enhancement and more hydro-
lysis reactions, which effectively increase with higher soil pH (Ahmad et al. 2014;
Kuzyakov et al. 2009). In contrast, a negative priming effect can occur when biochar
adsorbs dissolved organic carbon in soils, which lowers the decomposition rates and
subsequently reduces the microbial population.

10.6 Future Challenge

Short-term experiments (less than a few years) confirm the potential use of organic
sludge biochars as soil amendments and sorbing materials in the environment.
However, based on pot experiments in small scale, more studies on field sites are
necessary to confirm if similar outcomes are achieved. Also, long-term studies of
sludge biochar application in soils are lacking and yet to be further developed. The
studies on the longevity of microbial activities in soil-biochar systems and the
durability effect of organic sludge biochar in the long-term should be further
achieved as well as the regeneration of aged biochar.

Acknowledgements This chapter emanated from the PhD thesis entitled “Biochars from solid
digestates as sorbing materials for metal(loid)s removal from water” by Suchanya Wongrod. The
authors are thankful to the financial support by the Marie Sklodowska-Curie European Joint
10 Biochar Produced from Organic Waste Digestate and Its Potential. . . 287

Doctorate in Advanced Biological Waste-to-Energy Technologies (ABWET) under the European

Union’s Horizon 2020 framework (grant agreement N 643071).

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 Part V
Integration of AD in Biorefineries
Chapter 11
Integration of Bio-electrochemical Systems
with Anaerobic Digestion

M. M. Ghangrekar, S. M. Sathe, and C. N. Khuman

Abstract Anaerobic digestion is a well versed technology for treating organic waste
and converting the organic matter into biogas to be subsequently used as fuel or
converted into electricity. Over the years, anaerobic digestion has been widely used
for the treatment of industrial and domestic wastewaters as well as organic solid
waste, while simultaneously generating biogas. On the other hand,
bio-electrochemical systems (BES) are one of the prominent and upcoming ways
of harnessing organic matter into bioelectricity and industrially valuable products,
such as hydrogen, methane, biofuels, and hydrogen peroxide. This chapter reviews
the hybridization of BES and anaerobic digestion for enhanced pollutant removal
and pollutant monitoring in the form of nutrient removal and recovery, polishing of
anaerobically treated effluent, biogas upgrading, application of biosensors, and
merger of BES in anaerobic digestion for emerging contaminant removal. Addition-
ally, a brief discussion on the future prospect of the synergy of these two technol-
ogies has also been highlighted.

Keywords Anaerobic digestion · Bio-electrochemical systems · Bioenergy ·

Microbial fuel cell · Resource recovery

M. M. Ghangrekar (*)
Department of Civil Engineering, Indian Institute of Technology Kharagpur, Kharagpur, West
Bengal, India
School of Environmental Science and Engineering, Indian Institute of Technology Kharagpur,
Kharagpur, West Bengal, India
e-mail: [email protected]
S. M. Sathe
Department of Civil Engineering, Indian Institute of Technology Kharagpur, Kharagpur, West
Bengal, India
C. N. Khuman
School of Environmental Science and Engineering, Indian Institute of Technology Kharagpur,
Kharagpur, West Bengal, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 295
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_11
296 M. M. Ghangrekar et al.

11.1 Introduction

The urge of finding alternative and sustainable energy sources to replace ever-
depleting fossil fuels is a major global issue for sustainable development. The use
of fossil fuels is not environmentally friendly due to the emission of harmful gases
into the atmosphere. Therefore, the use of renewable energy sources such as solar,
wind, geothermal, and tidal are becoming increasingly popular now-a-days (Jingura
and Matengaifa 2009). Organic waste or biomass is also one of the promising
renewable energy sources, which is considered as an environmentally friendly
approach (Berndes et al. 2003; Lin et al. 2018; Stephen and Periyasamy 2018). It
is estimated that 1 kg of carbohydrates represents 1.06 kg of chemical oxygen
demand (COD), equivalent to 4.41 kWh of energy (Rabaey and Verstraete 2005).
Anaerobic digestion (AD) is one of the efficient and proven methods for the
treatment of organic matter present in solid waste and wastewater. It is specifically
preferred for the treatment of high strength waste in terms of COD due to its low
sludge generation, low energy requirement, and formation of valuable methane as
end product (Chen et al. 2008; Kiyasudeen et al. 2016). The AD of organic matter
takes place via hydrolysis, acidogenesis, acetogenesis, and methanogenesis,
resulting in the generation of biogas, which comprises of CH4 and CO2 and other
gases such as H2S, H2 and N2 (Gujer and Zehnder 1983; Zhang et al. 2014a).
Methane generated from the process can be either used as fuel or converted into
electricity. AD is a complex process involving a plethora of microorganisms, in
which the end-products of the initial bacterial reaction are often the substrate for
subsequent bioconversion. Hence, a balance between the microbial population and
the substrate concentration is vital for the stable operation of AD. In the initial three
stages (hydrolysis, acidogenesis, and acetogenesis), the organic matter is broken
down into smaller molecules via the formation of long chain volatile fatty acids
(VFAs) to finally short chain VFA and acetic acid; whereas, in the final stage
(methanogenesis), methane is produced by the methanogens (acetoclastic and
hydrogenotropic) (Gavala et al. 2003; Gude 2018).
The process of AD is vastly affected by variation in temperature, substrate pH,
hydraulic retention time (HRT), organic loading rate (OLR), and C/N ratio (Mao
et al. 2015). Apart from this, the failure of AD is often linked to toxic effects induced
by inhibitory substances. The presence of ammonia, sulfur, heavy metals, other ions
(Na+, K+, Mg2+, Ca2+, Al3+), and organic compounds (such as alkyl benzenes,
chlorophenols, surfactants, and alkanes) beyond the threshold limit can cause an
imbalance in the AD (Chen et al. 2008). Any imbalance in the process can be related
to reduced CH4 generation and deteriorated organic matter removal efficiency (Chen
et al. 2008). This necessitates the requirement of pre-treatments to make the waste fit
for treatment in AD.
Bio-electrochemical systems (BES) are a new generation of wastewater treatment
techniques capable of recovery of valuables and/or bioenergy production. In the
anodic and cathodic chambers of a BES, respectively oxidation and reduction takes
place. The organic matter from the waste is oxidized in the anodic chamber using
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 297

microorganisms; whereas, oxygen is reduced at the cathode to generate H2O or H2O2

(Logan et al. 2006; Fu et al. 2010). The BES in various forms is capable of removing
the nutrients and other organic compounds, which cannot be removed in AD. For the
industrial wastewaters that contain a high concentration of the aforementioned
pollutants, BES can be used as a pre-treatment prior to AD to reduce the concentra-
tion of pollutants, while for domestic wastewater BES can be used as a post
treatment to AD (Vrieze et al. 2018). Although, BES have been successfully applied
in lab scale investigations, upscaling a single reactor volume for field scale operation
is not a feasible option owing to a high mass transfer loss and possible scavenging
action by the anaerobic anodic microorganisms. Hence, from the view of upscaling,
stacking of multiple BES can be considered as a way forward.

11.2 Bio-electrochemical Systems

During AD, microorganisms decompose the organic matter into simpler molecules
with the release of energy in the form of electrons and protons. This energy can be
harvested using a novel technology known as BES. Compared to AD, the BES is a
recent technology that is capable of converting dissolved organic matter from
wastewater into bioelectricity or bioenergy. Though the first instance of electricity
generation using microorganisms was demonstrated by Potter in 1911 (Potter 1911),
the subsequent in depth research and widespread applications were initiated only in
the late twentieth century. Owing to the capability of one-step bioelectricity produc-
tion, BES become an attractive option for the conversion of biomass/organic matter
to electrical energy or by applying small external potential other value-added
products can be recovered.
A BES is an electrochemical system catalysed by the action of microorganisms
for the treatment of polluted soil and wastewater, while harvesting bioenergy or
synthesizing value-added products such as biofuels, H2O2, H2 and CH4. Addition-
ally, they have also been applied for the sequestration of CO2 with the concurrent
production of biochemical and biofuels in microbial electrosynthesis cells (Liu et al.
2005; Jeremiasse et al. 2010; Das et al. 2020). Majorly, BES may be galvanic as in
the case of microbial fuel cells (MFCs), which allow the conversion of chemical
energy of organic matter into electricity or electrolytic as in the case of microbial
electrolytic cells (MECs), where electricity is externally supplied to the system to
facilitate recovery of industrially valuable products (biohydrogen, biomethane, and
hydrogen peroxide).
In a nutshell, BES consists of a single pair of electrodes or multiple pairs of
electrodes, an ion exchange membrane (may not be necessary for a membrane-less
BES configuration), an external load (in case of MFC), or an external power supply
(in case of MEC), and microorganisms especially electrogens (Logan et al. 2006).
The electrogens are capable of electron transfer to the electrodes from their cell
(known as exoelectrogens) and from the electrodes into their cell (known as
endoelectrogens or electrotrophs) (Semenec and Franks 2015; Kang et al. 2021).
298 M. M. Ghangrekar et al.

11.2.1 Microbial Fuel Cell (or Galvanic Bioelectrochemical

System)

In a typical double chamber MFC, the anode is dipped inside wastewater, while the
cathode is placed in air-saturated water (aqueous cathode) or exposed to air (air
cathode). The anode and the cathode are connected to an external load. The anolyte
(wastewater) and the catholyte (air-saturated water or air) are separated by a proton
exchange membrane (PEM) or cation exchange membrane (Fig. 11.1a). Oxidation of
the organic matter occurs in the anodic chamber under anaerobic conditions using
microorganisms, thereby releasing electrons, protons, and CO2. The presence of
terminal electron acceptors (generally oxygen) in the catholyte attracts the electrons
towards the cathodic compartment. However, the PEM allows only the passage of
protons and prohibits the flow of electrons and organic matter towards the cathode.
The electrons flow towards the cathode through the external circuit and create a
charge imbalance between the anolyte and the catholyte. To maintain charge equi-
librium the protons flow through the PEM towards the cathode to combine with O2
and electrons forming H2O or H2O2 (Eqs. 11.1 and 11.2). Thus, electricity is
harvested due to the flow of electrons through the external circuit (Kim et al.
2004; Liu et al. 2005).

O2 þ 4Hþ þ 4e ! 2H2 O ðE0 ¼ 0:805 V, vs:standard hydrogen electrode, SHE at pH of 7Þ

ð11:1Þ
þ  0
O2 þ 2H þ 2e ! H2 O2 ðE ¼ 0:328 V, vs:SHE at pH of 7Þ ð11:2Þ

Power supply
e− External Load e− e− e−
a b

−
Substrate e− Substrate e e−
Separator

e−
Separator

O2
H2
Anode
Anode

H2O
Cathode
Cathode

H+ H+ H+ H+
CO2 CO2

Microorganisms Microorganisms
Anodic chamber Cathodic chamber Anodic chamber Cathodic chamber

Fig. 11.1 (a) Typical dual-chambered microbial fuel cell and (b) dual-chambered microbial
electrolysis cell
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 299

11.2.2 Microbial Electrolysis Cell (or Electrolytic

Bioelectrochemical System)

In contrast to MFC, electrical power is externally supplied into the electrolytic BES
(Fig. 11.1b). At the anode, oxidation of organic matter catalysed by microorganisms
and water hydrolysis takes place. The electrons flow toward the cathode through the
external power circuit, while the protons migrate to the cathode through the PEM,
similar to that of MFC. The MECs are capable of producing industrial valuable
products, such as H2, H2O2 and organic acids, in cathodic chamber along with
simultaneous removal of organic matter from wastewater in anodic chamber
(Jeremiasse et al. 2010; Jafary et al. 2015). However, the energy required for
bioconversion of organic matter into industrial valuable products is lesser in MEC
than what is required in conventional electrochemical methods due to the availability
of electrons and protons released during microbial decomposition of organic matter
(Zhang et al. 2014b; Kuntke et al. 2018).

11.3 Integration of Anaerobic Digestion

with Bio-electrochemical Systems

11.3.1 Integrating Bio-electrochemical System

with Anaerobic Digestion for Nutrient Removal

BES have been widely explored for the removal and recovery of nutrients (nitrogen,
phosphorus, sulfur) from the wastewater that cannot be completely removed using
AD. These nutrients have agricultural use and since the recovery of nutrients is
possible from the waste source; the BES perfectly fits into the concept of waste to
wealth.

11.3.1.1 Nitrogen Removal Techniques

The presence of nutrients and common elements is vital for AD and depending on
the particular concentration it can benefit or harm the digestion process. In some of
the high strength waste streams in terms of COD, such as concentrate from waste-
water sludge, food waste, and leachate, the concentration of total ammonia nitrogen
is often considerably higher than that detected in the domestic wastewater. It was
observed that a high concentration of total ammonia causes excessive inhibition of
methanogenesis (Sheng et al. 2013). The digestion process in the anaerobic digester
is most commonly disturbed by the sudden high ammoniacal nitrogen concentration
in the influent waste. High ammonia concentrations in the influent cause a consid-
erable increase in the VFA production resulting into reduced methane production
(Borja et al. 1996). With the rapidly increasing VFA, the digester might not have
300 M. M. Ghangrekar et al.

enough buffering capacity to maintain the pH in the working range of 6.8–7.2 that is
required for the methanogens, and can thus lead to the failure of AD. In such cases
(high ammoniacal nitrogen in the influent), instead of removal of ammonia in the
form of N2 gas, its recovery (as fertilizer) can be a beneficial option, considering the
valuable applications of the recovered products (ammonium sulfate, ammonium
bicarbonate) in the agricultural sector.
For such wastes, the application of a specific pre-treatment technique prior to AD,
which is targeted for ammonia removal, might be the best option (Kim et al. 2015a).
Over the years, several techniques for the removal of nitrogen from the wastewater
have been developed and implemented, which include nitrification and denitrifica-
tion, anammox process, chemical precipitation, and ion exchange (Peng and Zhu
2006; Zhang et al. 2009b; Lin et al. 2016; Ma et al. 2016). For the wastes that have
high nitrogen content, recovery instead of removal can be a feasible option to utilize
the nitrogen for beneficial uses. In this regard, BES offers an advantage of nitrogen
recovery from the waste at a lower operating cost compared with conventional
systems (Zamora et al. 2017).
In the cathodic chamber of BES, the formation of hydroxyl results in an increase
in pH. The transfer of NH3 and NH4+ through the cation exchange membrane to the
cathodic chamber of BES is due to diffusion and migration, respectively, wherein
increased pH causes conversion of NH4+ to NH3 in the cathodic chamber (Kim et al.
2015b). In this way, NH4+ ions are removed from the anolyte (waste stream). The
formed NH3 can be successively recovered in the form of ammonium sulfate or
ammonium bicarbonate that can be used as a fertilizer. The ammonium sulfate can be
obtained via passing gaseous NH3 through sulfuric acid; whereas the catholyte can
be further concentrated via the use of a tri-chambered system with cation and anion
exchange membranes to obtain solid ammonium bicarbonate (Nancharaiah et al.
2016; Jermakka et al. 2018). The BES have been used for nitrogen removal and its
successive recovery from various wastewater sources, such as urine (Kuntke et al.
2012), landfill leachate (Qin et al. 2016), effluent of sludge dewatering process
(Wu and Modin 2013), and the liquid fraction of pig slurry (Sotres et al. 2015).
The ammonia recovery depends on the imposed electrical potential and it was
observed that under the applied potential (MEC mode), ammonia recovery is higher.
Though the supplemented external energy increases the operation cost, it is still
lower than electrochemical ammonia recovery methods.
In a scaled-up MEC system operated with urine as anolyte (2.6 L of each
catholyte and anolyte), 31% of the total ammonia nitrogen recovery was achieved
under the applied potential of + 0.5 V (against Ag/AgCl reference). The electrical
energy required for the total ammonia nitrogen recovery in the aforementioned
investigation was 4.9 MJ kg N1 (Zamora et al. 2017). On the other hand, consid-
ering only the running electricity and fossil energy requirements, the conventional
biological nitrification and denitrification process requires about 45 MJ kg N1 of
energy in wastewater treatment plants (Maurer et al. 2003). Though complete
removal of the nitrogen fraction is not possible using BES, it can reduce the nitrogen
concentration so that the effluent can be suitable for AD, without negatively affect-
ing the digestion process.
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 301

In one of the investigations, MFC was implemented along with AD for NH4+
removal. In between the recirculation loop of AD, a MFC was installed with Nafion
as a separator. Substrate with 77–124.4 mg of N g1 of total solids was used as a feed
for anaerobic digester. The MFC resulted in a reduction of the NH4+ concentration
from 654 to 436 mg L1. The addition of a MFC caused an increase in methane yield
from 136 to 173 mL CH4 L1 day1. The reduced NH4+ might have improved the
stability of methanogenic consortia leading to an increased methane yield (Inglesby
and Fisher 2012). This shows that integration of BES with AD could reduce the
nutrient load thus it can be a feasible solution to prevent the failure of anaerobic
digestion.

11.3.1.2 Phosphorus Removal Techniques

Phosphate removal from wastewater is accomplished commonly via chemical pre-

cipitation or biological treatment processes. The use of BES gives an edge over other
biological processes due to the capability of BES to recover phosphate via the
struvite precipitation at the cathode. The struvite precipitation on the cathode is
possible due to a high localized pH near the cathode. Using a MFC, iron sulfate from
dried sludge was reduced to orthophosphate in the cathodic chamber. Stoichiomet-
rically calculated amounts for magnesium chloride and ammonium hydroxide were
externally added to the catholyte, which results in precipitation of struvite at the
cathode (Fischer et al. 2011). In another investigation, an air cathode MFC was
capable of simultaneous wastewater treatment (COD removal of 76–91%) and
70–82% of phosphorus removal while attaining the maximum power density of
1–2.3 W m2. The precipitate on the aqueous side of the cathode was struvite, as
confirmed by X-ray diffraction analysis. The results confirmed the capability of
MFC to simultaneously treat wastewater while attaining the removal and recovery of
phosphorus in the form of struvite (Ichihashi and Hirooka 2012). However, in a
follow-up investigation the same research group observed that the electricity gener-
ation in MFC deteriorated with an increase in struvite precipitation on the cathode.
The reduced electrical output might be attributed to the mass transfer limitation at the
cathode, caused by a layer of struvite precipitates (Hirooka and Ichihashi 2013). A
possible solution for this issue can be the operation of a two-stage system comprising
of a separate chamber for struvite precipitation, in order to prevent the deteriorated
electrical performance of BES in the long run (Jain and He 2018).
In a different investigation, the effluent from an anaerobic digester was used as a
substrate for a MEC with a fluidized cathode electrode for nutrient recovery. With an
applied voltage of 1 V, the total phosphorus concentration was reduced to 13 mg L1
compared with 46 mg L1 in the open circuit condition. The phosphorus removal
was due to struvite precipitation in the cathodic chamber of MEC (Cusick et al.
2014). A BES developed by Zhang et al. (2014b) showed a capability of 83.4% and
52.4% removal of ammonium nitrogen and phosphate, respectively, under the
applied voltage of 0.8 V. The removal was observed to be considerably higher
compared with open circuit conditions, which contributed in 3.6 and 21.1% removal
302 M. M. Ghangrekar et al.

of ammonium nitrogen and phosphate, respectively. This confirmed that the current
generation is important for nutrient removal and higher current due to externally
applied voltage can improve the nutrient removal efficiency in BES (Zhang et al.
2014b).

11.3.1.3 Sulfur Removal Techniques

Many of the industrial wastewaters are the prime source of sulfate. Chemical
precipitation, membrane filtration and biological treatments using sulfate reducing
bacteria are a common way for the removal of sulfate from sulfate laden wastewater.
Although domestic wastewater has a low sulfate concentration, the converted
sulfide remains dissolved in the anaerobically digested effluent that lowers the
overall performance. Hence, a post treatment is also essential to eliminate the sulfide
from the wastewater. Although sulfate in low concentrations does not contribute to
toxicity, a high concentration of sulfate can impart salinization of freshwater bodies
(Lens et al. 1998; Sharma and Kumar 2020). During the AD of wastewater, sulfate is
reduced to sulfide by sulfate reducing bacteria. The generated sulfide can be inhib-
itory to the methane producing bacteria, which reduces the rate of methanogenesis,
and decreases the methane yield by competing with the available carbon and/or
hydrogen (McCartney and Oleszkiewicz 1991). Hence, the sulfate concentration in
the influent wastewater needs to be controlled to prevent the inhibitory effect on
AD. The BES have been considered as among the promising technologies for the
removal of sulfate.
For the removal and recovery of sulfur in MFC, the sulfate reducing bacteria
should work along with electrogens in the anodic chamber. The sulfate present in the
wastewater is reduced to sulfide by sulfate reducing bacteria and it is subsequently
oxidized by sulfide oxidising bacteria to form elemental sulfur (Lee et al. 2014). This
has been demonstrated for the first time by Rabaey et al. (2006) using a MFC while
simultaneously harvesting a power output of 101 mW L1 volume of net anodic
compartment. Granular deposits containing 190 mg of sulfur, corresponding to about
9% of sulfide dose, were observed on the anode. When the same setup of MFC was
coupled with an up-flow anaerobic sludge blanket (UASB) reactor, a total of 98%
sulfide removal was achieved (Rabaey et al. 2006). The coupling of MFC shows that
sulfide can be successfully removed from the anaerobically treated effluent, thus
accomplishing a polishing treatment to the effluent. Another investigation showed
that the performance of a MFC deteriorated when the COD:sulfate ratio was dropped
to less than 0.8. The maximum COD removal efficiency of 79% at a power density of
1136.8 mW m3 was demonstrated using MFC at a COD: sulfate ratio of 0.8
(Ghangrekar et al. 2010).
Chatterjee et al. (2017) used a dual chamber MFC fed with sulfate (200 mg L1)
along with acetate as a carbon source. In the anodic chamber of a MFC, sulfate was
reduced to sulfide, which was observed via an increase in sulfide concentration from
0 to 55 mg L1 at an HRT of 48 h. However, after an additional 24 h, the sulfide
concentration dropped to 3 mg L1. In the absence of oxygen and with reducing
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 303

sulfate concentration, it was concluded that sulfate was reduced to sulfide, which
was subsequently oxidized to elementary sulfur. Energy dispersive X-ray spectros-
copy confirmed the presence of elemental sulfur on the anode with a weight
percentage of 27.45% of the entire surface area (Chatterjee et al. 2017). In the
same investigation, Co3O4 nano-octahedron was used as a cathode catalyst, whereas
a separate MFC without catalyst was operated under the same operating conditions,
to analyse the effect of improved electrical performance and reduced oxygen reduc-
tion reaction overpotential of the sulfide removal. However, it was observed that
except for increased power density and coulombic efficiency, the cathode catalyst
had no significant role in improving the sulfide oxidation (Chatterjee et al. 2017).
In a recent investigation, a tri-chamber MFC comprising of an oxic cathode and
anoxic cathode sharing the same anode was used for nitrogen and sulfate removal.
The system achieved total nitrogen and sulfide removal at rates of 10.0 g N m3 day1
and 206.5 g S m3 day1, respectively (Zhang et al. 2018). The available investi-
gations confirm the efficacy of BES for the removal of sulfate from wastewater,
which can be coupled with AD to aid in the overall treatment process. In the
conventional aerobic biological processes, sulfide is oxidized to sulfate; however,
in the secondary clarifier due to the oxygen depleting environment, the sulfate can
again be reduced to sulfide. On the other hand, using MFC, sulfide is converted to
elemental sulfur, thus offering a better way of addressing sulfide removal (Chatterjee
et al. 2017).
The use of BES in MFC or MEC mode had demonstrated promising results in
terms of removal and recovery of nutrients from the wastewater. The inability of AD
to eliminate nutrients creates need for coupling AD with suitable nutrient removal
techniques. Hence, integration of BES with AD can be a low-cost solution, which
can not only improve the performance of AD, but also be an effective way of nutrient
recovery.

11.3.2 Biogas Upgrading

The biogas produced from AD mainly consists of CH4 (55–75%) and CO2
(30–45%). Besides these, other gases such as H2S, N2, H2 along with H2O (moisture)
can also be present in the biogas (Igoni et al. 2008). However, CH4 is the only
component of interest in biogas as a source of fuel. The presence of CO2, H2S, and
H2O leads to a lower calorific value of biogas. Hence, biogas needs to be processed
before utilising it as a fuel source. Biogas processing is done in two broad processes,
viz. biogas cleaning (removal of impurities such as H2O and H2S) and biogas
upgrading (removal or transformation of CO2 into CH4 to increase the CH4 content
in the biogas) (Angelidaki et al. 2018; Fu et al. 2020).
Biogas upgrading is more concerning than cleaning as CO2 contributes a large
percentage of the total biogas generated. Biogas can be upgraded through physical
absorption, chemical absorption, cryogenic separation, and membrane separation
(Miltner et al. 2017; Rafiee et al. 2021). Biological methods of biogas upgrading
304 M. M. Ghangrekar et al.

such as chemoautotrophic biogas upgrading, photosynthetic biogas upgrading, and

use of BES have recently gained considerable interest. Depending on the configu-
rations of the upgrading system, the bioconversion process of CO2 to CH4 may be
either in-situ or ex-situ.
In-situ bioconversion of CO2 to CH4 occurs via three pathways namely: (1) direct
hydrogenotrophic methanogenesis, where methanogenic archaea reduce CO2 into
CH4 using externally injected H2 as electron donor; (2) indirect (acetoclatic
methanogenesis) pathway, where CO2 is converted into acetate by homoacetogenic
bacteria, and the acetate is converted into CH4 by acetoclastic methanogens and
(3) direct electron transfer (DET) pathway, where CO2 is reduced at the cathode of
BES operated in electrolytic mode (Angelidaki et al. 2018; Fu et al. 2020). Ex-situ
bioconversion may also be achieved by mixing compressed biogas and H2 in an
external digester containing methanogens (preferably hydrogenotrophic
methanogens to exhibit direct methanogenesis) or by feeding the biogas into an
independent microbial electrosynthesis unit operated in electrolytic mode. Either
way, H2 is necessary for the bioconversion of CO2 into CH4. It has to be supplied
from an external source into the upgrading system itself (in-situ method) or mixed
with CO2 in a completely independent external digester (ex-situ method), which is a
costly approach. Further, it might not be practical for large scale setups to use an
external supply of H2 for the bioconversion of CO2 into CH4, because the heating
value of hydrogen is more than methane (Lay et al. 2019). The external power
supply in electrolytic BES serves as the source of electrons. Moreover, the abioti-
cally produced H2 (or H+ ions) from water hydrolysis may present as an excellent
replacement for the H2, which is externally injected in other in-situ or ex-situ
methods (Cheng et al. 2009; Xu et al. 2014). Hence, in-situ biogas upgrading
using BES is a more sustainable approach compared to other biological methods.
Xu et al. (2014) performed both in-situ and ex-situ biogas upgrading using MFC.
It was observed that in-situ biogas upgrading was more effective than ex-situ biogas
upgrading although in both cases the CO2 content was kept below 10% of the total
upgraded biogas in batch as well as continuous operations (Xu et al. 2014). In-situ
upgrading is more advantageous than ex-situ upgrading, because CO2 formation and
reduction takes place simultaneously in the cathodic chamber; while in the ex-situ
method, CO2 reduction into CH4 is limited by gas-liquid transfer of CO2 into the
cathodic chamber. Jin et al. (2017) conducted an ex-situ biogas upgrading experi-
ment using an innovative three-chambered microbial electrolytic capture, separation,
and regeneration cell to facilitate simultaneous biogas upgrading, CO2 recovery, and
wastewater treatment. Synthetic biogas (60% CH4 and 40% CO2) was supplied into
the cathodic chamber using a column diffuser at a flow rate of 19.6 mL h–1. The CH4
content in the upgraded biogas was raised to 97.0 (0.20)% within a gas retention
time of 5.1 h, while the CO2 content was below 5%. Additionally, without the use of
external voltage (control), the CH4 content was 85.5 (0.3)% indicating a substan-
tial increment in the biogas quality in MFC mode (Jin et al. 2017).
In a different investigation, a MEC with 500 mV (vs. SHE) of applied potential
caused > 90% CH4 upgrading from 70%; in addition to 8.2% CO2 conversion into
CH4. It was observed that the Methanothrix and Azonexus species were enriched on
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 305

the cathode. Further, the transcriptomic analysis revealed that Methanothrix uses the
CO2 reduction pathway that stimulates direct electron transfer in MEC for enhancing
the CH4 content of biogas (Liu et al. 2019). These investigations showed that BES
presents a potentially sustainable means for in-situ or ex-situ biogas upgrading.

11.3.3 Post Treatment to the Effluent of Anaerobic Digestion

Using Bio-electrochemical Systems

11.3.3.1 Polishing Treatment to Anaerobic Digestion

The AD as a sole unit cannot be used as a stand-alone treatment for wastewater. For
high strength waste in terms of COD, the effluent of AD still contains a considerable
amount of residual organic matter, thus requiring the need for a second stage of
treatment. BES can be used in such cases as a follow-up treatment for organic matter
removal and energy recovery. On the other hand, for low strength wastewater in
terms of COD, the effluent contains nutrients and pathogens; thus, BES can be used
as an add-on secondary treatment unit or as a polishing treatment unit or both for the
effluent of AD.
Zhang et al. (2012a) reported treatment of organic matter and sulfate in an UASB
reactor and MFC. The combined system of UASB followed by MFC achieved 69.9
and 81.8% of COD and sulfate removal, respectively. The MFC harvested a max-
imum power density of 989.6 mW m2 at a COD/sulfate ratio of 2.3 and HRT of
54.3 h (Zhang et al. 2012a). In another investigation, four MFCs in series were used
as a polishing treatment for the biohydrogen and biomethanation process. The MFCs
were operated with varied OLR of 0.036–6.149 g COD L1 day1, resulting in a
COD removal efficiency of 35.1–4.4%. The highest coulombic efficiency of 60%
was attained at an OLR of 0.572 g COD L1 day1 simultaneously producing a
maximum power density of 3.1 W m3 (Fradler et al. 2014).
In another combination, an UASB-MFC-biological aerated filter was developed
for the molasses wastewater treatment. The UASB reactor was used for COD
removal and the MFC for generating bioelectricity simultaneously degrading COD
as well as oxidation of the generated sulfide; whereas the colour removal and phenol
degradation was accomplished in the biological aerated filter. The overall removal
efficiency of 53.2, 52.7, and 41.1% for COD, sulfate, and colour, respectively, was
achieved in the combined system. The MFC in this combined system produced a
maximum power density of 1410.2 mW m2 (Zhang et al. 2009a). Various combi-
nations have been tried to integrate BES as downstream treatment to AD in order to
meet the discharge norms for the disposal of the treated effluent. Additionally, the
harvested bioelectricity from the MFCs can be used for on-site applications. The
follow-up treatment in BES also offers an advantage for not only the removal of
carbonaceous organic matter but also nutrient removal and recovery as explained in
Sect. 11.3.1.
306 M. M. Ghangrekar et al.

11.3.3.2 Emerging Contaminants Removal

Even after secondary biological treatment targeted for the removal of carbonaceous
organic matter and nutrients from the wastewater, pathogens and emerging contam-
inants (ECs) persist in the effluent. The BES can be implemented to eliminate these
pollutants from the secondary or tertiary treated effluents. The role of downstream
BES-based technology is of paramount importance because AD is incapable of
eliminating the ECs, which could result in discharge of ECs into the receiving
water bodies. Pharmaceuticals, dyes, personal care products, estrogens, aromatic
hydrocarbons, surfactants, and pesticides are amongst the ECs that have been
successfully removed using BES (Chakraborty et al. 2020; Wu et al. 2020). Three
different possible approaches for degrading the ECs from the wastewater using
BES are: (1) anodic degradation; (2) cathodic degradation; and (3) anodic followed
by cathodic degradation.
The ECs can be used as a co-substrate by the anodic microbial consortia along
with organic matter. However, for the low concentration of ECs (generally detected
in the domestic wastewater), cathodic degradation is considered as an attractive
option (Fig. 11.2); wherein, the degradation is achieved using in-situ generated H2O2
or •OH. In an air-cathode MFC when penicillin (50 mg L1) was used with glucose
(1000 mg L1) as a feed, a maximum power density of 101.2 W m3 was generated.
The MFC achieved 98% penicillin removal in 24 h of operation. The maximum
power generated was 6.8-fold higher than that of MFC operated using solely glucose
(1000 mg L1) as a feed. This might be due to the direct electron transfer from the
bacterial cell to the anode by increasing the electron permeability of the bacterial cell
membrane (Wen et al. 2011b). Similarly, the addition of ceftriaxone and Tween-80
in the anodic chamber caused an increase in the electrical performance of MFC,

Recirculation of anolyte
to cathodic chamber

MFC/MEC

e−
Outlet
CO2 C
A A H2O2
Anaerobic N
O
T
H
O 2e− Treated
digester D
E D
E
effluent
OM O2
H+
Inlet
OM: organic matter

Fig. 11.2 Bio-electrochemical system as post treatment to anaerobic digester (second stage
biological treatment followed by tertiary treatment in cathodic chamber)
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 307

simultaneously achieving its removal (Wen et al. 2011a, c). However, not all ECs
showed a positive correlation with power generation. The addition of metronidazole
(10 mg L1) as feed along with glucose caused a reduction in the maximum power
density from 141.94 to 99.23 mW m2 in a dual chamber MFC. However, the MFC
demonstrated 85.4% of metronidazole removal in 24 h (Song et al. 2013).
Due to complexity, the ECs require advanced treatment processes for their
elimination from the wastewater. In the cathodic chamber of BES, the advanced
oxidation processes can be initiated for the removal of ECs. In BES, the Fenton
reaction can be carried via in-situ H2O2 synthesis supplemented with the use of ferric
ions. The first instance of the cathodic Fenton reaction in BES was undertaken by
Zhu and Ni (2009) for the degradation of p-nitrophenol using scrap iron as Fe source.
At a retention time of 12 h, 85% of TOC was removed simultaneously generating
143 mW m2 of maximum power density (Zhu and Ni 2009). In another investiga-
tion, sulfamethoxazole was removed in the anodic chamber followed by cathodic
treatment in a MFC. The combined process demonstrated 94.66% of sulfamethox-
azole removal in 48 h, in which 56.28% removal was in the anodic chamber and
remaining in the cathodic chamber due to Fenton oxidation achieved from carbon
nanotubes, stainless steel mesh, and γ-FeOOH cathode (Li et al. 2020). The bio-
electro-Fenton has been used for the degradation of dyes, antibiotics, pharmaceuti-
cals, and estrogens (Kahoush et al. 2018). It can be used as a polishing treatment to
effluents from an anaerobic digester for the removal of ECs, which cannot be
removed by the AD.

11.3.3.3 Hybridizing Anaerobic Digestion with Bioelectrochemical

Systems

The BES has an edge over conventional AD for the removal of ECs due to the
presence of the anode, which acts as an inexhaustible electron sink. Therefore, the
merger of BES in an anaerobic digester might be one of the options for bioremedi-
ation of recalcitrant contaminants from wastewater. In experiments conducted by Liu
et al. (2011b), a zero valent iron bed with graphite plate electrodes was embedded in
an anaerobic reactor. With the increase in applied voltage, the COD and colour
removal was improved. The bacteriological analysis confirmed the occurrence of azo
dye degrading microbes in the anaerobic reactor (Liu et al. 2011b). Similar obser-
vations of improved COD and azo dye removal were reported in an anaerobic reactor
packed with a pair of Fe-graphite plate electrodes. With 83.4% of COD and 84.7%
dye removal, the Fe-graphite electrode embedded anaerobic reactor outperformed
the sole anaerobic reactor, in which the COD and dye removal efficiency was 76.4
and 50.7%, respectively (Zhang et al. 2012b).
More recently, Shen et al. (2014) tried integration of BES in a UASB reactor for
removal of p-nitrophenol in which a graphite felt cathode and anode were horizon-
tally placed inside the UASB reactor. The results demonstrated that the p-
nitrophenol removal rate in the BES-UASB setup (6.77 M m3 day1) was higher
compared to the UASB reactor only. Also the p-aminophenol (final degradation
308 M. M. Ghangrekar et al.

product of p-nitrophenol) formation efficiency was reduced from 99.1 to 90.2% in

BES-UASB compared to reduction from 98.9 to 53.2% in the sole UASB reactor
when the p-nitrophenol loading rate was increased from 1 to 6.8 M m3 day1 (Shen
et al. 2014). A follow-up study was conducted for the degradation of
2, 4-dinitrochlorobenzene, which demonstrated the ability of BES-UASB to handle
shock loads in a better way compared to the UASB solely. At an applied voltage of
1.4 V, 95.78% of 2, 4-dinitrochlorobenzene removal was achieved in the
BES-UASB reactor. The enhanced pollutant removal was mainly attributed to the
diverse microbial community and presence of electroactive species (Jiang et al.
2016). These lab-scaled investigations validated that the integration of BES into
AD might be an innovation hybridization for simultaneous removal of organic
matter and ECs in a single setup.

11.3.3.4 Pathogen Removal/Disinfection

Apart from the removal of ECs, the cathodic chamber of BES has been used for
disinfection as well. In an investigation conducted by Jadhav et al. (2014), anolyte
was circulated in the cathodic chamber of the MFC; wherein sodium hypochlorite in
different dosage (0.67–3 g L1) was used as catholyte to facilitate disinfection.
Irrespective of the sodium hypochlorite dose, about 99.5% of disinfection was
achieved with the initial bacterial count of 2.8  106 colony-forming units per
100 mL of wastewater. The addition of sodium hypochlorite in 3 g L1 dose
achieved a 85% increase in sustainable power density (80 mW m2) compared to
the 0.67 g L1 dose of sodium hypochlorite. This states that the addition of
chemicals (such as disinfectants) with high redox potential in the catholyte not
only achieved disinfection but also increased the power output of MFC (Jadhav
et al. 2014).
Instead of externally adding the disinfectant, the cathodic disinfection can be
achieved in BES via in-situ synthesis of H2O2 at the cathode. Using oxygen as a
terminal electron acceptor, the cathodic reaction can be progressed via 2e or 4e
based on the cathode material and/or cathode catalyst. Although the 4e pathway is
preferred in BES in terms of electrical performance, the 2e pathway has the
advantage of in-situ H2O2 synthesis at the cathode. The treated anolyte of BES
can be circulated into the cathodic chamber for disinfection or catholyte (high
concentration of H2O2) and treated anolyte can be transferred to a separate offline
chamber for disinfection. Of these, the second option of offline disinfection is better,
as direct circulation of the anolyte into the cathodic chamber can lead to biofouling
on the cathode. The biofouling on the cathode reduces the availability of dissolved
oxygen for the cathodic reaction, thereby increasing the overpotential losses and
reducing the electrical performance (Noori et al. 2019). Nevertheless, while targeting
the two electron pathway of ORR the power produced from the MFC will reduce due
to a lower redox potential of this ORR, however it will offer the advantage of
improved wastewater treatment.
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 309

For the in-situ cathodic H2O2 synthesis different carbon-based cathodes such as
carbon felt (Liu et al. 2011a), graphite plate (Li et al. 2017), graphite (Sim et al.
2015), nitrogen-doped graphitic carbon (Asghar et al. 2017) have been implemented.
Additionally, different metal-based and metallic nanoparticles ranging from Pd/C
(Wang and Wang 2007), Au-Pd (Edwards and Hutchings 2008), and Ni-Pd (Gupta
et al. 2020) have also been successful for cathodic synthesis of H2O2 in BES. The
H2O2 production can be achieved in MFC as well as MEC. However, MEC has an
edge over MFC in terms of the rate of H2O2 generation (Chung et al. 2020).
Over the years, several researchers have tried altering the operating conditions,
such as pH, catholyte, anolyte, imposed potential and cathode catalysts in BES to
maximize the yield of H2O2 in the cathodic chamber (Fu et al. 2010; Sim et al. 2015;
Gupta et al. 2020). Arends et al. (2014) developed a wastewater treatment system
comprising of a wetland with a BES, capable of synthesizing cathodic H2O2. It was
observed that after 1 h of contact time, the produced H2O2 of 0.1% strength was able
to reduce the total coliforms to less than <75 CFU per mL (Arends et al. 2014). In
one of the investigations, Zhou et al. (2018) used bio-electro-Fenton for disinfection
of wastewater in the cathodic chamber and monitoring was performed in terms of
E. coli inactivation. The disinfection capability was optimized by varying the applied
voltage, iron concentration, and catholyte pH. Under acidic conditions (pH of 3),
4-log scale removal of E. coli was achieved at an applied voltage of 0.2 V (against
SHE) and Fe2+ concentration of 0.3 mM. The production of •OH caused microbial
cell membrane destruction, which was identified as a prime reason for the inactiva-
tion of E. coli (Zhou et al. 2018).
In another investigation, when Ni-Pd was used as cathode catalyst in a dual
chamber MEC, the highest H2O2 yield of 233 mg L1 day1 was observed at an
imposed potential of 0.8 V (against SHE). When treated anolyte with the most
probable number of 5.2  106 (9.3  104) per 100 mL was circulated into the
cathodic chamber, 5 ( 1) log-scale bacterial removal was achieved in 3 days of
contact time (Gupta et al. 2020). These investigations showed that the secondary and
tertiary treatment of wastewater can be achieved using BES by removing organic
matter in the anodic chamber and pathogens in the cathodic chamber. Presently
considerable lab-scaled investigations have been conducted on in-situ H2O2 synthe-
sis in the cathodic chamber of BES. However, it was reported that for upscaling, the
H2O2 production rates were significantly lower than lab-scaled investigations. It
might be due to the overpotential losses as well as H2O2 decomposition, which
should be addressed in upcoming research. Also, reactor design aspects ranging
from cathode size, electrode spacing, catholyte, and anolyte volume need to be
optimized for H2O2 generation and a complete downstream treatment scheme for
anaerobically treated effluent needs to be taken up (Sim et al. 2018; Chung et al.
2020).
310 M. M. Ghangrekar et al.

11.3.4 Bioelectrochemical System as a Biosensor to Monitor

the Anaerobic Digestion Process

The process of AD is interdependent on the interaction of a few microorganisms and

the products formed because of microbial interaction. Any imbalance caused in the
system due to induced toxicity should ideally be detected within a shorter time to
prevent the malfunction of the digester. Factors such as ammonia, hydrogen sulfide,
heavy metals, long chains fatty acids, and VFA have an inhibitory effect on
digestion. Of these, VFA is one of the factors resulting in the instability of digestion,
if not in suitable concentrations (Chen et al. 2008). The conventional lab-based
off-line protocols (gas chromatography, high-performance liquid chromatography)
of VFA monitoring are tedious and time consuming. Hence, the installation of in-situ
real-time sensors could be a useful strategy to monitor VFAs during AD (Fig. 11.3).
MFCs have been used as a biosensor for the detection and monitoring of
pollutants from the wastewater including VFAs (Sevda et al. 2020). In an investi-
gation conducted by Kaur et al. (2013), the correlation between the VFA concen-
tration and the resulting current/voltage response was estimated using MFC. It was
observed that cyclic voltammetry showed a linear correlation between the VFA
concentration and peak current up to a concentration of 40 mg L1 in a response time
of 1–2 min (Kaur et al. 2013). A three-chamber microbial desalination cell (MDC)
system was used as biosensor, in which VFAs produced from anaerobic digester
were dosed in the central chamber separated by an anion and cation exchange
membrane on either side. The VFAs in the central chamber could pass through the
anion exchange membrane to the anodic chamber, wherein they can be utilized by
exoelectrogens on the anode for producing electrons. Two distinct linear relation-
ships were observed for VFA concentrations of 1–30 mM and 30–200 mM against
the current densities of 0.04–8.50 mA m2 and 8.50 mA m2 to 10.80 mA m2,

Fig. 11.3 Bio-electrochemical system-based biosensor for volatile fatty acids monitoring
11 Integration of Bio-electrochemical Systems with Anaerobic Digestion 311

respectively after 5 h of contact time. Further, the VFA quantification by the

MDC-based biosensor showed no significant difference when compared with VFA
quantification based on gas chromatography, thus showing the accuracy of the
system for real time monitoring (Jin et al. 2016).
A single chamber air cathode membrane-less MFC was demonstrated by
Schievano et al. (2018) for monitoring the VFA, produced from the anaerobic
digester using four substrates (cheese whey, kitchen waste, citrus pulp, and fishery
waste). Although the electrical signals of MFC were not proportional to the VFA
concentrations up to 1000 mg L1, the negative peaks of current for VFA concen-
trations above 4000 mg L1 can be used as early-warning shock-sensors (Schievano
et al. 2018). The BES-based biosensors showed promising results for monitoring the
VFA production in the AD. In this way, BES can be integrated with anaerobic
digester to monitor the digestion process. Such integrations can be a quicker and
cheaper alternative compared with off-line monitoring. However, further research is
necessary for monitoring the long-term operability and capability of real time
monitoring of these BES-based biosensors.

11.4 Future Scope

AD is one of the most proven and proficient techniques of energy recovery from the
waste; whereas, BES is a relatively newer concept and offers an advantage of
one-step bioelectricity recovery when used as MFC. The process of AD is suscep-
tible to various factors such as pH, VFA, and ammonia concentration. The BES can
be effectively used for the recovery of nutrients and removal of ECs or it can be
integrated with AD as biosensor for better process control. Unlike AD, for BES a
single large reactor is not suitable due to deteriorated electrical performance. Instead,
stacking of multiple smaller BES have been recommended and used by the
researchers for the wastewater treatment at field scale (Feng et al. 2014; Walter
et al. 2018).
The cost is yet another governing factor for the integration of AD and BES.
Typically in BES, more than 60% of the overall cost is associated with the electrodes
and separator (Rozendal et al. 2008). Metal electrodes have higher conductivity;
however, they are susceptible to corrosion in anaerobic conditions. Additionally,
metal electrodes lack high surface area and can be potentially toxic to few microor-
ganisms (Noori et al. 2020). For the pilot-scale and field-scale applications, low-cost
electrodes and separators need to be developed and integrated into the hybrid
system. At the same time, these components should be effective and durable for
long term operations. For the use as a separator, ceramic-based membranes show
better mechanical and structural properties than polymeric membranes at much
reduced cost; hence it can be an ideal choice for separators to be used in scaled up
BES (Ghadge and Ghangrekar 2015; Ieropoulos et al. 2015).
The application of BES also needs to be explored in the field-scale setups,
augmented with already operated anaerobic digesters to assess the potential of
312 M. M. Ghangrekar et al.

BES in real life scenarios in the upcoming research to help in confirming the
feasibility of the integration. Furthermore, for the integrated systems, the environ-
mental impact in terms of energy balance as well as material balance also needs to be
quantified via the use of life cycle analysis tools that will aid in considering the
environmental viability of the integrated system. As discussed earlier, BES have an
immense potential to complement AD for improving wastewater treatment effi-
ciency. However, more experience is to be gained by operating real life systems to
evaluate the efficacy of this integration and to come up with suitable medications in
system configurations to gain real advantage of this integration.

Acknowledgement This research work is funded by Department of Science and Technology,

Ministry of Science and Technology (International Multilateral and Regional Cooperation Divi-
sion), Government of India, under the project SARASWATI-2.0 (file no. DST/IMRCD/India-Eu/
Water Call 2/Saraswati 2.0/2018/C).

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2008.11.023
Chapter 12
Use of Biogas for Electricity-Driven
Appliances

Afshin Davarpanah

Abstract Biogas can be considered as one of the primary renewable energy sources
to generate electricity regarding the grid connection and feed-in tariffs in industrial
plants. The present study is focused on introducing biogas systems on electricity
production and what environmental policies should be assessed to minimize the air’s
biogas component emission. Micro-gas turbine (MGT) systems, combined heat and
power (CHP) systems, solid oxide fuel cells (SOFC), and organic Rankine cycle
(ORC) systems are the most applicable renewable energy systems that can utilize
biogas. Combinations of technologies as a hybrid and novel system give the
engineers chance to optimize the biogas conversion to electricity for specified
industrial purposes. Moreover, environmental features of biogas emissions from
industrial plants were discussed and explained.

Keywords Biogas · Electricity Generation · Environmental Features · Full Cells

12.1 Introduction

Due to approaching depletion of the fossil fuels and out dependence on them in
recent decades, it is recommended to move towards renewable energy and green
technologies. Electricity generation is essential for industrial purposes and genera-
tion of electricity from green energy sources such as solar and wind energy should be
considered (Andalib-Bin-Karim et al. 2017; Hu et al. 2020; Ardebili 2020). Gener-
ating electricity from green energy sources is economically and environmentally
beneficial. Bioenergy resources are considered one of the preferable renewable
power generation resources due to their sustainable and productive clean electric
power (Chang and Zhao 2012; Effah and Boampong 2015; Qadrdan et al. 2018).
Right now, the electric power generation from renewable sources is about 8% and

A. Davarpanah (*)
Department of Mathematics, Aberystwyth University, Aberystwyth, UK
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 319
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_12
320 A. Davarpanah

this should be improved by implementing novel and efficient techniques to generate

electricity from bioenergy resources (REN21 2018).
Biogas technology is the combustible gas generation that can be supplied from
the anaerobic digestion of biomass. Therefore, to move towards green energy
implementation, most of the industrialized countries have intended to convert biogas
to electricity for their industrial purposes (Abbasi and Abbasi 2010; Balsora et al.
2017; Inayat et al. 2019). This is advantageous due to the lower operational
performance expenditures and the need for minimal energy investment in the
industrial sectors. Municipal solid waste, organic waste from industrial sources,
and housing (domestic) residual wastes are considered as the common substrates
to produce biogas (Pöschl et al. 2010; Kiran et al. 2016; Wu et al. 2016). Nowadays,
biomethane that is the upgraded biogas administered in petrochemical plants as a
feedstock, especially in gas pipelines (Arthurson 2009).
The biogas composition consists of 50–75% methane, 25–45% CO2, and 2–8%
of water vapor. Other components such as hydrogen sulfide, ammonia, and siloxane
that are present in biogas, considered as hazardous materials for the environment and
should be treated carefully to reduce the environmental impact (Abatzoglou and
Boivin 2009). In theory, fuel cells can be used to convert the biogas to electricity.
However, this process needs expensive instrumental setup and extremely clean gas.
Proper design and implementation of electric generators is considered as one of the
most important factor for converting biogas to electricity (Liu et al. 2012;
Makareviciene et al. 2013; Mao et al. 2015).

12.2 Electrical Power Generation from Biogas

The environmental friendly features of biogas in comparison to fossil fuels is the

main argument for its use for electricity generation which can revolutionize indus-
trial plants worldwide. Electricity production from biogas systems can be in the
range of 8–54% energy conversion efficiency from biogas processed through biogas
upgrading and methane reformation. Therefore, the main procedure of biogas
utilization would be related to combined heat and power units. The methods below
are the most well-known techniques for converting biogas to electricity (Chen et al.
2017; Barzegaravval et al. 2018).
Solid oxide fuel cell coupled with anaerobic digestion is one of the hybrid
conversion systems studied by Saadabadi et al. (2019). A high AD temperature
condition is applied to enhance the biogas production volume. In this hybrid system,
an external pre-reformer is applied in the system to enhance the electricity genera-
tion’s total efficiency. This procedure is schematically depicted in Fig. 12.1
(Baldinelli et al. 2017; Saadabadi et al. 2019).
A combined heat and power system was used by Kang et al. (2014) to econom-
ically evaluate the biogas conversion to electricity efficiency by implementing a
micro-gas turbine (MGT) (Fig. 12.2). The feedstock for this system was biogas or
natural gas. They concluded that the use of natural gas increased the cost of heat and
12 Use of Biogas for Electricity-Driven Appliances 321

Fig. 12.1 Solid oxide fuel cells for the conversion of biogas to electricity (Baldinelli et al. 2017)

Removal
Digestion Plant vessels
Feedstock COMP COMP

Air
Fuel
COMB
Removal
G COMP GT vessels

MC
Chiller

Heat NG
Demand
Exhuast
EVAP ECON Gas

HX
HRSG

Heat to Digester

Fig. 12.2 Micro-gas turbine (MGT) system for converting biogas to electricity (Kang et al. 2014)

electricity production. Therefore, the electricity and heat price was 0.1436 $/kWh
and 0.0738 $/kWh (Kang et al. 2014). Moreover, according to Kim et al. (2017),
electricity and heat production is estimated at 7.4 MWh/year and 8.5 MWh/year for
MGT systems in combined heat and power (CHP) and organic rankine cycle (ORC)
322 A. Davarpanah

Air

Water Useful heat

T C
Additional biogas
HX1 HRSG 1.013 bar
Burner
373 K
HX2
1073 K
Δploss = 3%
423 K
CATHODE
Electrolyte Steam
Inverter
ANODE HX3
Biogas

1073 K

Air

Fig. 12.3 Solid oxide fuel cells coupled with micro gas turbine and combined heat and power
system for the conversion of biogas to electricity (Wongchanapai et al. 2013)

systems. Combining the MGT and ORC systems has provided better results than the
MGT-CHP system (Kim et al. 2017).
Wongchanapai et al. (2013) developed a novel hybrid system containing solid
oxide fuel cells, micro gas turbine, and a combined heat and power system to convert
biogas to electricity (Fig. 12.3). In this hybrid system, an internal reformation unit
was implemented to convert the methane to hydrogen.

12.3 Environmental Features of Biogas Conversion

to Electricity

As biogas is considered as renewable energy resources to manage the energy wastes

such as disposal wastes, farming wastes, and landfill processes. Environmental
features of biogas conversion in operational processes should be taken into consid-
eration in every industrial procedure. These environmental aspects considered in
engineering designation include hazardous materials, wastewater treatment, CO2
emissions, and maintenance procedures. To implement engineering consideration
for biogas conversion, this process’s advantages and disadvantages should be
explicitly distinguished before any operational performance. The primary utilization
of biogas conversion has been done for industrial purposes. Its disadvantages
contained extreme noise levels during the operations, air pollution due to the release
of CO2 in the plant, and machines rapture through the separators.
In contrast, biogas to electricity has some advantages that should be preferred to
implement its utilization. As the process has been continued throughout the system,
more novel systems could be added and replaced, or in some conditions, they can be
12 Use of Biogas for Electricity-Driven Appliances 323

combined to increase the power generation efficiency. Thereby, waste gases have
been removed through the system circulation, and there are less hazardous emis-
sions. According to the measurements of biogas emissions, CO2 emissions range
from 0.1 to 0.4 kg CO2/kWh. Another biogas emission gas is NOx, which depends
on the nitrogen content of the produced biogas. One of the crucial factors in
environmental conservation is the leakage of hazardous materials that increase
global warming. As AD of waste has fugitive ammonia and greenhouse emissions,
efficient controlling methods to avoid leakages need to be designed (Sommer 1997;
Börjesson and Berglund 2006; Karapidakis et al. 2010; Senbayram et al. 2014;
Carreras-Sospedra et al. 2016).

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Chapter 13
Syngas Fermentation for Bioenergy
Production: Advances in Bioreactor
Systems

Arindam Sinharoy, Kannan Pakshirajan, and Piet N. L. Lens

Abstract Renewable energy resources, particularly biofuels, are gradually

replacing the fossil fuel sources for energy. This will eventually enable an energy
platform with reduced environmental pollution and greenhouse gas emissions. The
present technologies and raw materials available for biofuel production have, how-
ever, not yet reached commercial scale due to high costs and other economic
constraints. In this context, gasification of waste resources to syngas and its subse-
quent bioconversion to a wide range of useful products, including biofuels, could
provide significant advantages over conventional systems. A large number of
acetogens have been isolated that are capable of utilizing syngas as the substrate.
However, relatively poor gas-liquid mass transfer, CO toxicity to microorganisms
and low product yield are some major drawbacks of syngas fermentation. This
chapter reviews the literature of syngas fermentation with specific focus on different
bioreactor systems available for overcoming the limitations of syngas
bioconversions.

Keywords Bioreactors · Biofuel production · Synthesis gas · Syngas fermentation ·

Biorefinery

A. Sinharoy (*)
National University of Ireland Galway, Galway, Ireland
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati,
Guwahati, Assam, India
e-mail: [email protected]
K. Pakshirajan
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati,
Guwahati, Assam, India
P. N. L. Lens
National University of Ireland Galway, Galway, Ireland

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 325
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_13
326 A. Sinharoy et al.

13.1 Introduction

The majority of today’s energy demand is fulfilled by energy supplied from fossil
fuel resources, as the population and industrial growth progresses, the demand for
energy is going to increase furthermore (Johnsson et al. 2019; Sinharoy et al. 2020a).
This is one of the main reason for the exhaustion of fossil fuel reserve, which have
created many global problems and can potentially leads to future energy crisis (Roy
et al. 2016). Apart from that, environmental problems associated with fossil fuel
burning and their effect on human health have created worldwide concern and have
generated demand for clean energy (Chen et al. 2017). These factors combined with
the awareness about global warming have shifted our focus towards safeguarding the
environment and creating a more sustainable society by fulfilling our energy
demands from renewable sources.
The use of renewable energy sources can provide an alternative to the fossil fuel
and simultaneously avoid the environmental degradation associated with fossil fuel
to a larger extent (Soeiro and Dias 2020; Jurasz et al. 2020; Sinsel et al. 2020).
Biofuels, such a renewable energy source, have received a lot of attention during the
recent years. Biofuels are available mainly in liquid form, such as bioethanol,
biobutanol and biodiesel, or in gaseous form, such as biogas (mainly methane) and
biohydrogen (Bórawski et al. 2019). These fuels are mainly derived from biomass as
the raw material or produced via biological or biochemical conversion processes
(Sinharoy et al. 2020a). Due to the conflict with food crops, limited availability of
agricultural land and large water requirements for production of first generation
biofuels, the recent focus is towards the production of biofuels utilizing waste
resources (Bhatia et al. 2017). Solid as well as liquid wastes such as municipal
solid waste, lignocellulosic biomass, agricultural and forest residues and different
industrial wastewaters have been used for production of various types of biofuels
(Hassan et al. 2019). In contrast, the potential of gaseous industrial wastes for
production of biofuel or other useful products has not yet been fully explored. In
this context, the use of synthesis gas or syngas as a low cost substrate for the
production of biofuel or other value added products is attractive. This gas fermen-
tation approach is another way of producing second generation biofuels from waste
biomass.
Synthesis gas is a mixture of carbon monoxide (CO), hydrogen (H2), carbon
dioxide (CO2), nitrogen (N2) and some higher hydrocarbons. The CO concentration
in syngas ranges from 5 to 60%. CO can be steam reformed to enrich the H2 content
of the syngas (Couto et al. 2013). Biomass gasification, which is the main route for
syngas production, is an endothermic process and requires heat energy as an input as
is carried out at 750–800
C (Molino et al. 2016). Lignocellulosic biomass and even
municipal solid waste can be feedstocks for biomass gasification (Ahmad et al.
2016). For an efficient gasifier operation, a certain amount of feedstock homogeneity
should be maintained. If the feedstock is heterogeneous then the product composi-
tion may vary widely (Sikarwar et al. 2016). To overcome this bottleneck,
pre-treatment and post-treatment steps are included in the gasification process,
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 327

which increase the operation cost of the process (Cheah et al. 2016). Homogeneous
lignocellulosic materials, such as timber industry waste, paper mill waste and
agricultural residues can be used as feedstock for biomass gasification (Sikarwar
et al. 2016; Burra and Gupta 2018). Although, municipal solid waste can be a
suitable substrate for biomass gasification as well, it is still under consideration as
it also contains a variety of other (non-combustible) materials (Zheng et al. 2018).
Syngas can be used as the substrate for production of liquid fuel either by the
thermochemical catalytic route with a metallic catalyst or by the biochemical route
with acetogenic microorganisms (Griffin and Schultz 2012). A number of thermo-
chemical catalytic processes such as Fischer–Tropsch (FT) synthesis and the water
gas shift reaction have been applied for converting syngas to valuable products.
These operate at high temperature and pressure, and require costly metal catalysts
(Subramani and Gangwal 2008). Though the thermo-catalytic process has been
commercialized and works successfully in installed plants, it has several disadvan-
tages such as high operating costs, limited choice of metallic catalysts, perishable
nature of the catalyst, huge requirement of heat energy and environmental concerns
(Griffin and Schultz 2012). Furthermore, these catalyst based processes are often
prone to catalyst poisoning due to the emanation of unwanted gases, mainly hydro-
gen sulfide and other inhibitory substances (Subramani and Gangwal 2008). These
disadvantages have resulted in the need for alternative technologies, including
biochemical conversion of syngas. Biological methods offer certain advantages
such as high tolerance to trace contaminants, high product specificity, in addition
to being sustainable, environmental friendly and cost effective (Henstra et al. 2007).

13.2 Syngas Fermentation

13.2.1 Alcohol and Acetate Production

Some acetogens are capable of producing alcohols along with volatile fatty acids
from syngas (Table 13.1). These bacteria are obligate anaerobes and can utilize
CO/CO2 as the carbon and energy source (Henstra et al. 2007; Mohammadi et al.
2011). Examples of these solventogenic bacteria are Clostridium ljungdahlii, Clos-
tridium carboxidivorans, Clostridium autoethanogenum, Clostridium ragsdalei,
Alkalibaculum bacchi, Eubacterium limosum, Butyribacterium methylotrophicum,
Clostridium thermoaceticum and Clostridium formicoaceticum (Mohammadi et al.
2011; Bengelsdorf et al. 2013). Most of these microorganisms can grow on multiple
carbon sources, but their potential to utilize gaseous substrates such as CO, CO2 and
H2 and to produce useful products is commercially attractive. Some strains such as
Clostridium ragsdalei and Clostridium propionicum produce propanol from syngas
(Isom et al. 2015). The biochemical pathway used by these bacteria for the produc-
tion of ethanol is the reductive acetyl-CoA pathway or Wood-Ljungdahl pathway
(Mohammadi et al. 2011). This pathway is present in several organisms including
acetogenic bacteria (Schuchmann and Müller 2016) and methanogenic archaea
Table 13.1 Alcohol production from syngas in different bioreactor systems
328

Any other Ethanol

Temp Agitation carbon concentration Other


Microorganism Reactor C pH (rpm) Feed gas composition substrate /yield products Reference
Clostridium CSTR 37 NR 450 10% CO2, 15% Ar, Fructose NR Acetic acid Aghbashlo
ljungdahlii 20% H2, 55% CO et al. (2016)
Clostridium Batch 37 6.15 120 80% CO, 20% CO2 NR 2.6 g/g of Acetic acid Hurst and
carboxidivorans stirred cells Lewis
P7 reactor (2010)
Clostridium Monolithic 37 6.0 NA 20% CO, 5% H2, 15% Fructose 0.17 mole/ Acetic acid Shen et al.
carboxidivorans biofilm CO2, 60% N2 mole of CO (2014b)
P7 reactor
Clostridium HRPBR 37 6.0 NR 20% CO, 5% H2, 15% Fructose 7 g/L and Acetic acid Shen et al.
carboxidivorans CO2, 60% N2 6.7 g/L/day (2017)
P7
Clostridium BCR 37 6.0 NR 25% CO, 15% CO2, NR 0.15 mole/ Butanol and Rajagopalan
carboxidivorans 60% N2 mole of CO acetic acid et al. (2002)
P7
Clostridium CSTR 30 6.0 250 100% CO Xylose 7.143 g/L Acetic acid Abubackar
autoethanogenum (under et al. (2016)
mixotrophic
condition)
Clostridium CSTR 30 6.0 250 30% CO, 10% CO2, NR 2.22 g/L Acetic acid Lagoa-Costa
autoethanogenum 20% H2, 40% N2 and et al. (2017)
2,3-butanediol
Clostridium CSTR 30 6.0 250 100% CO NR 907.72 mg/L Acetic acid Abubackar
autoethanogenum et al. (2015)
Clostridium Two stage 32 6.0 NR 40% CO, 30% H2, NR 14.74 g/g of Acetic acid Kundiyana
ragsdalei CSTR 30% CO2 cells et al. (2011)
A. Sinharoy et al.
 13

Clostridium CSTR 37 6.1 150 20% CO, 5% H2, 15% NR 9.6 g/L Acetic acid Maddipati
ragsdalei CO2, 60% N2 et al. (2011)
Clostridium TBR 37 5.8 NR 38% CO, 28.5% CO2, NR 5.7 g/L Acetic acid Devarapalli
ragsdalei 28.5% H2, 5% N2 et al. (2016)
Clostridium CSTR 37 6.0 150 20% CO, 5% H2, 15% NR 25.26 g/L 2-propanol, Kundiyana
ragsdalei (pilot scale CO2, 60% N2 Acetic acid, et al. (2010)
100L) 1-butanol
Alkalibaculum BSB—fed 37 8.0–8.5 150 20% CO, 15% CO2, NR 1.75 g/L Acetic acid Liu et al.
bacchi batch mode 5% H2, 60% N2 and (2012)
40% CO, 30% CO2,
30% H2
Alkalibaculum CSTR 37 8.0 NR 20% CO, 5% H2, 15% Corn steep 8 g/L Acetic acid, Liu et al.
bacchi CO2, 60% N2 liquor n-propanol (2014)
and n-butanol
Anaerobic mixed BSB 37 6.0 100 PCO ¼ 2 kg/cm2 NR 2.2 g/L Acetic acid Singla et al.
consortium (2014)
NR, not reported; NA, not applicable; BSB, batch serum bottle; CSTR, continuous stirred tank reactor; HRPBR, horizontal rotating packed bed reactor; BCR,
bubble column rector; TBR, trickle bed reactor
Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems
329
330 A. Sinharoy et al.

Fig. 13.1 Wood-Ljungdahl pathway or reductive acetyl-CoA pathway utilized for syngas fermen-
tation. Abbreviations: ACS, acetyl-CoA synthetase; AckA acetate kinase; [CO], enzyme-bound
carbon monoxide; CODH, carbon monoxide dehydrogenase; CoFeSP, corrinoid sulfur protein;
FDH, formate dehydrogenase; FHS, formyl-THF synthetase; [H], reducing equivalents; HS-CoA,
coenzyme A; MET, methyltransferase; Pi, inorganic phosphate; PTA, phosphotransacetylase; THF,
tetrahydrofolic acid [reused from Kiefer et al. 2021; Copyright (2021) with permission from
Elsevier]

(Borrel et al. 2016). It consists of two branches, eastern branch and western branch
(Fig. 13.1). The eastern branch has a number of reductive steps where CO2 is
reduced to produce the methyl group of acetyl-CoA. Whereas in the western branch,
CO obtained from CO2 or directly from the source forms the carbonyl group for the
acetyl-CoA synthesis (Diender et al. 2015). There are two ways to generate reducing
equivalents by this pathway. Most commonly, a hydrogenase enzyme supplies the
reducing equivalents from H2 as per the following reaction (Eq. 13.1) (Diender et al.
2015):

H2 Ð 2Hþ þ 2e ð13:1Þ

If the hydrogenase enzyme is inhibited or the amount of H2 gas present in the

medium is insufficient, the CODH enzyme generates reducing equivalents by
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 331

oxidation of CO to CO2 as per the following reaction (Eq. 13.2) (Diender et al.
2015):

CO þ H2 O Ð CO2 þ 2Hþ þ 2e ð13:2Þ

These microorganisms are mostly mesophilic, although thermophilic conversion

of syngas using anaerobic consortia has also been reported (Shen et al. 2018). The
pH of the medium plays a vital role in their product formation from syngas. In most
cases, a low pH range tends to favour solventogensis over acetogenesis (Abubackar
et al. 2018).

13.2.2 Hydrogen Production

Similar to the catalytic water gas-shift reaction, carboxydotrophic hydrogenogenic

microorganisms use CO as the sole source of carbon and energy to form equimolar
amounts of CO2 and H2. Over the past decade, a number of such microorganisms
have been described (Table 13.2), which are mainly classified into three different
groups: mesophilic Gram-negative bacteria, thermophilic Gram-positive bacteria
and thermophilic archaea.
Gram-negative mesophilic bacteria are facultative anaerobes, but produce H2
from CO under strict anaerobic conditions. These are mostly non-sulfur purple
photosynthetic bacteria, including Rhodopseudomonas gelatinosa (Mörsdorf et al.
1992), Rhodocyclus gelatinosa (Klasson et al. 1993), Rubrivivax gelatinosus
(Henstra et al. 2007), Rhodopseudomonas palustris (Oelgeschläger and Rother
2008) and Rhodospirillum rubrum (Najafpour et al. 2003). Among these bacteria,
the phototrophic R. rubrum is well studied, its genetic map and enzyme system have
been described and it has been employed in bioreactors. The optimum temperature
and pH of this microorganism are in the range of 34–40
C and 6.5–7.5, respectively
(Klasson et al. 1993). In general, high levels of CO are inhibitory to this bacterial
species and its light requirement causes additional costs. Other than these
phototrophic organisms, certain non-phototrophic Gram negative anaerobes are
capable of converting CO to H2. These are Citrobacter strain Y19, isolated from
an activated sludge plant (Jung et al. 2002), an anaerobic microbial consortium with
predominantly Petrobacter sp. (Pakshirajan and Mal 2013) and anaerobic granular
sludge consortia from a full-scale upflow anaerobic sludge blanket reactor with
predominantly Acinetobacter sp. (Sinharoy et al. 2015).
A large number of carboxydotrophic organisms are extremophiles, isolated from
extreme environments such as hot springs and submarine hot vents. This group of
bacteria contains mainly anaerobic prokaryotes of the Bacillus/Clostridium subclass,
such as Carboxydothermus hydrogenoformans (Henstra and Stams 2011),
Carboxydobrachium pacificum (Sokolova et al. 2001), Thermincola carboxydophila
(Zavarzina et al. 2007), Carboxydocella sporoproducens (Slepova et al. 2006) and
Table 13.2 Hydrogen production from syngas in different bioreactor systems
332

Any other
Temp Agitation carbon H2 production


Microorganism Reactor C pH (rpm) Feed gas composition substrate rate /yield Reference
Rhodospirillum CSTR 30 6.5 NR PCO ¼ 0.55 atm Acetate 16 mmol H2/h Najafpour et al.
rubrum (2003)
Rhodospirillum CSTR 30 6.9 350–800 55% CO, 15% H2, 20% Malic acid/ 9.6 mmol H2/h Ismail et al.
rubrum Ar, 10% CO2 sodium (2008)
acetate
Rhodospirillum CSTR 30 6.9 150–500 55% CO, 15% H2, 20% Malic acid/ 80% mol/mol Younesi et al.
rubrum Ar, 10% CO2 sodium of CO (2008)
acetate
Rhodospirillum CSTR 30 7.0 400–750 17.2% CO, 16.3% Acetate 0.75 mol H2/ Do et al. (2007)
rubrum CO2, 8.8% H2, 56.0% day
N2
Thermoanaerobacter BSB 60 7.5 NR 100% CO NR 45 mmol/L Weghoff and
kivui Müller (2016)
Rubrivivax BSB 30 7.0 250 20% CO Sodium 0.9 mmol/ min/ Vanzin et al.
gelatinosus malate g cell (2002)
Rhodopseudomonas BSB 30 7.0 NR 60% CO, 10% CO2, Sodium 7 mmol/L Pakpour et al.
palustris P4 20% H2, 10% Ar acetate (2014)
Rhodopseudomonas CSTR 30 7.0 500–700 20% CO, 80% Ar NR 41 mmol/g Oh et al. (2005)
palustris P4 cell/h
Citrobacter sp. Y19 Two stage Batch 30 7 300–700 20% CO, 80% Ar Sucrose 20 mmol/g Jung et al.
stirred tank reactor cell/h (2002)
Carboxydothermus GLR 70 6.8–7.2 NR 100% CO Bacto- 95% mol/mol Haddad et al.
hydrogenoformans peptone of CO (2014)
Carboxydothermus HFMBR 60–70 6.8–7.0 Nil 100% CO NR 92% mol/mol Zhao et al.
hydrogenoformans of CO (2013)
A. Sinharoy et al.
 13

Carboxydothermus BSB 65 7.0 100 100% CO NR NR Henstra and

hydrogenoformans Stams (2011)
Carboxydothermus BSB 50–70 6.0–6.5 NR 100% CO Ferric citrate NR Yoneda et al.
pertinax (2012)
Moorella stamsii BSB 65 7.5 NR 100% CO NR NR Alves et al.
(2013)
Clostridium BSB NR NR NR 100% CO NR 7 mmol/L Martin et al.
thermoaceticum (1983)
Anaerobic sludge BSB 30 7.0 120 100% CO Acetic acid 60.8 mol Pakshirajan and
biomass (during Mal (2013)
startup)
Anaerobic granular UASB 55 7.5 NA 45–90 mmol/day NR 45.3 % (v/v) Liu et al. (2016)
sludge biomass biogas
produced
Anaerobic sludge GLR 30 7.0 NA 100% CO NR 30.7 mmol/L Sinharoy and
biomass Pakshirajan
(2020)
Anaerobic sludge MBBR 30 7.0 NA 100% CO NR 19.5 mmol/L Sinharoy and
biomass Pakshirajan
(2021)
NR, not reported; NA, not applicable; BSB, batch serum bottle; HFMBR, hollow fiber membrane bioreactor; GLR, gas lift reactor; UASB, upflow anaerobic
sludge blanket reactor; MBBR, moving bed biofilm reactor
Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems
333
334 A. Sinharoy et al.

Thermosinus carboxydivorans (Sokolova et al. 2004). The optimum temperature and

pH for these bacteria is in the range of 50–90
C and 6.8–8.0, respectively
(Table 13.2).
Though a large number of archaea have been isolated from extreme temperature
environments, only a few of these are capable of chemolithotrophic growth on CO
and H2 production, which include Thermococcus AM4 and Thermofilum
carboxyditrophus (Sokolova et al. 2009). The use of thermophilic microorganisms
is more advantageous than mesophilic ones for syngas fermentation, as the temper-
ature of syngas exiting from a gasifier is around 700–800
C and the cost of reducing
the temperature of raw syngas to a moderate range can be avoided (Griffin and
Schultz 2012). However, the high energy requirement of maintaining a large scale
reactor at an elevated temperature is still not economically feasible.

13.2.3 Methane Production

Production of methane from syngas can be advantageous, particularly in case of

highly lignified biomass or biomass containing pathogenic microorganisms (Li et al.
2020). Further, biomass gasification followed by syngas biomethanation can also
avoid generation of unwanted end products such as digestate produced in case of
anaerobic digestion of lignocellulosic substrates. Although biochar is produced as
byproduct during biomass gasification, quantity wise it is much less (<1% of the
biomass) than digestate produced during anaerobic digestion (Swart 2013).
Only a few methanogenic archaea are capable of utilizing CO as the sole carbon
and energy source. CO is not a very efficient substrate for methane production, as
only three pure strains produce methane while growing on CO, namely
Methanobacterium thermoautotrophicus, Methanosarcina acetivorans and
Methanosarcina barkeri (Oelgeschläger and Rother 2009).
The direct CO conversion to methane can be achieved by the following reactions
(Eqs. 13.3 and 13.4):

CO þ 3H2 Ð CH4 þ H2 O ð13:3Þ

4CO þ 2H2 O Ð 3CO2 þ CH4 ð13:4Þ

An excess amount of H2 in the syngas is required for methane production to take

place using Eq. (13.3). In case of Eq. (13.4), CO is first converted to other products
(H2 or acetate) from which methane is produced by hydrogenotrophic or aceticlastic
methanogenesis (Guiot et al. 2011; Li et al. 2020). The H2 to CO ratio plays a very
important role in this case and by increasing the H2 concentration methane produc-
tion can be improved. For example, the methane content in the biogas increased from
66.37 to 73.35% by changing the H2/CO ratio from 3:1 to 5:1 (Li et al. 2020).
In some microorganisms, such as Methanococcus jannaschii the gene coding
carbon monoxide dehydrogenase (CODH) is located on the same operon of
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 335

hydrogenase, signifying its potential for hydrogenogenic carboxydotrophic metab-

olism (Bult et al. 1996). In spite of having one or more CODH enzyme systems in
most of the methanogenic organisms, many of them have never been tested for CO
utilization (Ferry 2010). Hence, their growth potential on CO or product formation
capabilities are mostly unknown.
Many researchers have studied anaerobic mixed consortia for their capability of
CO utilization and methane production (Table 13.3). For example, Sinharoy et al.
(2015) found that anaerobic granular sludge from three different upflow sludge
blanket reactors treating municipal sewage were capable of biological CO conver-
sion with methane as the main product along with low concentrations of H2 and
acetic acid. To illustrate the methane production pathway using CO at mesophilic
conditions (35
C), two selective inhibitors namely 2-bromoethanesulfonate (BES)
and vancomycin were used (Navarro et al. 2014). Carboxydotrophic methanogenesis
was found to be a two-step process in this case, where CO is first converted to
acetate, from which methane is subsequently produced.
Sipma et al. (2003) studied CO conversion using seven different sludges sampled
from anaerobic wastewater treatment plants. All the sludges were capable of
converting CO to methane and acetate at 30
C, and this methane production
occurred via acetate, not H2. At a higher temperature (50
C), CO was rapidly
converted into hydrogen or methane, produced with H2 as the intermediate. The
increase in temperature has a positive impact on the methane production from
syngas. Guiot et al. (2011) reported a more than fivefold increase in methane
productivity from CO by increasing the incubation temperature from 35 to 60
C.
This could be attributed to the change in microbial community to enrich
carboxydotrophic hydrogenogenic bacteria at elevated temperature resulting in
higher CO bioconversion rates (Grimalt-Alemany et al. 2018).
Methane production from syngas has been realised even at extreme thermophilic
(70
C) conditions with 83–97% CO conversion to CH4 (Bu et al. 2018). The
carboxydotrophic methanogenic archaea Methanothermobacter
thermoautotrophicus was the predominant strain at this elevated temperature. CO2
was used preferentially during hydrogenotrophic methanogenesis and once it was
exhausted, methanogens started to consume CO.

13.3 Conventional Bioreactors for Syngas Fermentation

13.3.1 Reactor Consideration

Proper design and operation of a bioreactor are important considerations for suc-
cessful scale up of syngas bioconversion to useful products (Yasin et al. 2015). The
key parameters necessary while selecting a suitable bioreactor are mainly related to
gas-liquid mass transfer, which is governed by agitation speed, impeller design,
power consumption, temperature, pressure conditions and bioreaction kinetics
(Munasinghe and Khanal 2010a; Yasin et al. 2015). Both batch and continuous
Table 13.3 Methane production from syngas in different bioreactor systems
336

Any
other
Temp Agitation carbon CH4 production


Inoculum Reactor C pH (rpm) Feed gas composition substrate rate /yield Reference
Anaerobic granular sludge GLR 35.2 7.1 NA CO loading ¼ 15–122- NR 0.49–4.77 mmol/ Guiot et al.
mmol/g VSS/day g VSS/day (2011)
Enriched TBR 37, 60 7.0 NA 45% H2, 25% CO2, NR 30–70% CH4 in Asimakopoulos
mixed microbial consortia 20% CO, 10% N2 biogas et al. (2021a)
Enriched thermophilic TBR 60 7.0 NA 45% H2, 25% CO2, NR 17.6  0.6 mmol/ Asimakopoulos
mixed microbial consortium 20% CO, 10% N2 L/h et al. (2021b)
Anaerobic sludge CSTR 37, 55 8.5 400–500 10% CO2, 40% CO, 50% NR 66–73% CH4 in Li et al. (2020)
H2 biogas
Anaerobic granular sludge UASB 37 7.28–7.65 NA 100%, 2.5 L CO/day Glucose 1709–2628 mL/ Jing et al.
L/day (2017)
Anaerobic sludge RMBR 55 7.0 NA 55% CO, 20% H2, 10% NR 186 mL/L/day Westman et al.
CO2 (2016)
Anaerobic sludge TBR 37, 60 7.0 NA 45% H2, 25% CO2, 20% NR 8.49 mmol/L/h Asimakopoulos
CO, 10% N2 et al. (2020)
Anaerobic sludge TBR 25, 50 7.0 NA 80% H2, 20% CO2 NR 13.1 m3/m3/day, Strübing et al.
97.5% CH4 in (2018)
biogas
Anaerobic sludge TBR 55 7.0 NA 80% H2, 20% CO2 NR 15.4 m3 CH4/m3/ Strübing et al.
day (2017)
Enrichment anaerobic Sealed 37, 60 7.2 100 PH2 ¼ 1 atm, NR 18.8 mmol/g Grimalt-
cultures flasks PCO ¼ 0.2–0.8 atm VSS/day Alemany et al.
(2020)
Anaerobic granular sludge BSB 35 7.5 200 20–100% CO NR 1 mmol/g Sancho Navarro
VSS/day et al. (2016)
A. Sinharoy et al.
 13

Thermophilic mixed culture FMBR 55 8.0 NA 20% H2, 55% CO, 10% NR 34.41 mmol/L/ Chandolias
CO2 day et al. (2019)
Sewage sludge CSTR- 37 7.6 NA 2% H2, 8% NR 1322 mL/day Wang et al.
HFM CO biogas produc- (2013)
tion, 98.8% CH4
Anaerobic mixed cultures BSB 70 7.5 NA 80% H2, 5% CO, 15% NR 78.6–89.5 mL/ Bu et al. (2018)
CO2 day
Co-culture of CSTR 65 7.2 NR 66.66% H2, 33.33% CO Acetate 4.91 mmol/L/h Diender et al.
Carboxydothermus (2018)
hydrogenoformans and
Methanothermobacter
thermoautotrophicus
NR, not reported, NA, not applicable; BSB, batch serum bottle; CSTR, continuous stirred tank reactor; RMBR, reverse membrane bioreactor; FMBR, floating
membrane bioreactor; GLR, gas lift reactor; UASB, upflow anaerobic sludge blanket reactor; TBR, trickle bed reactor; HFM, hollow fiber membranes
Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems
337
338 A. Sinharoy et al.

operation modes using a few bioreactor configurations have been applied for syngas
fermentation to useful products. Continuous stirred-tank reactor (CSTR) is the most
commonly used bioreactor system for syngas fermentation, but other types of
reactors such as bubble column reactor (BCR), trickling bed reactor (TBR), packed
bed bioreactor (PBR), gas lift reactor (GLR) and membrane bioreactor (MBR)
(primarily hollow fibre membrane bioreactor) have also been examined under
different modes of operation and experimental conditions (Asimakopoulos et al.
2018). Table 13.4 compares the commonly used reactors for gas fermentation along
with their volumetric gas to liquid mass transfer coefficient (KLa) values. The
different types of bioreactor configurations studied for syngas fermentation to useful
products are depicted in Fig. 13.2 and discussed further in the following
sub-sections. Table 13.5 overviews the relative advantages and drawbacks of the
different types of bioreactors for syngas fermentation.

13.3.2 Continuous Stirred Tank Reactor

The continuous stirred tank reactor (CSTR; Fig. 13.2a) is the most commonly used
bioreactor type employed for syngas fermentation research as it offers good mixing.
Mixing of gaseous substrates in the liquid media inside the reactor is achieved by
impellers and baffles which enhance mass transfer between the substrate and micro-
organisms inside the reactor (Munasinghe and Khanal 2010b). The hydrodynamic
shear generated by a high level of agitation or mixing creates small sized bubbles
from bigger ones, which increases the interfacial area for mass transfer and subse-
quent enhancement in the bioavailability of gaseous substrates (Bredwell et al.
1999). Besides, the slow rising velocity of the finer gas bubbles lead to a prolonged
gas retention time in the liquid medium, resulting in high mass transfer rates. In this
type of reactor, gaseous substrate is supplied continuously and liquid medium
containing the nutrients is fed into the bioreactor to support microbial growth and
metabolism (Bredwell et al. 1999; Sinharoy et al. 2020a). Products formed in the
reactor by microbial conversion are drawn out from the system at the same flow rate
as the feed to maintain steady state profiles under continuous mode of operation.
The effect of different operational parameters, such as agitation speed, medium
flow rates and composition as well as syngas flow rate on ethanol and acetate
production by Clostridium ljungdahlii was studied using a CSTR (Aghbashlo
et al. 2016). The sustainability and renewability of the process was also evaluated
by a thermodynamic model. From the calculated model parameters, the maximum
exergetic productivity index was 6.82 and 6.90 using, respectively, the conventional
exergy and eco-exergy concepts at an optimum condition of 450 rpm agitation
speed, 0.55 mL/min liquid medium flow rate and 8 mL/min syngas (55% CO,
20% H2, 10% CO2 and 15% Ar) volumetric flow rate. A six fold increase in ethanol
concentration by Clostridium ragsdalei was achieved in a pilot scale (100 L) stirred
tank reactor compared with that in a serum bottle (Kundiyana et al. 2011). The
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 339

Table 13.4 Commonly used bioreactor systems for biological CO conversion and their volumetric
mass transfer coefficients
Volumetric
Agitation mass transfer
speed coefficient KLa Gaseous
Reactor type (rpm) (h–1) feed Products Reference
Continuous 200 14.2 CO NR Bredwell
stirred tank et al. (1999)
reactor
(CSTR)
CSTR 300 35 Syngas NR Bredwell
et al. (1999)
CSTR 450 101 Syngas NR Bredwell
et al. (1999)
CSTR 500 86.4 CO H2 ¼ 9.6 mmol/h Ismail et al.
(2008)
CSTR 450 153 CO NR Ungerman
and Heindel
(2007)
CSTR 600 154.8 CO NR Riggs and
Heindel
(2006)
CSTR 700 192.8 Syngas NR Kapic et al.
(2006)
CSTR 300 14.9 CO NR Klasson
et al. (1993)
CSTR 400 21.5 CO NR Klasson
et al. (1993)
CSTR 700 35.5 CO NR Klasson
et al. (1993)
Trickle bed NA 137 Syngas NR Cowger
reactor et al. (1992)
(TBR)
TBR NA 121 Syngas NR Bredwell
et al. (1999)
Bubble col- NA 72 CO Acetate ¼ Chang et al.
umn reactor 15.6–90 mM, butyrate (2001)
(BCR) ¼ 0.7–1.3 mM
Hollow fiber NA 400 CO NR Munasinghe
membrane and Khanal
reactor (2012)
(HFMR)
HFMR NA 1096.2 CO Acetate ¼ 8.2 g/L, Shen et al.
ethanol ¼ 23.93 g/L, (2014a)
butanol ¼ 0.45 g/L
HFMR NA 385 CO NR Lee et al.
(2012)
Packed bed NA 21 Syngas NR Bredwell
reactor et al. (1999)
(continued)
340 A. Sinharoy et al.

Table 13.4 (continued)

Volumetric
Agitation mass transfer
speed coefficient KLa Gaseous
Reactor type (rpm) (h–1) feed Products Reference
Gas lift reac- NA 129.6 CO NR Munasinghe
tor (GLR) and Khanal
(2014)
GLR NA 1.5–2.0 CO H2 production ¼ Haddad et al.
74.6–96.7% of CO (2014)
(mol basis)
GLR NA 91.08 CO NR Munasinghe
and Khanal
(2010b)
GLR NA 153, 122 CO, H2 Acetate ¼ 7.2 mM, Riegler et al.
ethanol ¼ 32.3 mM, (2019)
1-butanol ¼ 5.1 mM,
1-hexanol ¼ 1.0 mM
GLR NA 80.28 CO Acetate ¼ Park et al.
328–711 mg/L, etha- (2013)
nol ¼ 44–498 mg/L,
butyrate ¼
83–389 mg/L, butanol
¼ 10–251 mg/L
Stirred tank 200 90.6 CO NR Bredwell
with et al. (1999)
microbubble
sparger
Stirred tank 300 104 CO NR Bredwell
with et al. (1999)
microbubble
sparger
NA, not applicable, NR, not reported

ethanol concentration reached a maximum value of 25.26 g/L along with the
co-products 2-propanol, 1-butanol and acetic acid.
A two-stage CSTR for ethanol production from syngas was studied using Clos-
tridium carboxidivorans, in which the pH of the first reactor was maintained at pH
6 for acidogenesis and the second one was maintained at pH 5 for solventogenesis
(Abubackar et al. 2018). This two-stage approach achieved a high biomass concen-
tration in the first stage and increased the ethanol production in the second stage.
Bioconversion of CO rich syngas to hydrogen using a CSTR with the anaerobic
photosynthetic bacterium Rhodospirillum rubrum, capable of performing the bio-
logical water-gas shift reaction, was studied (Younesi et al. 2008). The best perfor-
mance in terms of hydrogen production rate and conversion efficiency of 16 ( 1.1)
mmol/g cell/h and 87 ( 2.4) %, respectively, was achieved at an agitation speed and
gas flow rate of, respectively, 500 rpm and 14 mL/min. In order to achieve a high gas
liquid mass transfer a CSTR equipped with a dual-impeller agitation and
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 341

Fig. 13.2 Schematic of various commonly used bioreactor systems for syngas fermentation. (a)
Stirred tank reactor, (b) bubble column reactor, (c) packed bed reactor, (d) gas lift reactor and (e)
membrane bioreactor

microsparger system was utilized (Najafpour et al. 2003). A very high H2 production
rate of 16 mmol/g cell/h and 80% conversion efficiency on CO by R. rubrum at a
syngas flow rate of 14 mL/min was achieved. The maximum cell density during
steady state operation reached 1.3–1.4 g/L with a specific growth rate of 0.0225/h.

13.3.3 Bubble Column Reactor

A bubble column reactor (Fig. 13.2b) offers a high gas-liquid mass transfer due to
the high surface area of bubbles and increased turbulence as the bubbles rise through
the liquid column. Small-sized finer bubbles produced in this type of reactor have
high surface-to-volume ratios and low rise velocities through the liquid, resulting in
improved contact times in the reactor. The short gas residence time and high pressure
drop in the bubble reactor are some drawbacks limiting its scale-up and commer-
cialization. Other drawbacks of this bioreactor type include back-mixing and
342 A. Sinharoy et al.

Table 13.5 Relative advantages and disadvantages of different bioreactors used for syngas
fermentation
Reactor type Advantages Disadvantages
Continuous • Simple to operate • High agitation causes shear
stirred tank • Mixing improves gas-liquid mass transfer stress on microbes
reactor • Gaseous substrates are more accessible to • Large power requirement
microbes due to breaking of large bubbles • High operating cost
into finer ones by increasing rotational speed
of impellers
Bubble col- • High gas-liquid mass transfer rates • Back mixing and coalescence
umn reactor • Low operating and maintaining cost
• Low shear stress
Trickle bed/ • Gas and liquid flow could be either in • Clogging of bed
packed bed co-current or counter-current direction
reactor • No need of mechanical agitation
• Power requirement is less
Membrane • Gases easily diffuses through wall of the • Not yet commercially used for
bioreactor membranes CO conversion or syngas fer-
• Membrane wall provides excellent support mentation
for microbial growth and improves biomass • High installation cost
retention • Clogging problems
• High product yield, high reaction rate, high
tolerance to toxic elements
Gas lift • Mechanical simplicity • Not suitable for viscous broths
reactor • Low energy requirement • Minimum process volume
• No focal points for energy dissipation • Dead zones inside the reactor
• Low shear stress • Insufficient mixing at high
• Suitable for process with variable gas feed- biomass densities
ing requirements
Moving bed • Good biomass retention • Prolonged startup time
biofilm • High surface area for biofilm formation • Loss of biomass support over
reactor • Auto-control of biomass due to shear and time
tear during mixing • Troubleshooting is difficult
• Less area requirement due to complicated design

coalescence in the bubble column (Datar et al. 2004). But the main attraction of this
type of reactor is the ease of obtaining the desired amount of biomass in the reactor.
Syngas fermentation by Clostridium carboxidivorans was studied in a continu-
ously operated bubble column bioreactor (4.5 L volume): ethanol, butanol, and
acetic acid yields were 0.15, 0.075 and 0.025 g/mol CO, respectively, under steady
state condition (Rajagopalan et al. 2002). The authors further suggest different
approaches such as cell recycle to increase the cell concentration without altering
the ethanol yield, H2 addition in the gas stream to improve the CO conversion to
ethanol and optimization of nutrient supplements to the medium to further improve
the bioreactor performance.
Amos (2004) evaluated the performance of a bubble column reactor for CO
bioconversion to H2 with or without gas recycle. The reactor with added gas recycle
loop performed better with an overall conversion efficiency of over 50% for a 10:1
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 343

recycle-to-feed ratio. The performance of the bioreactor was attributed to a

prolonged residence time, high cell mass, buffering capacity of the liquid and low
inlet CO concentration to the reactor.

13.3.4 Packed Bed Bioreactor

The trickle-bed bioreactor or trickling filter (Fig. 13.2c) contains a tubular reactor
with solid support for biomass to grow and attach to. The cells can either be
immobilized on the solid packing or suspended in the liquid medium (Bredwell
et al. 1999). Generally, such bioreactor system is operated under counter current flow
of liquid to gas, where gaseous substrate rises upwards and the water flows down-
wards through the packed bed (Stoll et al. 2020). Special care needs to be taken to
maintain a low water flow rate to prevent flooding in the column. The liquid flow is
mainly provided to keep the cells moist along with nutrient supplementation for cell
growth (Sinharoy et al. 2020c). Various packing materials, including wood, acti-
vated carbon, lava rock, plastic and porous ceramic supports have been tested for
biofilm formation (Amos 2004). The primary advantage of this bioreactor configu-
ration is the improved gas transfer area with a minimum pressure drop. Also, it is
easy to enhance the mass transfer by controlling the liquid flow rates (Sinharoy et al.
2020c).
The effect of the reactor packing material on syngas fermentation by the photo-
synthetic bacterium Rubrivivax gelatinosus was examined using two geometrically
similar reactors, but with different volumes (1 and 5 L) and non-porous glass beads
of two different diameters (3 and 6 mm) as the support material (Wolfrum and Watt
2001). The CO conversion efficiency with the glass beads of 3 mm diameter at
different superficial liquid velocities was better than with the 6 mm diameter glass
beads, which was attributed to a high mass transfer with the smaller diameter
particles owing to their high surface area to volume ratio. However, the reactor
volume did not significantly affect the CO conversion, indicating consistency in
performance of this bioreactor configuration which is helpful in predicting its
performance.
A trickle bed bioreactor with 6 mm size soda lime glass beads as packing material
was studied for ethanol fermentation from syngas (Devarapalli et al. 2016). CO
inhibition could be overcome by a high amount of biomass available in the form of
biofilm formed over the beads, resulting in 1.9 times enhanced H2 uptake and
conversion. The final ethanol and acetic acid concentrations inside the reactor
reached a maximum value of 5.7 and 12.3 g/L, respectively. It was also found that
the co-current mode of gas and liquid flow in the reactor reduced the gas bypass and
reactor flooding problems that were encountered in counter-current operation.
Apart from passive immobilization methods to grow biomass on support mate-
rials, active immobilization techniques to entrap the anaerobic biomass inside
polyvinyl alcohol (PVA) or sodium alginate beads have also been studied (Kumar
et al. 2018). PVA addition to the sodium alginate beads improves their strength and
344 A. Sinharoy et al.

recycle potential for long term use. High CO utilization (90%) efficiencies were
achieved using a CO fed PBR operated at an HRT of 48 h. The superior performance
of the PBR containing immobilized beads is attributed to the excellent gas-liquid
mass transfer due to counter current flow of the liquid and the feed gas. Moreover,
the use of biomass immobilization in PVA-alginate beads can prevent CO toxicity to
the anaerobic biomass by avoiding direct exposure of the biomass to high CO
concentrations (Kumar et al. 2018).
Methane production from syngas using a trickle bed reactor was studied to
understand the effect of temperature on the process efficiency (Asimakopoulos
et al. 2020). Thermophilic conditions (60
C) were best suited for methane produc-
tion with a maximum methane productivity of 8.49 mmol/L/h. The microbial
community change at mesophilic conditions resulted in high accumulation of acetate
in the reactor and lowering of the final methane productivity. The absence of
acetoclastic methanogens in both the mesophilic and thermophilic reactor indicates
that the methane production occurred through the hydrogenotrophic route. A scale
up study using a 7.5 L volume reactor was also carried out during which a very high
methane productivity of 17.6 mmol/L/h was achieved at a 0.33 h empty bed
residence time (Asimakopoulos et al. 2021b). The reactor showed a maximum of
100% H2 and 92.4% CO utilization without any process inhibition during the
continuous bioreactor operation.

13.3.5 Gas Lift Reactor

A gas lift reactor (GLR; Fig. 13.2d) is similar to the bubble column reactor, but it
differs by the fact that it contains a draft tube, through which syngas flows (Sinharoy
et al. 2020b). The draft tube is either an inner tube (called gas lift bioreactor with an
internal loop) or an external tube (called gas lift bioreactor with an external loop)
which improves circulation and gas liquid mass transfer and equalizes shear forces in
the reactor (Negi et al. 2020; Sinharoy et al. 2020c). The major advantages of a gas
lift bioreactor are its simple design with no moving parts or agitators, low energy
requirement as well as homogeneous distribution of nutrients and shear force
(Riegler et al. 2019). The gas stream facilitates the exchange of material between
the gas phase and the liquid phase, thus enhancing effective mass transfer. For
example, a very high mass transfer coefficient of 91.08/h for CO was reported in a
gas lift reactor combined with a bubble diffuser compared to other reactor configu-
rations (Munasinghe and Khanal 2010b).
A CO and H2 fed GLR with a high gas-liquid mass transfer coefficient
(kLa ¼ 80.28/h) was used to enrich acetogenic microorganisms from different
animal faeces to isolate strains capable of syngas fermentation (Park et al. 2013).
Ten out of 42 strains isolated from this enriched animal faeces were able to utilize
CO/H2 and produce alcohols and volatile fatty acids. The enriched consortium
isolated from chicken faecal samples showed the highest product formation from
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 345

syngas, i.e. 711 mg/L acetate, 498 mg/L ethanol, 389 mg/L butyrate and 251 mg/L
butanol (Park et al. 2013).
CO conversion to H2 by Carboxydothermus hydrogenoformans in a gas lift
reactor (35 L volume) was examined to study the effect of different operating
conditions, i.e. gas recirculation rate, CO feeding rate and addition of bacto-peptone
to the medium (Haddad et al. 2014). The ratio of gas recirculation over CO feeding
rate was the most important parameter affecting the reactor performance, which
kinetically limited both the CO conversion and H2 production rates.
A high amount of biohydrogen (20.5 mM) was produced from 36 mM CO using
an anaerobic microbial consortium in a continuously operated GLR (Sinharoy and
Pakshirajan 2020). The hydrogen production was further enhanced to 30.7 mM with
addition of biologically synthesized iron nanoparticles to the bioreactor. The CO
utilization by the anaerobic consortium was well above 80% for the low CO
concentrations of 7.6 and 15 mM, but gradually depleted with further increase in
the CO concentration.
Immobilized cells of Butyribacterium methylotrophicum have been studied in a
GLR system for syngas fermentation by immobilizing it on different biosupports,
namely celite, molecular sieves, alumina, activated carbon, wood, and ion exchange
resins (Chatterjee et al. 1996). Among the different materials, molecular sieves were
the best suited for cell growth, whereas celite was better for cell attachment than the
other materials. Moreover, the product formation from syngas measured in terms of
total electron content was higher in case of molecular sieves based system.
In addition to GLR operated under mesophilic conditions, its performance has
also been evaluated under thermophilic conditions. For example, hydrogenogenic
CO conversion for biological sulfate reduction was studied using a gas lift reactor
and the effect of different HRT was investigated under thermophilic (55
C) condi-
tions (Sipma et al. 2007). The authors reported that at a high retention time (>5.5 h)
the CO conversion resulted in hydrogenotrophic methane production, whereas H2
production was higher at a short HRT (4 h). Overall, a CO conversion of 85% was
achieved (Sipma et al. 2007).

13.3.6 Membrane Bioreactor

A membrane bioreactor (MBR) consists of a membrane module that is partially or

completely submerged in liquid media (Fig. 13.2e). The syngas is diffused through
the walls of the membranes without forming bubbles. Biomass grows on the outer
wall of the membranes as a biofilm, which converts the syngas to useful products.
Several studies on hollow fibre membrane (HFM) reactors have suggested that the
MBR can potentially replace the universally used bioreactors with high gas liquid
mass transfer rate for syngas bioconversion (Gunes 2021). A number of choices for
membrane materials are available, among which hydrophobic membrane materials,
e.g. polypropylene (PP), polyethylene (PE) and polyvinylidene fluoride (PVDF), are
the most commonly used (Cho et al. 2018). Advantages of MBR over conventional
346 A. Sinharoy et al.

bioreactor types are effective gas liquid mass transfer and low energy requirement.
Moreover, a MBR provides a high yield, high reaction rate, and increased tolerance
to toxic compounds (e.g. tar, acetylene, NOx and O2) (Campanario and Ortiz 2017).
Besides, this reactor type can operate at high CO partial pressure (PCO). Main
disadvantages are clogging and biofouling of the membrane due to excessive
biomass growth (Munasinghe and Khanal 2010a; Yasin et al. 2015).
Ethanol fermentation from syngas by Clostridium carboxidivorans P7 was stud-
ied in a hollow fibre membrane biofilm reactor (Shen et al. 2014a). The KLa value
(1096.2/h) was higher than most of the commonly used bioreactors. A very high
ethanol concentration of 23.93 g/L was achieved with an ethanol to acetic acid ratio
of 4.79.
CO bioconversion to H2 by Carboxydothermus hydrogenoformans was investi-
gated in a hollow fiber membrane bioreactor under thermophilic (70
C) conditions
(Zhao et al. 2013). The reactor demonstrated consistent performance under different
operational conditions such as liquid flow rate, temperature, CO pressure and CO
loading rate. The CO utilization was 0.44 mol CO/g VSS/day for a partial CO
pressure of 2 atm along with a maximum H2 yield of 92% (mol basis).
A thermophilic reverse membrane bioreactor with a flat plain hydrophilic
polyvinylidene fluoride (PVDF) membrane was used for biomethane production
from CO (Westman et al. 2016). The maximum methane production rate reached
186.0 mL/L/day with a very high syngas consumption, i.e. 7.0, 15.2 and 4.0 mL/L/
day for H2, CO and CO2, respectively. In a separate study, a thermophilic floating
membrane bioreactor was examined for methane production from syngas
(Chandolias et al. 2019). The maximum methane productivity amounted to
34.41 mmol/L/day along with H2 and CO utilization rates of 22 and 50 mmol/L/
day. The results obtained using the membrane bioreactor system for H2 and CO
utilization were, respectively, 38% and 28% higher than the values obtained using a
free cell suspended bioreactor.

13.4 Novel Bioreactors for Syngas Fermentation

In order to improve the process efficiency of syngas fermentation, a few novel

bioreactor configurations have been studied. These are biofilm based bioreactor
systems with innovative designs to increase the gas-liquid mass transfer (Fig. 13.3).

13.4.1 Horizontal Rotating Packed Bed Reactor

The horizontal rotating packed bed (HRPB) reactor is one such novel bioreactor used
for syngas fermentation (Shen et al. 2017). This bioreactor (Fig. 13.3b) is a combi-
nation of two distinct types of bioreactors, viz. the rotating biological contactor and
the packed bed reactor. In this system, similar to the rotating disks in a conventional
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 347

Fig. 13.3 Schematic of some selected novel bioreactor systems for syngas fermentation. (a)
Monolithic biofilm reactor [reused from Shen et al. (2014b), Copyright (2014) with permission
from Elsevier], (b) horizontal rotating packed bed reactor [reused from Shen et al. (2017),
Copyright (2017) with permission from Elsevier], (c) bulk-gas-to-atomized-liquid reactor [reused
from Sathish et al. (2019), Copyright (2019) with permission from Elsevier] and (d) moving bed
biofilm reactor [reused from Sinharoy and Pakshirajan (2021), Copyright (2021) with permission
from Elsevier]

rotating biological contractor, a packed bed column with biosupport materials is

fixed on the rotating horizontal shaft. The internal packed bed column configuration
is similar to a cage where biofilm carrying support materials are retained inside,
whereas the liquid phase can move freely in and out of the column during rotation
(Shen et al. 2017). In this reactor, a portion of the packing material is submerged,
whereas the remaining portion is exposed to the headspace. Due to continuous
rotation the biofilm is in alternate contact with the liquid phase and the headspace
gas. The mass transfer coefficient and available surface area for biofilm growth is
very high in this system (Shen et al. 2017). Due to proper packing in the internal
rotating column and the rotation of the packed column, bed clogging is completely
348 A. Sinharoy et al.

avoided in this bioreactor configuration. This novel bioreactor type was studied for
ethanol production from syngas by Clostridium carboxidivorans P7 strain and a high
ethanol titre and productivity of, respectively, 7.0 g/L and 6.7 g/L/day was achieved
(Shen et al. 2017).

13.4.2 Monolithic Biofilm Reactor

Ethanol production from syngas by C. carboxidivorans P7 was carried out using a

monolithic biofilm reactor (MBFR) (Shen et al. 2014b). Monoliths are structures
made up of parallel straight channels with thin walls separating them (Fig. 13.3a).
Using the MRFR at optimum process conditions, i.e. 300 mL/min of syngas flow
rate, 500 mL/min of liquid flow rate and 0.48/day of dilution rate, ethanol concen-
tration and productivity values were 4.89 g/L and 2.35 g/L/day. The values of syngas
utilization and product formation were much higher than those obtained using a
conventional bubble column reactor. This is due to high kLa values of the MBFR in
comparison to the bubble column reactor (BCR), due to the slug flow pattern in the
monolithic channels present in the MBFR.
The major advantage of the MBFR is that the pressure drop in this reactor is much
smaller than in other biofilm-based reactor configurations, such as the trickling-bed
or packed bed reactors, since the flow in the channel does not have bends and
obstructions. However, a critical factor that influences the performance of this
reactor type is the biofouling or sloughing of the biofilm attached on the monolithic
channel wall due to fluid flow through the channel. Although the monoliths have
been previously used extensively as catalyst support for multi-phase reactions such
as hydrogenation and oxidation in chemical industry, their application in biological
systems is limited, in particular as biofilm support. In comparison to other biofilm
reactors, the MBFR provides added benefits such as high surface area for biofilm
formation and good mechanical stability to the formed biofilm (Ebrahimi et al.
2006).

13.4.3 Bulk-Gas-to-Atomized-Liquid Contactor Reactor

Another novel bioreactor used for enhancing gas-liquid mass transfer during syngas
fermentation is the bulk-gas-to-atomized-liquid (BGAL) contactor reactor (Sathish
et al. 2019). The BGAL reactor consists of a packed bed with biosupport materials to
allow biofilm growth, and syngas saturated liquid micro-droplets are dispersed into
the packing inside the reactor. The syngas-rich liquid droplets then percolate through
the bio-bed and CO is converted to ethanol by the biofilm present on the support
material. The study reported a high ethanol productivity of 746 mg/L/h with an
ethanol to acetic acid ratio of 7.6 from syngas using Clostridium carboxidivorans P7
strain (Sathish et al. 2019).
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 349

In this bioreactor system, liquid is supplied as discrete minute size droplets which
causes a significant increase in the liquid and bulk gas interface (Fig. 13.3c). The
superior mass transfer was demonstrated with oxygen as a model gas, which resulted
in a very high oxygen transfer rate (OTR) of 569 mg/L/min and a KLa value of 2.28/
s. These values are 100 times higher than the values obtained using any other
bioreactor configuration.
Kaldness® biosupport material was used for biofilm formation due to its large
surface area. Use of immobilized biomass in this reactor configuration is more
advantageous than suspended biomass as dispersion of the syngas containing liquid
droplets into the bulk liquid in suspended biomass system (such as CSTR) causes
dilution of the dissolved gases, which in turn reduces the mass transfer and conver-
sion efficiency. The overall energy consumption for transferring the gas to the liquid
phase was also much lower (four-fold) in this BGAL bioreactor in comparison to
a CSTR.

13.4.4 Moving Bed Biofilm Reactor

The moving bed biofilm reactor (MBBR; Fig. 13.3d) is another biofilm based reactor
configuration with specially designed biofilm carriers, known as Kaldness®
biosupport material, which offer a large surface area for biofilm formation (Sinharoy
et al. 2020c; Sinharoy and Lens 2020). Although it has not been studied for liquid
biofuel production from syngas, CO bioconversion for H2 production has recently
been reported (Sinharoy and Pakshirajan 2021). A very high H2 production value of
19.5 mmol/L along with ~2 mmol/L of acetate were obtained for 36 mM inlet CO
concentration in the MBBR. The CO utilization was better (>70%) at low inlet CO
concentrations (in the range 9.05–15 mM). Methane production could be completely
avoided in the reactor by 2-Bromoethanosulfonate supplementation to the reactor
during its continuous operation.
The main advantages of the MBBR include high biomass retention, high treat-
ment efficiency, resistance to shock loading conditions and small footprint (Sinharoy
et al. 2019; Sinharoy et al. 2020c). In addition, bed clogging, which is mostly
observed with other attached growth bioreactor configurations, is uncommon in a
MBBR due to continuous mixing. Furthermore, due to the gaseous feed, mixing
inside the reactor can be easily achieved without the need for supplying a separate
(additional) gas stream for mixing, thus keeping the process costs low.
350 A. Sinharoy et al.

13.5 Bioreactor Strategies to Overcome Gas Liquid Mass

Transfer Problems

In a bioreactor system involving gaseous substrates, e.g. syngas, a high product yield
directly depends upon its bioavailability to the microorganisms, which in turn can be
limited by the high mass transfer rate of the substrate (Liu et al. 2014). A high cell
concentration necessary for the high yield also depends on mass transfer rate of the
gaseous substrates: a low mass transfer rate leads to reduced cell concentrations in
the fermentation media (Yasin et al. 2015). The most important factor limiting the
bioconversion of gaseous substrates is gas-to-liquid mass transfer, similar to oxygen
in aerobic fermentations (Worden et al. 1997). However, limitation due to gas liquid
mass transfer is more problematic for synthesis gas fermentations compared to that
in aerobic fermentations, as the solubility of CO (0.0225 g/kg water) or H2
(0.0015 g/kg water) is less than O2 (0.033 g/kg water) solubility (on a mass basis,
at 30
C) (Abubackar et al. 2011; Munasinghe and Khanal 2012). The different
strategies suggested to improve syngas solubility and gas-liquid mass transfer are
discussed below.

13.5.1 Agitation Speed and Impeller Configuration

An easy way to improve the gas liquid mass transfer is by enhancing the agitation
speed (rpm) in a bioreactor. Bredwell et al. (1999) reported a high KLa value of
101/h for CO at 450 rpm agitation speed. In another study, an increase in the
agitation speed up to 700 rpm is reported to enhance the CO liquid mass transfer
(Klasson et al. 1993). For achieving a high mechanical agitation speed, a high power
input is required, and, therefore, a high ratio of power required per reactor volume
makes this strategy economically weak for large scale industrial reactors, merely
because of the excessive power costs (Yasin et al. 2015).
A different strategy to improve the mass transfer is by employing different
impeller design (Ungerman and Heindel 2007). The effect of six different impeller
configurations, namely Rushton-type, Philadelphia Mixing concave (hollow blade)
turbine, Philadelphia Mixing pitched blade turbine (PBT), Philadelphia Mixing LS
hydrofoil, Lightnin A315 fluidfoil and Lightnin A310, on the gas liquid mass
transfer rate was analyzed. The Rushton-type performed better compared to the
other five impeller types. Also, the dual Rushton type showed a 27% higher gas
liquid mass transfer compared to the conventional single Rushton type impeller.
However, its performance in terms of the ratio of volumetric mass transfer coeffi-
cient to power input was poor. Hence, it should be emphasized that improved gas
liquid mass transfer in a stirred system demands a high power consumption and cost
associated with it. Also, the increase in the agitation speed to enhance the gas liquid
mass transfer may be damaging to shear sensitive microorganisms, thereby hamper-
ing microbial growth in the bioreactor (Munasinghe and Khanal 2012).
13 Syngas Fermentation for Bioenergy Production: Advances in Bioreactor Systems 351

13.5.2 Additives

The use of additives such as electrolytes, surfactants, alcohols and nanoparticles

(with or without surface modification) have been reported to enhance the gas liquid
mass transfer (Wainaina et al. 2018). The effect of various electrolytes such as
sulfate, nitrate, and chloride on the gas-liquid mass transfer was studied, and the
results show an enhancement of 3.4–4.7 times using the sulfate electrolytes (Cu, Co,
Mn, Ni and Fe) compared to a system without any added electrolytes (Zhu et al.
2009). Among the different metal ions, viz. copper, cobalt, nickel, iron, manganese
and magnesium, copper containing electrolyte is the best for enhancing the volu-
metric gas liquid mass transfer (Zhu et al. 2009). This is attributed to an increase in
the gas-liquid interfacial area and a reduction in the gas bubble coalescence in the
liquid phase. These findings on enhancement in the gas liquid mass transfer in water
due to the addition of electrolyte or metallic salts are also valid for aerobic systems.
For example, Zuidervaart et al. (2000) reported a 250% enhancement in the oxygen
mass transfer rate when metal sulfate based electrolytes [e.g., CuSO4, FeSO4,
ZnSO4, and Al2(SO4)3] were added to the liquid media. Use of surfactants such as
Tween (polyoxyethylene sorbitans) and Brij (polyoxyethylene alcohols) for enhanc-
ing the gas-liquid mass transfer is also reported (Bredwell et al. 1997).
The increase in gas-liquid mass transfer due to the addition of nano- or micro-
particles was reported by Zhu et al. (2008). Nanoparticles made of mesoporous silica
materials and coated with different hydroxyl, mercaptopropyl or organic groups
enhanced the gas-liquid mass transfer. The mercaptopropyl group grafted
nanoparticles yielded the maximum gas-liquid mass transfer (upto 1.9 times
increase). The presence of functional groups on nanoparticles and their hydropho-
bicity significantly enhanced the gas-liquid mass transfer (Zhu et al. 2008). Biolog-
ically synthesized iron nanoparticles enhanced the CO aqueous solubility by 56%
(Sinharoy and Pakshirajan 2020). The enhancement in CO solubility was attributed
to the presence of hydrophobic functional groups, such as amides, polyphenols and
proteins obtained from green tea extract, which was used as the reducing agent for
the iron nanoparticle synthesis.
The different mechanisms of gas liquid mass transfer enhancement due to small
particle addition include physical adsorption, homogeneous reactions in a slurry,
dissolved particles and suspended particles in heterogeneous reactive systems
(Beenackers and Van Swaaij 1993). The small particles transport an additional
amount of gas to the bulk liquid through gas adsorption at the gas liquid diffusion
layer followed by desorption in the bulk liquid (Zhu et al. 2008), which is referred to
as the shuttle effect as depicted in Fig. 13.4 (Cheng et al. 2019). This method of
enhancement is greatly influenced by the size and surface characteristics of the
particles.
352 A. Sinharoy et al.

Fig. 13.4 Schematic of nanoparticle mediated enhancement of the gas-liquid mass transfer

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 Part VI
Life Cycle Analysis
Chapter 14
Up and Downstream Technologies
of Anaerobic Digestion from Life Cycle
Assessment Perspective

Seyedeh Nashmin Elyasi, Hadis Marami, Li He, Ali Kaab, Junting Pan,
Hongbin Liu, and Benyamin Khoshnevisan

Abstract The development of anaerobic digestion (AD) plants has been considered
as a solution to overcome the depletion of fossil resources and the increasing
environmental pollution caused by the over-use of fossil fuels. However, there are
some challenges which may undermine the sustainability of the AD including its
associated up- and downstream strategies. In order to sustain the development of AD
plants it is necessary to calculate the environmental hotspots through their whole life
cycle and represent the possible solutions to mitigate the potential pollution. Among
different methods, life cycle assessment (LCA) is widely employed to quantify and
evaluate the environmental impacts of AD and its related up- and downstream
technologies. This chapter comprehensively summarizes the environmental impacts
of AD plants from an LCA point of view and proposes advanced strategies to
alleviate the negative impacts and bring biogas plants into sustainable circular
bio-economy.

Keywords Anaerobic digestion · Life cycle assessment · Biorefinery ·

Sustainability

Seyedeh Nashmin Elyasi and Hadis Marami contributed equally.

S. N. Elyasi · H. Marami · A. Kaab

Department of Mechanical Engineering of Agricultural Machinery, Faculty of Agricultural
Engineering and Technology, College of Agriculture and Natural Resources, University of
Tehran, Tehran, Iran
L. He
Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute
of Ministry of Agriculture and Rural Affairs, Chengdu, P. R. China
J. Pan · H. Liu · B. Khoshnevisan (*)
Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural
Sciences, Ministry of Agriculture and Rural Affairs, Beijing, P. R. China
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 361
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_14
362 S. N. Elyasi et al.

14.1 Introduction

In the last decades, the development of anaerobic digestion (AD) plants have
contributed to achieving both environmental pollution mitigation and energy inde-
pendency by utilizing waste streams, such as livestock manure and organic fraction
of municipal solid waste (OFMSW) to produce biogas and biofertilizer (Duan et al.
2020). Biogas and nutrient-rich digestate produced by the AD plants bring about
significant environmental savings, specifically, in terms of mitigation of greenhouse
gas (GHG) emissions (Fusi et al. 2016). However, properly designed AD plants
consist of upstream and downstream technologies for the appropriate treatment of
the feedstock and suitable biogas and digestate conditioning. Some upstream and
downstream processes rely on the consumption of considerable amounts of
chemicals as well as energy; thereby they could significantly undermine the envi-
ronmental sustainability of the AD plants (Khoshnevisan et al. 2016, 2018b;
Tsapekos et al. 2021). Furthermore, taking the environmental savings into consid-
eration, biogas utilization and digestate management are leading criteria to assess the
sustainability of AD facilities (Ardolino et al. 2020; Kohlheb et al. 2020). Therefore,
to sustain the processing of any type of feedstock in AD plants, the environmental
footprints of every constitution of the AD processes should be concerned.
In this regard, life cycle assessment (LCA), covering energy flow, mass flow,
GHG emissions, and other environmental impacts, is a well-established method
which can be employed as a standard tool to evaluate the sustainability of any
given system through its whole life cycle (Finkbeiner 2014). LCA enables the
quantification of the environmental impacts of the systems under consideration
and find the environmental hotspots and possible opportunities for future improve-
ments (Dastjerdi et al. 2021). In this context, considerable studies have already been
published under the framework of LCA to assess the environmental impacts of the
AD plants and their related subsystems. This chapter aims to comprehensively
review the state-of-the-art progress in the sustainability of AD plants by scrutinizing
the environmental impacts caused by both up and downstream processes (i.e.,
pretreatment technologies, biogas desulfurization and upgrading, as well as biogas
and digestate utilization) to show the recent advancements and the roadmaps for
future work.

14.2 LCA to Determine the Sustainability of AD Systems

LCA has long been used as a valuable tool to assess the environmental footprints of
AD systems. Since AD has been considered as a waste management strategy along
with a decentralized energy technology, LCA has been widely employed during the
past decades to scrutinize the sustainability of AD plants and their associated
sub-systems as part of the waste management system and waste to energy scenario
(De Jong et al. 2017). The AD technology has been in use for over 100 years and is
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 363

known as an effective approach to reduce the environmental impacts of fossil-based

energy sources (McCarty 1982). Moreover, it has been considered as a more
sustainable solution compared to other waste management strategies such as landfill
or incineration (Rao et al. 2010). Hence, numerous studies have been conducted to
compare either the environmental impacts of biomethane production via AD with
fossil based energy carriers, or different waste management strategies for a given
waste stream. These LCA studies have highlighted various aspects of the AD
technology, such as the use of AD for food waste management and bio-energy
production (Chiu and Lo 2016; Piao et al. 2016; Righi et al. 2016), utilization of
municipal solid and liquid wastes for bioenergy production with a focus on algal
biofuel production (Chiu and Lo 2016), biogas production in Europe (Hijazi et al.
2016) and sustainable management of agricultural residues (Bacenetti et al. 2016).
Table 14.1 summarizes some of the previous studies conducted in this context.

14.3 LCA of Upstream Technologies

14.3.1 Pretreatment Requirement

Organic waste, agricultural waste, manure, and energy crops are the most frequently
used biomass in AD plants (Aziz et al. 2019). The environmental performance of AD
systems largely depends on the feedstock used within the plants and the amount of
biogas exploited (Montgomery and Bochmann 2014). However, some specific types
of biomass such as lignocellulosic materials are critically slow to be degraded during
the AD process to produce reasonable amount of biogas (Lee et al. 2020). Accord-
ingly, prior to being fed into anaerobic digesters, specific pretreatment technologies
are needed depending on the feedstock used and the AD technology (Carrere et al.
2016).
AD consists of four main steps, i.e., hydrolysis (formation of sugar, long chain
fatty acid, amino acid), acidogenesis (acid formation i.e., alcohols and carbonic
acid), acetogenesis (acetic acid formation, i.e., CH3OH, NH3, H2S, and CO2), and
methanogenesis (biogas) (Koupaie et al. 2019). Pretreatments target enhancing the
hydrolysis step to facilitate the breakdown of the complex molecules, shortening the
biodegradation time, and enhancing biomethane formation (Tabatabaei et al. 2020).
Pretreatment technologies are divided into physical, chemical, biological, and com-
bined process (Gnaoui et al. 2020). Further information and subcategories of main
classes of pretreatment methods are illustrated in Fig. 14.1.

14.3.2 Biological Pretreatment Methods

From the LCA point of view, any pretreatment technology performed upstream of
AD, apart from the effectiveness for increasing the biomethane yield, should be
Table 14.1 Overview of studies related to LCA of AD systems
364

System Boundary
Feedstock
production
Surveyed and Biogas Product Impact assessment
location Goal and Scope collection AD Upgrading use method Feedstock type Reference
Italy Co-digestion of sewage No Yes No Yes ILCD Metrics Co-digested fruit and veg- Di Maria et al.
sludge and fruit and vege- etable waste with waste (2016)
table wastes mixed sludge from
municipal wastewater
treatment plants
USA Compare areal productiv- No Yes No Yes No Dairy waste water Ciliberti et al.
ities of AD and wind farms (2016)
France LCA of microalgae AD Yes Yes Yes Yes ReCiPe 2008 Microalgae Shimako et al.
(2016)
Spain AD of sewage sludge No Yes No Yes ReCiPe v.1.08 Sewage sludge Blanco et al.
(midpoint) (2016)
Sweden AD of food waste and No Yes No Yes CML 2001 (GHG Municipal solid waste Eriksson et al.
sewage sludge results only) (2016)
Germany Electricity production Yes Yes Yes Yes ReCiPe v.1.06 Co-digestion of maize Junne and
from agriculture feedstock (midpoint) and CED (or sunflower, horse Junne (2016)
v.1.08 manure and biogenic
waste), grass, rye silage,
and chicken manure
Germany Compare organic WM Yes No No No A method with sim- Organic waste Jensen et al.
systems ilar indicators as (2016)
specified in ReCiPe
midpoint metrics
Austria Biogas from pretreated Yes No Yes No ReCiPe v.1.08 Maize silage Kral et al.
maize silage (mid-point) and (2016)
ILCD 2011
S. N. Elyasi et al.
 14

USA Comparative analysis of No Yes No No IMPACT 2002+ and Wastewater Pericault et al.
TRACI 2.0 (2017)
Turkey AD of agricultural farm No Yes No Yes EDIP 2003 Agricultural wastes Nayal et al.
waste (2016)
Australia Co-digestion of sewage No Yes Yes Yes CML-IA Sewage sludge and sorted Edwards et al.
sludge and municipal food food waste (2017)
waste
Germany Electricity production Yes Yes Yes Yes ReCiPe v.1.06 Co-digestion of marine Ertem et al.
from microalgal feedstock (midpoint) Microalgae or maize, (2016)
grass, rye silage, and
chicken manure
France AD of sewage sludge and No Yes Yes Yes GHG Emissions; Sewage sludge Collet et al.
methanation ReCiPe v.1.08 (2017)
(endpoint)
Denmark Different strategies for No Yes Yes No IMPACT 2002+ Household waste Khoshnevisan
energy and nutrient recov- et al. (2018b)
ery from source sorted
organic fraction of AD
Denmark Environmental impacts of Yes Yes Yes Yes IMPACT 2002+ Grass Tsapekos et al.
biogas production from (2021)
grass
Denmark Biowaste biorefinery No Yes Yes Yes IMPACT 2002+ Municipal solid waste Khoshnevisan
concepts et al. (2020b)
China LCA of AD of pig manure No Yes No Yes IMPACT 2002+ Pig manure Duan et al.
(2020)
Adapted from Rajendran and Murthy (2019)
Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . .
365
 366

Fig. 14.1 Pretreatment technologies utilized in AD plants. LS, lignocellulosic. Characteristics taken from Chiu and Lo (2016), Shrestha et al. (2020), Torres and
Lloréns (2008), Wang et al. (2015) and Zhang et al. (2011)
S. N. Elyasi et al.
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 367

eco-friendly in terms of water, energy, and chemical consumption with less envi-
ronmental burdens (Ravindran et al. 2018). Table 14.2 summarizes the previous
studies with focus on pretreatments prior to AD. Among the existing technologies
for the pretreatment of bio-feedstock, biological routes have low energy demand and
could take part in the AD plants at low temperature in the absence of chemical
additives (Chiu and Lo 2016; Tabatabaei et al. 2020). The enzymatic pretreatment
has shown high efficiency to significantly improve the biogas production potential
while being independent of water consumption and has minor environmental
impacts (Asgher et al. 2014). Besides, fungi, specifically white-rot fungi, enable
removal of the environmental pollutants from solid and liquid waste before being
used for AD (Jayachandra et al. 2011; Montgomery and Bochmann 2014).
Biological pretreatment technologies, such as micro-aeration and anaerobic
methods, are carried out too slowly compared with physical and chemical processes
(Bochmann and Montgomery 2013). Therefore, some of the organic matter is
degraded to CO2 instead of being converted into methane in the subsequent AD
process (Chiu and Lo 2016). In order to alleviate this bottleneck to some extent, it is
recommended to combine microaeration with an anaerobic pretreatment reactor and
thereby enhancing the methane yield (Montgomery and Bochmann 2014). Apart
from the mentioned advantages and disadvantages, biological methods are com-
monly used in combination with other physical-chemical technologies to tackle the
aforementioned limitations (Khoshnevisan et al. 2018b; Tabatabaei et al. 2020).
Accordingly, the requirements for auxiliary pretreatment technologies have
undermined the wide industrial application of biological methods in AD systems
(Tabatabaei et al. 2020).

14.3.3 Physical Pretreatment Methods

As illustrated in Fig. 14.1, physical methods have a number of subcategories which

are different in terms of operation and energy demand as well as types of
biofeedstock for which they are best suited (Mudhoo 2012). The most prevailing
types of physical pretreatment technologies are mechanical methods in which large
and unwanted objects are properly separated and the size of the bulk substrate is
reduced (Chiu and Lo 2016). Mechanical pretreatment technologies, which are
mostly applied for conditioning of food waste, and lignocellulosic biomass normally
have a high power demand and consume large amounts of energy (Hernández-
Beltrán et al. 2019; Khoshnevisan et al. 2018b; Montgomery and Bochmann
2014). Meanwhile, they can lead to higher energy recovery which compensates
the background environmental burdens from the electricity consumption (Chiu and
Lo 2016). Moreover, by incorporating more renewable power into the electricity
grids, the GHG intensity of electricity could be alleviated to a great extent (Prasad
et al. 2016).
Among the mechanical facilities and from LCA viewpoint, pulping technology is
the most preferred method for the pretreatment of food waste due to appropriate
Table 14.2 Studies conducted over the LCA of pretreatment technologies of AD plants
368

Pretreatment method LCA

Chemical Functional
Feedstock Physical biological Combined unit Goal Investigated impact Reference
Sludge Thermal 1 t dry Evaluating the environmental impact GWP; POCP; EP; AP; Mills et al.
mass of of the configurations of AD fed ADP (2014)
sludge sludge
OFMSW Mechanical 1t Environmental impact assessment of GHG Carlsson et al.
OFMSW physical pretreatment (2015)
SSOHW Mechanical 1t LCA of upstream/downstream GWP; Human health; Khoshnevisan
SSOHW technologies Ecosystem quality; et al. (2018b)
Resources
Sludge Thermal Alkali & 1000 kg LCA evaluation pretreatment GWP Wang et al.
acid dry methods in methane generation (2020)
MSW Thermal & Biomass LCA of alternative management GWP; EP; AP; TP; ODP; Lima et al.
mechanical 1 t MSW options for MSW in Brazil OFP; PMF (2018)
Oil palm Chemical- 1 ml Analyzing the environmental impact GWP Lee et al.
empty fruit thermal methane of generating methane as a useful (2020)
bunches material with and without the
pretreatment technology
MSW Mechanical- 46,929 t Environmental performance of GWP; POF; PMF;TA; Beylot et al.
biological MSW mechanical biological pre-treatment ME,FSD;HT (2015)
MSW fed biogas plant
Sludge Thermal & 1 t mixed Environmental performance of Climate change, abiotic Cartes et al.
Ultrasound sludge (dry mechanical biological pre-treatment depletion, acidification (2018)
basis) MSW fed biogas plant and eutrophication
S. N. Elyasi et al.
 14

Microalgae Thermal 1 kWh Comparing the hydrothermal and GHG Xiao et al.
biogas solar (2020)
driven hydrothermal
GWP, global warming potential; TP, toxicity potential; EP, eutrophication potential; AP, acidification potential; GHG, greenhouse gas emissions; ODP, ozone
depletion potential; PMF, particulate matter formation; OFP, ozone formation potential; POCP, photo ozone creation potential; ADP, abiotic depletion; POF,
photochemical oxidant formation; TA, terrestrial acidification; ME, marine eutrophication; SFD, fossil resource depletion; HT, human toxicity; SSOHW, source
sorted organic fraction of household waste; OFMSW, organic fraction of municipal solid waste
Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . .
369
370 S. N. Elyasi et al.

sorting of food waste into a substrate and reject fraction as well as producing a
homogeneous feedstock for the AD process to have a higher methane recovery
(Khoshnevisan et al. 2018b). Furthermore, thermal pretreatment methods which falls
into physical pretreatment technology accelerates the hydrolysis of biofeedstock by
disintegrating cell membranes and facilitating the dissolution of recalcitrant organic
compounds, specially sewage sludge, microalgae, and food waste (Chen et al. 2012;
Gnaoui et al. 2020; Karuppiah and Azariah 2019). This method is normally
performed at very high temperature and thus requires a significant amount of heat
(Rajput et al. 2018). Although thermal pretreatment technology increases the meth-
ane yield of specific feedstock which otherwise have a low biodegradability during
the AD process, it may not increase the net environmental savings of AD systems
due to the high energy demand (Mills et al. 2014). It is worth mentioning that, if the
heat demand of thermal pretreatment methods could be supplied from renewable
sources such as solar systems, the environmental performance would be more
reasonable than using fossil-based heat sources. To illustrate, the total GHG emis-
sions from the production of biogas with hydrothermal pretreatment and solar-driven
hydrothermal pretreatment methods were estimated at –129.94, and –166.13 g CO2
eq/kWh biogas, respectively (Xiao et al. 2020).
Last but not least, ultrasound and electrokinetic disintegration are available
physical pretreatment technologies that are not applied for lignocellulosic substrate
and are only specified for sewage sludge (Dhar et al. 2012; Montgomery and
Bochmann 2014). Previous studies show that ultrasound and electrokinetic disinte-
gration could be useful technologies to improve the AD fed sludge by disordering
microbial cell walls in the liquid (Bochmann and Montgomery 2013; Cano et al.
2015; Cartes et al. 2018). However, generally, these pretreatment methods consume
electricity and reduce the net environmental profits since a part of exploited elec-
tricity from biogas should be utilized internally (Cano et al. 2015).

14.3.4 Chemical Pretreatment Methods

Chemical pretreatment approaches are popular for treating substrate with a high
share of lignocellulosic composition in which various types of chemicals such as
HNO3, H2SO4, HCl, H3PO4, KOH, NaOH, and CaO are applied to break the
complex structures and reduce the digester hydraulic retention time (Vannarath
and Thalla 2019; Zhen et al. 2017). The application of chemical pretreatment routes
could increase the methane yield up to 200% (Torres and Lloréns 2008; Wang et al.
2015; Zhang et al. 2011). For example, oxidative pretreatment by utilizing hydrogen
peroxide doubled the biogas production from rice straw (Song et al. 2012). However,
the use of chemicals for this purpose, specifically in the case of alkali and acid
methods, increases the concerns over the sustainability of chemical pretreatment
(Smullen et al. 2017, 2019). Accordingly, the chemical methods have not been
widely used in large scale biogas plants despite their vast utilization for the produc-
tion of ethanol (Carrere et al. 2016). Nevertheless, there are increasing attempts at
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 371

developing this pretreatment method whilst decreasing the environmental impacts

by recycling chemicals and residues (Smullen et al. 2019).

14.3.5 Combined Pretreatment Methods

In order to take the advantage of previously mentioned technologies and reduce their
bottlenecks to some extent, combined processes have been developed which cannot
be classified into the former categories (i.e., mechanical, thermal or chemical
pretreatment). Such pretreatment methods are more efficient since they are a com-
bination of two or more types of main pretreatment technologies (Montgomery and
Bochmann 2014). These methods are in forms of physicochemical, physical-
thermal, thermo-chemical, mechanical-chemical, and bio-chemical technologies.
Such integrated technologies require less energy, lower capital costs, and easy
operation compared to each individual technology (Shrestha et al. 2020). As a matter
of fact, by combining chemical methods with mechanical, thermal, and biological
technologies, the amounts of energy and chemical consumption by individual
technologies are reduced while the biomethane yield is increased. For example,
thermal-alkaline and microwave-alkaline pre-treatment have been shown to decrease
the consumption of alkali compounds whilst increasing the biogas production (Pilli
et al. 2020). As another example, some integrated thermal-based technologies such
as steam explosion and extrusion consume lower energy compared to individual
thermal and hydrothermal methods. Nonetheless, they are carried out at elevated
temperature with high positive effect on the methane yield on straw in AD system
(Martínez-Gutiérrez 2018).

14.4 Downstream Processes

In addition to the AD and its upstream processes, downstream technologies also play
a critical role in the sustainability of AD plants. Downstream processes include
biogas conditioning (i.e., purification and upgrading), biogas utilization, and
digestate management. In the following sub-sections, the effects of downstream
processes and technologies on the sustainability of AD plants are discussed in detail.

14.4.1 Biogas Conditioning

14.4.1.1 H2S Removal

The generated biogas in the AD process consists of 40–70% (v/v) CH4, 30–60%
(v/v) CO2, 0–1% (v/v) H2, 0–3% (v/v) H2S, and 0–5% (v/v) other gases (Tabatabaei
372 S. N. Elyasi et al.

Fig. 14.2 Physiochemical and biological desulfurization technologies from LCA perspective

and Ghanavati 2018). For further application of the biogas and the subsequent
production of heat and electricity in conventional conversion systems including
large turbines, gas micro-turbines, combined heat and power (CHP), and internal
combustion engines, the H2S content of biogas should be removed to avoid corro-
sions (Santos-Clotas et al. 2019). The removal of H2S, which is formed by microbial
reduction of sulfur compounds, is the most important downstream process for biogas
conditioning since the H2S content of biogas can cause corrosion or toxicity (Hauser
2017). There are various physicochemical and biological technologies available for
H2S removal. Figure 14.2 illustrates all types of available technologies employed for
H2S removal. Previous studies have widely characterized various types of desulfur-
ization technologies (Xiao et al. 2017; Zăbavă et al. 2019; Zulkefli et al. 2016).
Taking into account the desulfurization of biogas from LCA point of view, in all
the evaluated technologies so far, daily operation of desulfurization is the highest
contributor to the overall environmental impacts rather than the construction and
disposal of desulfurization units alongside their auxiliary equipment (Cano et al.
2018). Among various technologies commercialized, bio-desulfurization which is
normally performed in bioreactors are the most environmentally friendly and eco-
nomically favourable approach, especially compared with their physicochemical
competitors (Syed et al. 2006; Xinqiao et al. 2003; Zăbavă et al. 2019). This can
be attributed to the fact that bio-desulfurization technology normally doesn’t need a
high amount of energy and supplementary chemical compounds (Tabatabaei and
Ghanavati 2018; Zăbavă et al. 2019). Besides, the recovery of sulfur is also possible
so that the recovered sulfur in different forms (depending on the availability of
oxygen in the process) can substitute its chemical-based counterpart (Ramos et al.
2013). This brings more environmental savings and therefore can compensate the
environmental impacts caused by the consumption of energy resources.
Having considered the physicochemical biogas upgrading technologies, the use
of chemicals significantly contributes to the environmental impacts of the upgrading
technology. In other words, the background emissions caused by the production of
chemicals and materials are the most important contributors to the environmental
burdens of physicochemical biogas desulfurization technologies (Zulkefli et al.
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 373

2016). For instance, in chemical scrubbing and immobilized activated carbon desul-
furization, the H2O2 production stage as well as active carbon production and
preparation imposed the highest impacts on the biogas upgrading facility (Cano
et al. 2018).
In order to inject biogas into the natural gas grid or utilize it as transportation fuel,
apart from the H2S, the CO2 content of biogas should also be removed to a great
extent. The existing technologies for upgrading the biogas into the natural gas grade
biomethane, are chiefly divided into physicochemical methods and methanation
technologies (Sun et al. 2015). In the former, the CO2 content of biogas is separated
while in the latter the CO2 components of biogas react with H2 in a Sabatier reaction
either biologically or chemically and thereby forms additional CH4 (Angelidaki et al.
2018). Among the diverse types of physicochemical upgrading technologies, pres-
sure swing adsorption, water scrubbing, amine scrubbing, membrane separation, and
cryogenic separation are the most widely applied technologies which have been
commercialized and long been employed in biogas plants (Kapoor et al. 2019)
The previous LCA studies with focus on biogas upgrading technologies have
shown that biogas upgrading units are among the top two contributors to the
environmental impacts of AD systems (Hauser 2017; Wang et al. 2016). Corre-
spondingly, many studies have been conducted to compare and introduce the most
environmental friendly biogas upgrading technologies (Ardolino and Arena 2019;
Florio et al. 2019; Lombardi and Francini 2020; Starr et al. 2012). Table 14.3
summarizes some of those studies dedicated to the LCA of biogas upgrading
technologies. The results of LCA studies over the biogas upgrading technologies
demonstrated “win-win” situations compared to the fossil routes for the production
of transportation fuel and natural gas. However, comparing different forms of
upgrading technologies have shown that each technology has specific merits and
drawbacks over the others. This attributes to the difference between the input-output
analysis of each technology, including energy, material and emissions.

14.4.1.2 CO2 Removal

Methane slippage is the main contributor to the global warming damage category
among different biogas upgrading technologies (Ardolino et al. 2020). Chemical
absorption has the least methane leakage as illustrated in Table 14.4; however, this
facility requires high amounts of heat and further addition of chemicals such as
amines and alkali solutions (Angelidaki et al. 2018). Correspondingly, chemical
absorption would have high background emissions associated with the heat and
chemicals production. Generally, the results of LCA studies of physicochemical
technologies depend on hypothesis made by authors over methane slippage, energy
and chemical consumption. This makes it difficult to directly compare the results of
the previous studies (Ardolino et al. 2020; Hauser 2017; Lombardi and Francini
2020). Furthermore, another parameter which affects the LCA of physicochemical
upgrading technologies, is the possible recovery of CO2 to substitute its conven-
tional industrial counterpart. Although the CO2 released into the environment from
 374

Table 14.3 Overview of LCA studies on different biogas upgrading technologies

Physicochemical technologies Methanation Products and by products
OPS WS CA PSA MS CS Biological Chemical CO2 O2 Transportation fuel Natural gas Power Reference
✓ Cozma et al. (2013)
✓ ✓ Adelt et al. (2011)
✓ ✓ Rehl and Müller (2013)
✓ ✓ ✓ ✓ ✓ Morero et al. (2015)
✓ ✓ Leonzio (2016)
✓ ✓ ✓ ✓ ✓ ✓ ✓ Hauser (2017)
✓ ✓ ✓ ✓ Collet et al. (2017)
✓ ✓ Ardolino et al. (2018)
✓ ✓ ✓ ✓ Khoshnevisan et al. (2018b)
✓ ✓ ✓ ✓ ✓ Lombardi and Francini (2020)
✓ Kohlheb et al. (2020)
✓ ✓ ✓ ✓ ✓ ✓ Florio et al. (2019)
✓ ✓ ✓ ✓ ✓ Zhang et al. (2020)
✓ ✓ ✓ ✓ Lorenzi et al. (2019)
✓ ✓ ✓ Khoshnevisan et al. (2020b)
✓ ✓ Ardolino and Arena (2019)
✓ ✓ Tsapekos et al. (2021)
✓ ✓ ✓ ✓ ✓ Ardolino et al. (2020)
✓ ✓ ✓ Moioli et al. (2020)
OPS, organic physical scrubbing; WS, water scrubbing; CA, chemical absorption; PSA, pressure swing absorption; MS, membrane separation; CS, cryogenic
separation, biological methanation, chemical methanation
S. N. Elyasi et al.
Table 14.4 Specific characteristics of dominant and emerging biogas upgrading technologies
14

Physicochemical Biogas upgrading Methanation

Water Organic physical Cryogenic
Features scrubbing absorption Chemical absorption PSA Membrane separation separation Biological Chemical
Electricity 0.24–0.5 0.1–0.3 0.05–0.15 0.31–0.43a 0.15–0.3 0.2–0.79 nfe nfe
requirement
(kWh/Nm3
raw biogas)
Heat require- na < 0.2 0.55 na na na nff nff
ment
(kWh/Nm3
raw biogas)
CH4 loss (%) <3 1.5–4 < 0.1 1.6–4 0.5–13.5 0.5–3 nfg nfg
Purity (%) 95–9 93–98 96–99 95–99 96–98 97–99 97–99 97–99
Pre purifica- Not required Not required b Required Required Required Required b Required Required
b
tion
requirement
Simultaneous Yes Yes No Possible Possible Yes No No
H2S removal
CO2 recovery No Yes Yes c No Yes c Yes d na na
Consumable Antifouling, Organic solvents, Amines, alkali solu- Activated Silicone rubbers, cel- Glycol Inoculum Chemical
material drying agent polyethylene tions, antifouling, dry- carbon lulose acetate hollow refrigerant supplied catalyst
glycol ing agent zeolite fibers From
digestate
nf, not found
Data from: Adnan et al. (2019); Angelidaki et al. (2018); Ardolino et al. (2020); Hauser (2017); Hoyer et al. (2016); Kapoor et al. (2019)
Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . .

a
Value includes required electricity for upgrading, drying and compressing
b
H2S removal is required for high concentrations of sulfur (500 mg H2S/m3 biogas)
c
Recovering CO2 needs energy consumption
d
98% purified
375

(continued)
e
Indirect electricity consumption of 5.94 kWhe for the production of 0.11 kg hydrogen which is required for upgrading 1 kg biogas, and direct electricity
376

consumption for the main process

f
Process temperature for chemical and biological methanation, are, respectively, ~ 270  C and 60  C
g
Depends on the type of the reactor
S. N. Elyasi et al.
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 377

upgrading processes is accounted as biogenic, its reuse for industrial purposes brings
more environmental benefits. As illustrated in Table 14.4, recovering highly purified
CO2 from cryogenic separation technology without further energy demand for its
conditioning, could compensate the utilization of high amounts of electricity and
glycerol refrigerant (Hauser 2017).
Biogas methanation technologies referred to as “power to gas” have been intro-
duced as a promising approach to integrate biogas plants in the renewable electricity
sector, which could overcome the challenges of surplus electricity production from
renewable sources (Duan et al. 2020). This novel technology could form a closed-
loop cycle under the concept of circular bioeconomy in which: (1) hydrogen is
converted into biomethane with higher energy content, i.e., the energy content of
biomethane (36 MJ/m3) is almost three times as much as that of hydrogen (10.88 MJ/
m3) (Luo et al. 2012) and (2) the CO2 fraction of biogas is also transformed into an
energy product instead of releasing to the atmosphere without any implication.
However, the greenhouse gas (GHG) emission intensity of the electricity used for
the upstream water electrolysis technology supplying the hydrogen demand of the
methanation process plays a key role in the sustainability of this novel biogas
upgrading method (Zhang et al. 2020). Therefore, if the renewable share of electric-
ity utilized in electrolyzer units is less than 80%, the methanation process at the
downstream leads to higher impacts in all environmental damage categories, com-
pared to physicochemical biogas upgrading processes (Lorenzi et al. 2019).
Apart from the GHG intensity of the electricity, the type of feedstock used for the
production of biomethane at the initial stage is a predominant criterion since biomass
with higher methane potential decreases the hydrogen demand for biogas upgrading
and thereby increases the environmental benefits of the methanation process. More
specifically, in case of chemical methanation a part of the environmental burden is
attributed to the chemical catalyst, alumina supported Nickel-based catalyst
(Ni-α-Al2O3), utilized in the methanation reactor (Lorenzi et al. 2019). It is worth
noting that, if oxygen, which is produced as a co-product in electrolyzer units is
given credit, the net environmental impacts from the AD systems will decrease. This
is due to the fact that the oxygen demand is growing in municipal solid waste (MSW)
and wastewater treatment plants, as well as electric furnace and glass melting
industries and thereby utilizing high amounts of oxygen instead of its prevailing
conventional counterpart from cryogenic air separation that would provide eco-
nomic, environmental and energetic merits (Kato 2007; Khoshnevisan et al. 2020b).

14.4.2 Biogas and Digestate Utilization

14.4.2.1 Biogas Utilization

The final use of biogas and digestate and the products they substitute are key
parameters which can affect the overall LCA results. Some of the previous studies
demonstrated that if biogas is used as transportation fuel to substitute fossil-based
378 S. N. Elyasi et al.

fuel, better environmental impacts will be achieved compared to their combustion in

gas engines to produce heat and electricity (Ardolino et al. 2018; Khoshnevisan et al.
2020b). There are some studies which concluded that the utilization of biomethane
as transportation fuel is a more environmentally friendly than its application as
natural gas (Kampman et al. 2017; Pérez-Camacho et al. 2019). However, it can
vary from one country to another based on regionalized parameters such as source of
heat and electricity and the marginal transportation fuel which may be substituted in
this context. For instance, in a country like Denmark with dominant wind electricity,
the use of biogas for electricity and heat production will be constrained in the future
due to the high penetration of other types of renewable energy such as solar and
wind. Wind and solar energy are considered as the most environmentally friendly
electricity sources because of having no direct emissions at wind or solar farms
(Jacobson et al. 2015). Accordingly, the generated biogas in the forthcoming years
would be more likely to be used as transportation fuel or be injected into the natural
gas grid or valorized into value added products (Khoshnevisan et al. 2020a; Zha
et al. 2021). However, some studies discussed that the production of value added
products from biogas and digestate under the biorefinery concept still cannot com-
pete with the production of transportation fuels (Khoshnevisan et al. 2020b).

14.4.2.2 Digestate Utilization

Apart from biogas, the digestate remaining at the end of AD process contains
reasonable amounts of macro- and micro-nutrients and is thereby considered as a
substitution for chemical fertilizers (Styles et al. 2018). In fact, utilizing the
remaining slurry after AD as soil amendment not only avoids the generated emis-
sions from the production of chemical fertilizer but also from its application.
However, the limitations such as long storage time, emissions from storage systems,
oversupply of digestate on the farmlands, and the application out of cropping season
or at improper time undermine the sustainability of using digestate as a substitute for
chemical fertilizers (Khoshnevisan et al. 2021). In the previous LCA studies,
ammonia and methane emissions from digestate storage and application as well as
emissions caused by the transportation of such bulky materials imposed the highest
environmental burdens associated with digestate management (Tsapekos et al.
2021). Although transportation of digestate is still challenging, some studies dem-
onstrated that application of digestate on surrounding farmlands would still have
better environmental performance than their use for microalgae or powder
biofertilizer production (Duan et al. 2020)
Depending on the feedstock used for biogas production, the digestate may contain
heavy metals or soil contaminants such as Cu, Zn, and Mn, hence increase the risk of
soil contamination (Valeur 2011). Therefore, digestate may need some pretreatment
to remove contaminants and hazardous components from the digestate before field
application. Moreover, in regions with intensive biogas plants, if long distance
transportation is needed, composting and pelletizing are widely employed as post-
treatment of the digestate (Rehl and Müller 2011). The type of technology used for
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 379

digestate treatment is related to the composition of the remaining slurry from the AD
reactor (Styles et al. 2018). Normally digestate management is carried out through
mechanical, physicochemical or biological processes (Monfet et al. 2018). The
characteristics of each type of technology employed for the post-treatment of
digestate have been thoroughly described in the review paper by Monfet et al.
(2018). From an LCA point of view, the high energy demand of post-treatment
technologies has been found as the environmental hotspot. Mechanical treatment,
i.e., belt drying and thermal concentration have the highest energy demand which
affects the environmental performance of AD systems (Rehl and Müller 2011).
Furthermore, the consumption of specific amounts of chemicals such as sulfuric
acid, sodium hydroxide and powered polymers in physicochemical and biological
treatments would further increase the environmental damages caused by the post-
treatment of the digestate (Vázquez-Rowe et al. 2015).

14.4.3 AD in the Biorefinery Concept

The recent advancement in biotechnological sciences and rising environmental

awareness have encouraged the expansion of biogas plants with more attention to
the circular bio-economy. Attempts are made to produce more value-added products
and building block chemicals than biogas and digestate. Hence, some researchers
have studied different valorization pathways of bio-feedstocks and the effluents from
anaerobic digestion under the biorefinery concept (Elyasi et al. 2021). Figure 14.3
shows a simplified process design of an AD based biorefinery. Agricultural waste,
forestry residues, food waste, and industrial organic waste have shown great poten-
tial for being treated under the biorefinery approach to produce a wide range of
bioenergy and biomaterials (Nizami et al. 2017). The production of succinic acid,
ethanol, single cell protein, phenolic compounds, dietary fibres, animal feed,
enzymes, polymers, and lactic acid, among others, have been widely tested and
their sustainability has been addressed (Dermeche et al. 2013; Ertem et al. 2016;
Khoshnevisan et al. 2020b; Lam et al. 2018; Strazza et al. 2015).
The transition from conventional biogas plants toward biorefineries would bring
about controversial sustainability challenges such as energy efficiency, economic
printability, direct/indirect emissions which should be addressed in a standard
framework. Therefore, a number of well-established sustainability assessment
tools have to be implemented towards a successful multi-functional biorefinery
(Hetemäki et al. 2017). In this context, LCA as a standard sustainability assessment
tool could be employed to characterize the environmental impacts of feedstock
biorefining. Such evaluations lead to attaining more mature knowledge about oppor-
tunities to improve the circular production of bioproducts. This also enables
decision-makers to find out how such value chains have pros & cons over the
traditional biogas production. Table 14.5 illustrates some the LCA studies conducted
on the biorefining of different types of biomass.
380 S. N. Elyasi et al.

Fig. 14.3 A simplified process design of an AD based biorefinery. SCP, single cell protein; CHP,
combined heat and power generation; Ag waste, agricultural waste

It is worth noting that the biorefining approach can have a positive influence on
one aspect, but a negative point in another aspect. Holistically, investigating the
LCA and environmental impacts of biorefineries have shown that the cascading use
of biomaterials through integral bioprocesses would lead to economic growth,
positive environmental impacts, expansion of fossil-free bioprocesses, and positive
social effects in terms of improving the regional economy following the product
diversification (Bello et al. 2018; García-Bustamante et al. 2018; Husgafvel et al.
2017). Specifically, the production of various bioproducts through biorefinery path-
ways lead to climate mitigation compared with their chemical processes
(Khoshnevisan et al. 2020b; Sadhukhan and Martinez-Hernandez 2017; Seghetta
et al. 2016). Furthermore, under the concept of a biorefinery, the worthless materials
are returned back into the supply chain more efficiently. To illustrate, by applying
the digestate and biogas downstream for the production of single cell protein (SCP),
the usage of environmental-costly protein sources such as soybean meal would be
limited (Khoshnevisan et al. 2018b).
The exploitation of a wide range of bioproducts under the biorefinery concept
requires higher quantities of energy and additive chemicals/materials (Bello et al.
14 Up and Downstream Technologies of Anaerobic Digestion from Life Cycle. . . 381

Table 14.5 LCA studies focusing on the valorization of various types of biofeedstock to diverse
bioproducts
Type of
biomass Products Remark on LCA Reference
Seaweed Ethanol, proteins and liquid Environmental restoration & Seghetta et al.
fertilizer climate mitigation (2016)
The major environmental
hotspot is energy consumption
Residual Bioethanol, acetic acid, lig- Identified hotspot: Bello et al.
beech nin, and furfural; Glucose, Pre-treatment of biomass; (2018)
woodchip hemicellulose energy demands of the process
and enzyme production
Municipal Levulinic acid, electricity, Global saving of 0.4 kg CO2-eq Sadhukhan
solid waste fertilizer per kg levulinic acid and Martinez-
Hernandez
(2017)
Organic Succinic acid, lactic acid, Biorefining pathway and biogas Khoshnevisan
fraction of single cell protein, usage are the determinable et al. (2020b)
municipal biomethane, heat, electricity, effect on LCA results
solid waste biofertilizer
Vine shoots Lactic acid, and furfural Production of bio-based lactic Pachón et al.
acid or furfural has strong (2020)
environmental benefits
Castor Electricity, biodiesel, etha- Improvement effect on global Khoshnevisan
nol, heat, biomethane, warming and resources; Nega- et al. (2018a)
glycerol tive environmental impacts on
the Human Health and Ecosys-
tem Quality
Citrus peel Phenolic compounds, oil, Replacement of microwave and Joglekar et al.
methane, syngas, ethanol ultrasound-assisted technolo- (2019)
gies by conventional intensify-
ing technology improve the
environmental effects
Sugars and Succinic acid, adipic acid, The life-cycle GHG results are Cai et al.
lignin Biodiesel highly sensitive to co-product (2018)
handling methods
Wheat straw Bioethanol, lactic acid, elec- Net environmental saving com- Parajuli et al.
& alfalfa tricity, feed protein, pared to petrochemical compet- (2017)
biofertilizer, fodder silage itors of bioproducts
Energy consumption, specifi-
cally for the production of
enzymes in biorefinery was a
major hotspot
Olive wastes Biofuel, phosphate salts, Mitigation of environmental Khounani
natural antioxidant, and an impacts of foods et al. (2021)
oxygenated fuel additive Transition from olive agri-food
(triacetin) to agro-biorefinery is environ-
mentally favorable
382 S. N. Elyasi et al.

2018). For instance, the production of lactic acid under a biorefinery approach
requires high energy and chemical consumption (Liu et al. 2021). Thus, the choice
of biorefinery technologies in terms of energy and pollutant intensity have major
environmental effects (Bello et al. 2018; Seghetta et al. 2016). Such limitations are
the reason that the production of biofuel still outperforms the production of bio-
chemicals (Khoshnevisan et al. 2018a, 2020b). This is due to the low conversion
efficiency as well as the energy intensity of biorefining technologies (Bello et al.
2018; Khoshnevisan et al. 2020b; Seghetta et al. 2016). Overall, downstream biogas
usage, bioproduct substitutions, and the conversion rate are the most effective
parameters affecting the LCA of biorefineries (Khoshnevisan et al. 2020b). If
agricultural biomass is used in biorefineries, some other criteria such as crop
cultivation and land use change should not be neglected in the boundary of LCA
studies (Katakojwala and Mohan 2020; Liu et al. 2021)
Despite all the attempts made so far to investigate the LCA of biorefineries, it still
suffers from low transparency and clarity. Hence, future research should critically
evaluate the environmental impacts of the AD process under the biorefinery concept
to attain more sophisticated knowledge and thereby selecting the most suitable
interlocked bioprocesses for biogas production. Also, further attempts should be
made for process optimization to satisfy the environmental indicators. This would
lead to the production of more value-added products alongside bioenergy and thus
overcoming the environmental hazards of the petrochemical based production of
building block chemicals and materials.

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Chapter 15
Life Cycle Assessment of Anaerobic
Digestion Systems: An Approach Towards
Sustainable Waste Management

Marlia M. Hanafiah, Iqbal Ansari, and Kalppana Chelvam

Abstract The wastes generated from different human activities such as agricultural
or crop wastes, livestock, food wastes are biodegradable wastes, which can be
utilized for various purposes including biogas generation and production of other
value added products. The main aim of this chapter is to provide information on the
types of waste that can be used to produce biogas through anaerobic digestion
(AD) system. Anaerobic digestion is a process of conversion of biodegradable
waste into biogas by the action of microbial communities. The gases obtained during
AD mainly consist of methane, carbon-dioxide and some amount of water vapour.
Better understanding of the different processes, including the pretreatment and post-
treatment involved in the AD, is necessary to develop the green technology to
achieve sustainable development. In this regard, LCA is a great tool to study the
feasibility of the AD process for environmental sustainability. In this chapter, an
introduction to life cycle assessment (LCA) and description of previous studies on
the LCA of anaerobic digestion systems towards environmentally sustainable man-
agement practices are presented. The chapter also discusses LCA for improvement
of the performance of the anaerobic digestion process of various biodegradable
wastes for generation of biogas. The findings suggest that more future research is
needed for process improvement and to promote the wide application of the AD
process for generation of green energy.

Keywords Environmental LCA · Organic waste · Renewable energy · Waste-to-

energy · Circular economy · Sustainable management

M. M. Hanafiah (*)
Department of Earth Sciences and Environment, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, Bangi, Selangor, Malaysia
Centre for Tropical Climate Change System, Institute of Climate Change, Universiti
Kebangsaan Malaysia, Bangi, Selangor, Malaysia
e-mail: [email protected]
I. Ansari · K. Chelvam
Department of Earth Sciences and Environment, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, Bangi, Selangor, Malaysia

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 391
A. Sinharoy, P. N. L. Lens (eds.), Renewable Energy Technologies for Energy
Efficient Sustainable Development, Applied Environmental Science and Engineering
for a Sustainable Future, https://doi.org/10.1007/978-3-030-87633-3_15
392 M. M. Hanafiah et al.

15.1 Introduction

Biodegradable waste like agricultural and crop residue, food waste, livestock wastes,
sewage sludge, organic municipal waste are rich sources for energy production,
including biogas or biofuels (Appels et al. 2011; Banch et al. 2020b). Conversion of
organic wastes to energy and by-products has become increasingly important for
achieving the circular economy (Banch et al. 2020a). Utilization of such wastes for
bioenergy production provides many advantages such as low-cost of substrate,
minimizing environmental impacts by reducing waste generation, providing extra
revenue to the producers, reducing fossil fuel dependency and making energy
sufficient (Hanafiah et al. 2018; Nizam et al. 2020). Anaerobic digestion systems,
embracing the concept of waste-to-energy is one of the environmentally friendly
technologies for generating biofuel and biogas (Aziz et al. 2019; Vambol et al.
2019).
However, in many areas, solid wastes are being dumped directly to the environ-
ment without proper treatment and management (Ashraf et al. 2019). Issue related to
improper waste management affects human health and the environment (Al-Raad
et al. 2020; Ashraf and Hanafiah 2019; Hanafiah et al. 2020). A common disposal
method such as landfill consumes large amounts of land resources, energy, and
causes significant atmospheric greenhouse gas emissions (Banch et al. 2019). Apart
from that, landfills also produce a substantial amount of landfill leachate containing
high concentrations of organic and inorganic pollutants that poses hazards to the
environment. Therefore, in recent years, research in alternative renewable energy
generated from organic wastes is gaining more interest.
The waste-to-energy concept has been introduced to solve environmental degra-
dation related to waste disposal (Ashraf and Hanafiah 2017). Various technologies
and methods have been used to convert waste into bioenergy. However, this further
requires investigation on the impacts of these technologies on the environment,
human health and economical aspects. In this context, holistic and comprehensive
assessment of the impacts of anaerobic digestion (AD) systems used to convert
waste to energy is important. This will help to identify strategic and feasible
improvements in waste management and renewable energy generation towards
environmental sustainability (Aziz et al. 2020). This chapter provides useful infor-
mation to researchers, students, and policy makers on life cycle assessment (LCA) of
AD systems.

15.2 Anaerobic Digestion Systems

15.2.1 Anaerobic Digestion Process

Anaerobic digestion is a process by which biodegradable materials such as animal

waste, food, lignocellulosic waste and agricultural waste are broken down by
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 393

microorganism through the process of decomposition in the absence of oxygen to

produce biogas. Anaerobic digestion (AD) is a complex biodegradation process of
organic matter, by the synergistic action of a range of microorganisms (Kougias and
Angelidaki 2018). The main end products obtained during AD are biogas (CH4 and
CO2) and digestate (Carlsson et al. 2015). The schematic of the anaerobic digestion
process is shown in Fig. 15.1. The biogas produced during AD consists of CH4
(53–70%) and CO2 (30–50%) and some small amount of water vapor (5–10% or
higher in thermophilic phases), oxygen (0–5%), hydrogen sulfide (0–20,000 ppm),
siloxanes (<0.08–0.5 mg/g-dry), ammonia (< 100 ppm), hydrocarbons, carbon
monoxide, along with nitrogen (2–6%) (Mao et al. 2015). Biogas is considered to
be a green and renewable source of energy. Several techniques including
pre-treatment of substrate, co-digestion, additives and optimization of parameters
have been applied for improving biogas production (Gupta et al. 2012a).
Apart from that, biogas can also be produced by thermochemical conversion of
biomass that can be achieved through a process known as gasification. The
bio-gasification of vegetable waste could be a potential source of biogas generation
by using some indigenous microbes from natural sources (Gupta et al. 2010). In
general, biogas is generated from many other sources such as landfills, municipal
waste, green waste, energy crops, sewage, and industrial wastewater. Biogas is
treated to remove carbon dioxide to be used as renewable natural gas, which is
also known as biomethane. On the other hand, digestate, which is the co-product of
anaerobic digestion, is rich in ammonia and phosphate which are essential for the
growth of photosynthetic organisms like microalgae. These products can be further
refined through certain processes to meet specific requirements to aid the growth of
plants.
The efficiency of biogas production is mostly dependent on the biodegradation
process and the optimum conditions increase the process efficiency and rate (Li et al.
2017). According to Saraswat et al. (2019), factors such as temperature, carbon to
nitrogen (C/N) ratio, compression ratio, total solid concentration and the pH value
greatly influence the biogas production. The pH influences the activity of the
microorganisms during the process. Each class of microbes has a definite range of
pH where they perform optimal. For instance, a pH more than 6.5 is more suitable for
methanogenic bacteria/archaea; however, acidogenic bacteria work better at a pH
value of 5–6.5. Sitorus et al. (2013) have investigated the biogas production with
variations in temperature and pH throughout the process from mixed fruit and
vegetable wastes during AD. The study found great influence of these factors on
the biogas production rate as well as on the biogas composition.
The substrate composition is also important in the AD process, where the
degradability rate of the feedstock varies with its composition (Winichayakul et al.
2020; Hundal et al. 2019). For example, a higher carbohydrate content in the
substrate will result in a higher yield of biogas due to the high degradability rate
and rapid transformation of carbohydrates (Yang et al. 2015). Li et al. (2017) have
studied different types of food wastes and showed the effect of carbohydrates,
proteins and lipids on the AD process and the methane production. In the study,
 394

Anaer obic Digestive System

THE BIOCHEMISTRY

Energy crops Waste water Complex organic matter

(Carbohydrates, proteins, fats)

Livestock waste Food waste 1. Hydrolysis

Soluble organic molecules

(Sugar, amino acids, fatty acids)
Anaerobic Digester
2. Acidogenesis
Electricity
Biogas Digestate
Volatile fatty acids

Heat • Fertiliser
• Soil amendments Acetic acid 3. Acetogenesis H2 CO2
Biomethane • Livestock bedding

Fuel 4. Methanogenesis
Gas grid CH4 + CO2

Fig. 15.1 Overview of the anaerobic digestion process along with the applications of its products in different sectors
M. M. Hanafiah et al.
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 395

different food wastes contained different organic compositions which lead to a

change in methane production.

15.2.2 Products from Anaerobic Digestion Process

The biogas produced is mostly methane (CH4) and carbon dioxide (CO2) with a
minimal amount of water vapour and other gases. The carbon dioxide and other
gases can be removed leaving only methane which is the primary component of
interest. This biogas purification process will increase the energy value of the biogas
as a source of renewable energy to produce heat and electricity for power engines,
heat generation in digesters, boilers and heating furnaces, run alternative-fuel vehi-
cles and domestic and industrial usage as alternative to natural gases.
Digestate is the other product which is a wet mixture of the material that is left
after the anaerobic digestion. It is separated into solid and liquid which are rich in
nutrients and can be used as fertilisers for crops. Digestate can be applied directly on
the land and added into the soil to improve soil quality which will promote plant
growth. Moreover, some engineering techniques can be applied post-digestion to
recover the phosphorus and nitrogen present in the digestate and produce concen-
trated nutrient products such as struvite (magnesium-ammonium-phosphate) and
ammonium sulfate fertilisers. Other than that, digestate can also be made as bedding
for livestock and be used for soil amendments. Figure 15.2 illustrates the elements of
the biogas recovery system.

15.3 Biogas from Organic Wastes

15.3.1 Food Waste

Waste generation in recent times is quite high in amount and particularly for food
waste it is up to 32% from all food produced throughout the world (Morales-Polo
et al. 2018). This leads to a remarkable social and economic loss as well as
environmental issues (Browne and Murphy 2013; FAO 2011; Papargyropoulou
et al. 2014). According to the Ministry of Housing and Local Government
(MHLG) Malaysia, disposal of food waste directly to the landfill sites is one of the
main sources of greenhouse gases (GHG) emissions in Malaysia (Hoo et al. 2017).
Another research carried out by Paritosh et al. (2017) also states that the carbon
footprint of food waste is estimated to contribute to the GHG emissions by accu-
mulating approximately 3.3 billion tonnes of CO2 in the atmosphere annually.
Food waste makes up a large percentage of a landfill, where only a very minimal
amount of food waste is being recycled into soil improver or fertiliser. The remaining
food waste in landfills releases methane to the atmosphere as it breaks down.
However, the energy potential produced from food waste is very significant. During
396 M. M. Hanafiah et al.

Organic Waste Collecting System

Collects waste in way that is
compatible with biogas recovery

Organic Waste Hadling System

Store and pretreats organic waste(s)

Anaerobic Digester Coproducts

A system in which AD can occur to
Digestate (solid & liquid)
produce biogas and other co-products

Biogas
Biogas Handling System
Electricity,Fuel & Flare
Collects and treats biogas excess

Fig. 15.2 Steps in biogas production from organic wastes

recent years, due to the generation of tremendous amounts of food waste, it can be a
promising substrate for the AD process (Lin et al. 2013; Paritosh et al. 2017). Food
waste (FW) poses the characteristics of high moisture and organic contents due to
which it is easily degradable (Li et al. 2019a; Ren et al. 2018). During the AD
process, organic matter present in FW is converted into CH4, CO2 and other gases.
However, FW is characterized by a low C/N ratio (Akindele and Sartaj 2017).
Hence, many times the AD process of FW results in the accumulation of volatile
fatty acids (VFAs) in the reactor, due to the imbalanced nutritional level in the
substrates (Chukwudi et al. 2019; Yazdanpanah et al. 2018).
There are many studies on biogas generation using food waste (Table 15.1).
El-Mashad and Zhang (2010) investigated the production of biogas from food waste
in batch digesters under mesophilic conditions (35
C). The methane produced from
the food waste was 353 L/kg VS after 30 days of digestion. Al-Wahaibi et al. (2020)
carried out a techno-economic investigation of biogas production from different
types of food waste. The methane production during the fermentation period showed
a good correlation between the theoretical and experimental values with a coefficient
of determination R2 ¼ 0.99. The cumulative gas production from mixed food waste
samples was 1550 mL/1 g of dry matter at day 21. Jeong-Ik et al. (2018) have studied
the biogas production from food waste with wood chips via anaerobic co-digestion.
Use of wood chips as co-substrate increased the rate of methane and hydrogen
production in AD. 20 mL/g of methane and 13.9 mL/g of hydrogen were generated
at the food waste to wood chip ratio of 0.5, during 15 days at 35
C.
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 397

Table 15.1 Biogas production from different types of waste using different AD systems
Type of Quantity/
Waste/location Study Method biogas productivity Reference
Fruit and vege- Lab Anaerobic diges- Methane 50–60% in Maile et al.
table waste tion (biochemical volume (2016)
(FVW), Johan- methane potential),
nesburg mar- bioprocess
ket, AMPTS II
South Africa
Kitchen waste Both The batch diges- Methane Productivity Wang et al.
(KW) and fruit/ lab & tion in lab-scale in (0.725L CH4/g (2014)
vegetable waste pilot- 2-L conical flasks, VS)
(FVW), China scale Single-phase
co-digestion
A10-L completely
stirred tank reactor
(CSTR)
Fruit and vege- Lab Single-stage anaer- Methane Methane yield of Farhat et al.
table waste obic sequencing 340 L/kg volatile (2018)
(FVW), Tunisia batch reactors solids (VS)
(ASBRs) and con- Biogas (8% H2,
tinuously stirred 28.5% CO2 and
tank reactor 63.5% CH4)
(CSTR)
Municipal solid Lab Anaerobic Biogas Biogas 493.8 N Pavi et al.
waste (MSW) co-digestion and mL/g VS, meth- (2017)
and fruit and (batch) methane ane- 396.6 N
vegetable waste mL/g VS,
(FVW), Brazil
Fruit and vege- Lab Anaerobic diges- Biogas Biogas: Masebinu
table waste tion semi- and 0.87 Nm3/kg VS et al.
(FVW) Johan- continuous digester methane with 57.58% (2018)
nesburg Mar- methane
ket,
South Africa
Market waste Lab Anaerobic diges- Biogas 35 L/kg MW/day Ranade
(vegetables & tion 200L Biogas et al.
fruits), Pune, plant (1987)
India
Fruit and vege- Lab Anaerobic Methane MSS: 0.303 m3/ Arhoun
table waste co-digestion (kg VS) et al.
(FVW), munici- FVW: 0.403 m3/ (2019)
pal sewage (kg VS), average
sludge (MSS), methane content
Malaga, Spain of digester biogas
was about 62–
64%
Fish waste (FW) Lab Anaerobic Biogas Average biogas Bouallagui
Fruit and vege- co-digestion anaer- production rate et al.
table waste obic sequencing varied between (2009)
(FVW), Tunis, batch reactors 1.53 and 2.53 L/
Tunisia (ASBR) day
(continued)
398 M. M. Hanafiah et al.

Table 15.1 (continued)

Type of Quantity/
Waste/location Study Method biogas productivity Reference
Fruit and vege- Lab Lab-scale continu- Biogas Biogas at rate of Lin et al.
table waste ous stirred-tank and 2.17 m3/(m3 day) (2011)
(FVW) and food reactor at 35
C methane and methane at
waste (FW), 0.42 m3 CH4/kg
Tsinghua, China VS. Methane
production was
0.49 m3 CH4/kg
VS at the opti-
mum mixture
ratio 1:1 for
co-digestion
Fruit and vege- Lab Solid-state strati- Biogas Gas production Chanakya
table wastes fied bed (SSB) about 0.5 L/L/ et al.
(FVW), Banga- process day at a 2 g/L/day (2007)
lore, India feed rate
Fruit and vege- Lab Continuous culture Hydrogen H2 production Garcia-
table waste setup (glucose as (H2) rate (1.7 mmol/ Peña et al.
(FVW), Mexico substrate) and day), the highest (2013)
City, Mexico anaerobic reactor cumulative H2
volume of 3 L volume (310 mL)
KW (Kitchen Lab Acidogenic-phase Methane Highest methane Yang et al.
waste), fruit– reactor (APR) and daily capacity (2013)
vegetable waste methanogenic- 325 mL (50 %
(FVW) Hang- phase reactor KW)
zhou, China (MPR)
Fruit and vege- Lab Batch anaerobic Biogas Biogas produc- Garcia-
table waste (batch) digestion, stainless and tion: 0.42 m3/kg Peña et al.
(FVW), Mexico steel tubular Methane VS), methane: (2011)
reactor 0.25 (m3/kg TS)
Agroindustrial Lab Anaerobic Methane After 63 days, González-
wastes (AW), digestion 63.89 mL CH4 Sánchez
banana waste, per gram of et al.
Chiapas, chemical oxygen (2015)
México demand of the
waste
Anaerobic Lab Two-phase anaero- Methane Methane: Wu et al.
sludge, Beijing, bic digestion, up 261.4 mL/g COD (2016)
China flow anaerobic removed. High
sludge bed reactor methane-
348.5 mL/g VS
removed
Municipal bio- Pilot- Anaerobic Biogas Biogas produc- Liu et al.
mass waste scale co-digestion in tion @ rate of (2012)
(MBW), China Continuous stirred- 4.25 m3/(m3 day)
tank reactor at organic load-
(CSTR) ing rate of 6.0 kg
VS/(m3 day) and
hydraulic reten-
tion time of
20 days
(continued)
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 399

Table 15.1 (continued)

Type of Quantity/
Waste/location Study Method biogas productivity Reference
Fruit and Vege- Pilot Full-scale anaero- Biogas Average specific Scano et al.
table Wholesale scale bic digestion sys- and biogas produc- (2014)
Market of Sar- tem, Pilot scale Methane tion was about
dinia, Italy tubular reactor, 0.78 Nm3/kg VS,
Single waste with a specific
substrate methane yield of
about 0.43 Nm3/
kg VS
Garden waste Lab Anaerobic diges- Methane First enrichment Gupta et al.
(GW) CIMFR, tion produces meth- (2012b)
Dhanbad, India Glass apparatus ane: 19.74 cc on
that contained one 49th day
reaction vessel and Second enrich-
one reservoir for ment produces
the collection of methane:
gas by water dis- 107.90 cc on 49th
placement method day
Fruit and vege- Lab Anaerobic diges- Biogas Biogas yield of Viswanath
table wastes tion (60-liter 0.6 m3/kg VS et al.
(FVW), India digester) major yield (1992)
(74.5%) of gas
within 12 h of
feeding at a
16-day HRT
whereas at a
24-day HRT only
59.03% of the
total gas
Fruit and vege- Lab Co-digested with Methane FSS + FVW pro- Park et al.
table waste first stage (FSS) duced (2012)
(FVW) British and second stage 514  57 L CH4
Columbia, anaerobic digester kg/VS and
Canada sludge (SSS) sepa- SSS + FVW pro-
rately, in batch duced
reactors 392  16 L CH4
Waste activated Lab Two-stage anaero- Biogas Biogas yield of Dinsdale
sludge (WAS)/ bic co-digestion of 0.37 m3 kg/VS et al.
vegetable waste waste activated added. The bio- (2000)
Hyder plc. DL, sludge and fruit/ gas methane con-
UK vegetable waste tent was 68% and
using of inclined bicarbonate alka-
tubular digesters linity in the
methanogenic
stage was over
4000 mg CaCO3/
L
400 M. M. Hanafiah et al.

Gaby et al. (2017) studied the effect of the retention time and temperature on
microbial community composition and methane production in staged anaerobic
digesters fed with food waste. During this experiment, two anaerobic digestion
parameters were studied, namely the effect of 55 and 65
C as acidogenic reactor
temperature, and the effect of lowering the hydraulic retention time (HRT) from
17 to 10 days in the methanogenic reactor. Higher acetate and butyrate concentra-
tions were found in the 65
C acidogenic reactor. The CH4 production was increased
from ~3600 mL/day to ~7800 mL/day when the HRT was decreased to 10 days. The
effect of yeast on the performance of biogas production from food waste has been
studied (Gao et al. 2020). The result showed that biogas production increased by
520 and 550 mL with addition of 2.0% (of volatile solids; VS) of activated yeast on,
respectively, the 12th and 37th day of anaerobic digestion and the gas production
was relatively stable (Gao et al. 2020).
Pallan et al. (2018) reported that anaerobic digestion of organic wastes like
banana stalk (BS), banana peel (BP), vegetable waste (VW), spent tea waste
(TW) and food waste (FW) in different combinations can be utilized to produce
methane in lab scale fabricated digesters of one litre capacity. The combination of
vegetable waste (VW) and spent tea waste (TW) has produced a maximum biogas
yield of 3.75 L.
Viswanath et al. (1992) carried out a study on anaerobic digestion of fruit and
vegetable wastes for biogas production in a 60 L digester. A maximum biogas yield
of 0.6 m3/kg VS was obtained at a 20 day HRT and 40 kg TS/m3/day organic loading
rate. The biogas production was observed in the digesters operated at 16 and 24 day
HRT. The maximum biogas yield of 74.5% was observed within 12 h of feeding at a
16 day HRT, whereas at a 24 day HRT only 59.03% of biogas yield on substrate
could be generated. Kitchen waste (KW) and fruit/vegetable waste (FVW) could be a
potential candidate for methane production (Wang et al. 2014). The results of
lab-scale experiment showed a higher methane productivity of 0.725 L CH4/g
VS. Similarly, during the anaerobic digestion process of FVW in a batch reactor,
514 ( 57) L CH4/kg VS was produced (Park et al. 2012). Dinsdale et al. (2000)
studied two-stage anaerobic co-digestion of waste activated sludge and FVW using
inclined tubular digesters. The design system could achieve a high biogas yield of
0.37 m3/kg VS. The methane content in the biogas was 68% and bicarbonate
alkalinity in the methanogenic stage was over 4000 mg CaCO3/L.

15.3.2 Livestock Waste

Manure produced from dairy cows are primarily stored in holding tanks before being
applied to fields in rural areas. This leads to methane emissions in the environment as
the manure decomposes and also may contribute to excess nutrients release in
waterbodies. Biogas production from livestock manure through AD can minimise
such problems, and can also prevent other associated issues of odours and manure
pathogens.
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 401

Most of the rural areas of Asia is still facing problems of large quantities of
manure from dairy, pigs and poultry and the suitable treatment and management of
these waste is required (Vu et al. 2015). By utilizing these large wastes for energy
generation in the form of biogas will help to prevent direct discharge of animal
wastes into aquatic bodies, a common practice in various parts of the Asian region
that lack proper waste management systems (Anenberg et al. 2013; Vu et al. 2015).
Khalil et al. (2019) have reviewed the potential of sustainable biogas production
from animal waste in Indonesia. It was found that about 9597.4 Mm3/year of biogas
can be produced from animal waste in Indonesia and this will generate an electric
power up to 1.7  106 KWh/year.
Achinas and Euverink (2019) have reviewed the biogas production from the
anaerobic co-digestion of farmhouse waste and have studied their performance and
kinetics. El-Mashad and Zhang (2010) have investigated the effect of manure-
screening on the biogas yield from dairy manure in batch digesters under mesophilic
(35
C) conditions. The production of methane from the fine and coarse fraction of
screened manure were 302 and 228 L/kg VS, respectively, and for unscreened
manure it was 241 L/kg VS. Mähnert and Linke (2009) have studied biogas
production from whole-crop rye silage, maize silage and fodder beet silage along
with cattle slurry at mesophilic temperatures. The maximum biogas production and
the rate of biogas generation were in the range of 0.61–0.93 m3/kg volatile solids and
0.032–0.316 per day, respectively. Abubakar and Nasir (2012) have investigated the
effectiveness of cow dung for biogas production using a 10 L volume bioreactor in
batch and semi-continuous mode at mesophilic (53
C) temperature conditions. The
average cumulative biogas yield and methane content were 0.15 L/kg VS and 47%,
respectively. Their study showed that cow dung is an effective feedstock for biogas
production with stable performance.
The AD process of animal manure with other biomass increases the yield of
biogas and it provides advantages for the proper management of manure and such
organic wastes simultaneously (Nielsen et al. 2002). Biogas production from manure
and digestible organic wastes coming from food industry can be an example (Braun
and Wellinger 2003). Uzodinma and Ofoefule (2009) have studied biogas produc-
tion from cow dung, poultry dung, swine dung and rabbit dung blended with field
grass in a 50 L capacity digester. On blending with rabbit dung, cow dung, swine
dung and poultry yielded an average of 7.73  2.86, 7.53  3.84, 5.66  3.77 and
5.07  3.45 L of biogas per total mass of slurry, respectively. The biogas yield
improved during codigestion due to improvement in nutritional quality.
Ojolo et al. (2007) investigated biogas production from poultry wastes, cow dung
and kitchen waste in a 9 L volume reactor. The highest production of biogas was
observed on the 14th day with 85  10–3 dm3 of biogas. The biogas obtained from
poultry droppings, cow dung and kitchen waste were 0.0318 dm3/day, 0.0230 dm3/
day and 0.0143 dm3/day, respectively. In another study, Zhang et al. (2013) found
high biogas production from goat manure by adding with three crops residues at
35
C. The highest cumulative biogas production was 16,023 mL for different
co-substrates after 55 days of digestion.
402 M. M. Hanafiah et al.

15.3.3 Crop Residues

Crop residues include materials such as stalks, straw, and plant trimmings, which
needs further processing to dispose of after the cultivation. In general, the crop
residue can be left behind in the fields by which the organic and moisture content of
the soil can be improved. However, where multiple crop cultivations are carried out
successively, this method can cause hindrance to subsequent crops. Furthermore,
higher crop yields leads to higher amounts of residue that need to be taken care of,
creating more problem for the cultivators. Thus, converting such crop residues into
biogas promotes better environmental sustainability. Crop residues are usually
co-digested with other organic materials as it contains a high lignin content that
makes it difficult to break down. However, growing energy crops for the sole
purpose of bioenergy resources comes with a number of downsides such as require-
ment of high costs and large land use, water, and nutrient requirements. In this
context, the biogas obtained from wastes such as crop residues is more
advantageous.
Several agricultural residues in the form of agricultural biomass can be
categorised as the food based portion (oil and simple carbohydrates) of crops
(such as beets, corn and sugarcane) and the non-food waste (complex carbohydrates)
portion (such as rice husk, leaves, orchard trimmings, stalks, corn stover, wheat
straw and pearl millet stalk), perennial grasses and biogenic waste. Rice, maize, and
wheat are the world top three growing crops and their residues are potentially being
used as a good substrate for biofuel production (Sims 2004; Chandra et al. 2012).
Kumar et al. (2018) have reviewed the potential of some cereal crop residues in
India for enhanced biogas production via chemical pre-treatment. They have mainly
focused on chemical pre-treatment of cereal crop residues to generate biogas by
removing lignin. Li et al. (2019b) have evaluated the anaerobic digestion of vege-
table crop residues and their biochemical methane potential. Svensson et al. (2005)
have studied the financial feasibility and prospects of biogas production from crop
residues on a farm-scale level. The study revealed that the high-solids single-stage
fed-batch operation provides the best option for high biogas yield. The methane
yield, operational costs and degree of gas utilization had the strongest impact on the
financial success of the process. Kalra and Panwar (1986) have investigated husk
and straw of rice crops for the AD process in 190 L capacity digesters. 1 kg of rice
straw has yielded about 220 L of biogas during the batch digestion process. A ratio
of rice straw and cattle dung (1:1 dry weight basis) produced 9.1% more gas as
compared to rice straw alone.
In a study using a mixture of agricultural wastes with semi-solid chicken manure,
a maximum methane production of 502 and 506 mL/g VS obtained at 55
C and
35
C, respectively. However, an additional 42% increase in methane production
(695 mL/g VS) was also observed with pretreated (ammonia stripped) chicken
manure compared to other control (untreated) systems (Fatma et al. 2014). The
anaerobic co-digestion of swine manure mixed with crop residues produced 3.5 L/
day of biogas (Cuetos et al. 2011). The semi-continuous experiment was conducted
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 403

in mesophilic reactors having 3 L working capacity for 30 days. Elena et al. (2012)
showed the production of biogas from a mixture of cattle slurry and cheese whey as
the substrate with a maximum biogas production up to 79%.

15.4 Life Cycle Assessment Framework

Life cycle assessment (LCA) is an important decision making tool for determining
alternative methods to reduce the environmental impacts of any process and its
products (Aziz and Hanafiah 2020). Sustainability is the main priority when it comes
to LCA as this methodology suggests the most eco-friendly solution throughout the
process and always has room for improvement to reduce the environmental impacts
(Ismail and Hanafiah 2019a). LCA can help organizations and industries to evaluate
and understand the holistic environmental performance of their products or activities
depending on their sustainability goals towards product improvement, product
innovation or strategic marketing and business plan (Aziz et al. 2019). It allows
organizations and industries to involve in a decision-making process for
implementing and achieving sustainable development and green solutions and
adopt life cycle thinking into their business. Incorporating LCA into their practice
provides the best design and technology options for their products and also ensure
that it does not cause any adverse environmental impact (Ismail and Hanafiah 2020).

15.4.1 Steps in Conducting an LCA

Based on ISO 14040 series (ISO 2006a, b), there are four phases in the framework of
the LCA as shown in Fig. 15.3.
The first phase is to identify the goals and scope of a process. During this phase,
the type of product or service, functional units and system boundaries are identified.
Functional units (FU) are the quantity of products to be studied while the boundaries
of the system provide an overview to explain the processes covered in the evaluation.
A functional unit is one the most important aspects in modelling or constructing a
system of a product in LCA. FU is the measurement of a product that describes its
function and it is used in all the calculations throughout the assessment. Products
must have the same functional unit to make comparisons. A product’s feature can
vary from performance, aesthetics, technical quality and additional services up to
costs (Arzoumanidis et al. 2020). For instance, a material such as food waste can be
described as 1 tonne of food waste, whereas energy can be described as 1 kWh of
electricity. System boundaries determine the inputs that should be included or
excluded in the LCA assessment.
The second phase is the analysis of the life cycle inventory (LCI). It is a part of a
LCA where all the data of a system are collected. It is a complex portion of an
assessment as it tracks and analyses the entire flow of the input and output data of a
404 M. M. Hanafiah et al.

Goal & Scope Definion Inventory Analysis Impact Assessment

• Intended use • Input-output flow • Classificaon

• Product system • Data collecon • Characterizaon
• Funconal unit • Foreground data • Normalizaon
• System boundary • Background data • Grouping
• Allocaon procedure • Data validaon • Weighng

Interpretaon

• Provide conclusion, limitaon and recommendaon

• Evaluaon of completeness, sensivity and uncertainty
• Communicate the results

Fig. 15.3 Main phases of life cycle assessment (LCA)

product’s system. LCI can include material use, energy use, coproduct generation,
waste generation, raw material extraction, refining and processing, product
manufacturing, product use, and recycling or disposal of a product in a system
(DeRosa and Allen 2017)
The third phase in the LCA framework is the life cycle impact assessment
(LCIA). The environmental impact potential is identified using the results of the
life cycle inventory analysis that will be sorted into major impact categories which
are divided into three different groups: ecosystem impacts; human impacts; and
resource depletion. These categories will subsequently be assessed for decision
making. The fourth phase is the interpretation. In this phase, the results obtained
from the previous phases are interpreted in an informative form. In addition,
evaluations and recommendations to reduce the environmental impact of the pro-
duction of a product or service are also reported. The four phases in this life cycle
evaluation framework demonstrate a systematic process of providing relative and
quantitative information to measure the environmental load at each stage of the
product’s life cycle.
As illustrated in Fig. 15.4, LCA uses cradle-to-cradle or cradle-to-grave tech-
niques which analyse raw material extraction through materials processing, manu-
facture, distribution, product usage and disposal or end of life.

15.4.2 History and Evolution of LCA

LCA is a systematic method to determine and calculate environmental impacts

associated with all stages of the life cycle of a product or service (Aziz and Hanafiah
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 405

Materials Manufacturing Use

Preprosessing Distribu n End of Life

Fig. 15.4 An LCA cradle-to-grave approach

2021; Harun et al. 2020; Ismail and Hanafiah 2019b). Below are the historical
background of the introduction and application of LCA for products and systems.
(a) 1960–1990: decades of genesis
The initial studies that are now recognised as partial LCAs, date from the late
1960s to early 1970s. The scope of study during this era was limited to energy
analyses but gradually broadened to encompass resource requirements, emission
loadings and generated waste. LCA mainly focused on covering the alternatives
in this period. The first impact assessment method was introduced in the early
1980s, separating the airborne and waterborne emissions by semi-political
standards for those emissions and aggregating them into ‘critical volumes’ of
air and ‘critical volumes’ of water, respectively. From 1970s to 1980s, there was
a lack of international scientific discussion and information exchange on LCA
platforms. Thus, LCAs were performed using different methods and without any
common standard structure. This prevented LCA to be accepted more generally
as an analytic tool as there were great differences in the results obtained even
when the same materials were used (Guinee 2011).
(b) 1990–2000: decade of standardisation
The tremendous elevation in scientific and coordination activities world-wide
reflected in the increasing number of LCA guides and handbooks produced. The
International Organisation for Standardisation (ISO) has been involved in LCA
since 1994. Other than that, the first scientific journal papers began to appear in
the Journal of Cleaner Production; Resources, Conservation and Recycling;
International Journal of LCA; Environmental Science and Technology; Indus-
trial Ecology and other journals. Moreover, LCA became part of policy docu-
ments and legislation during these years. Several well-recognised impact
assessment methods such as CML 1992 environmental theme approach that
are still being used till today were developed in this period.
(c) 2000–till now: decades of expansion
The attention towards LCA began to grow in the first decade of the twenty-
first century. In 2002, the United Nations Environment Programme (UNEP) and
the society for Environmental Toxicology and Chemistry (SETAC) launched an
International Life Cycle Partnership known as the Life Cycle Initiative, where
their main aim was formulated as putting life cycle thinking into practice and
improving the supporting tools through better data and indicators. The European
406 M. M. Hanafiah et al.

Platform on Life Cycle Assessment was established in 2005, mandated to

promote the availability, exchange and use of quality assured life cycle data,
methods and studies for reliable decision support in (EU) public policy and
business. In the USA, the US Environmental Protection Agency started promot-
ing the use of LCA and simultaneously environmental policy gets increasingly
life cycle based all over the world. The decade 2000–2010 can be characterised
as the decade of elaboration. Over the years, several types of LCA have been
evolved such as environmental life cycle assessment (E-LCA) which is now
known as LCA, social LCA (S-LCA), and life cycle sustainability assessment
(LCSA). New approaches of LCA have also emerged such as consequential
LCA, attributional LCA, dynamic LCA and Input-output (IO) LCA, that specify
the need for the study to be carried out.

15.5 Life Cycle Assessment of Anaerobic Digestion Systems

Figure 15.5 illustrates the phases involved in the LCA of AD system. Some previous
studies on life cycle assessment of anaerobic digestion system are given in
Table 15.2.
Mezzullo et al. (2013) evaluated the environmental impacts of biogas production
and utilization through the framework of LCA. The production of biogas and
fertilizer (from digestate) from a cattle farm waste is compared with other alternative
forms of energy including fossil fuel. The biogas based system was much more
advantages in terms of greenhouse gas emission and fossil-fuel usage. In terms of

Fig. 15.5 Phases involved in LCA of AD system

15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 407

Table 15.2 Selected studies on life cycle assessment of anaerobic digestion process
Organic waste Impact
Location source Functional unit Software method Reference
Australia Food waste 1 kg of food SinaPro ReCiPe Opatokun et al.
waste v.8 (2017)
Italy Agricultural prod- 1 MWh of Gabi CML Fusi et al.
ucts and waste electricity LCA 2001 (2016)
v6.11
United Cattle waste 1 m3 of biogas SimaPro EI 99 Mezullo et al.
Kingdom (2013)
United Cow manure 1 kg of excreted GaBi 5 IPCC, Andres and
States manure 2006 Rebecca (2019)
Vietnam Pig manure 100 kg of solid ReCiPe, Vu et al. (2015)
pig manure 2008
1000 kg of liquid
pig manure
Norway Fuel 1 kg of CO2 SimaPro CMLIAb Lyng and
v8.5.2 v3.05 Brekke (2019)
IPCC
(2013)
Malaysia Palm oil mill 1 tonne of POME SimaPro ReCiPe, Aziz and
effluent (POME) v8.5 2016 Hanafiah (2020)
Italy Organic waste 1 tonne of CML Di Maria and
organic waste Micale (2015)

both environmental and energy impact, the system contributed negligible amounts
towards the whole life cycle impacts.
Fusi et al. (2016) evaluated the environmental impacts (through LCA) associated
with generation of electricity from biogas produced by AD of agricultural wastes in
five actual plants. The results suggest that the main contributors to the impacts are
the use of maize silage, the operation of the anaerobic digester, including open
storage of digestate. The small scale system using animal slurry is the best option
among the different plants considered in this study. The only impacts an animal
slurry based biogas plants has are marine and terrestrial ecotoxicity. However, in
comparison to other renewable sources and natural gases for electricity generation,
biogas based electricity performed poorly with higher environmental impacts.
According to the author, the environmental impacts could be mitigated by under-
taking better practices such as avoiding digestate storage, preventing biogas emis-
sions and reducing digestate application to land.
Vu et al. (2015) reported that loss of biogas from digesters as well as the
intentional release of biogas and emissions of CH4 from manure storage compromise
the beneficial effects of biogas use. However, from the sensitivity analysis, it is also
clear that biogas digesters can become a means of reducing global warming impacts
relatively easy if CH4 emissions into the environment can be kept low. In another
study, Lyng and Brekke (2019) stated that when applying life cycle assessment to
evaluate the environmental impacts from biogas as a fuel for transport, the results are
408 M. M. Hanafiah et al.

largely dependent on the underlying assumptions. Biogas appears to be one of the

fuels on the market with the lowest environmental impacts. Aspects that have
considerable impact on the results for biogas used as a fuel are system boundaries,
transport distances and methane leakages.
In Malaysia, Aziz et al. (2020) concluded that waste-derived biogas is a promis-
ing technology that can be used to meet the national goals for a sustainable
renewable energy and highlighted the sustainability of biogas production for a
proper waste management and energy recovery in the Malaysian context. There
are some previous studies that compared AD in terms of its sustainability with other
processes for their waste management capability using LCA. For example, environ-
mental impacts and benefits of three treatment scenarios for food waste management,
namely anaerobic digestion, pyrolysis and integrated anaerobic digestion followed
by pyrolysis have been investigated (Opatokun et al. 2017). The results revealed that
the integrated system and anaerobic digestion have similar high overall benefits and
low impacts in comparison to the pyrolysis process. In contrast, Di Maria and Micale
(2015) reported that incineration of organic waste leads to maximum environmental
benefits compared to anaerobic digestion and composting. Furthermore, anaerobic
digestion and composting were characterized by negative impacts due to high
gaseous emissions.

15.6 Sustainable Waste Management

Most countries deal with the wastes according to their respective waste hierarchy.
Although, the steps of the waste hierarchy of a country may slightly vary from one
another, its ultimate motive is to work towards sustainable waste management. In
general, the first step of a waste hierarchy is to reduce waste generation at the point of
source. Reducing waste needs to be considered from the beginning of designing any
sort of product in order to minimise the toxicity and the amount of waste generated
throughout the product’s life. This step emphasises the importance to carry out a
LCA before manufacturing a product. Apart from that, reducing waste can be
practiced in every household as this is the simplest way to curb problems related
to waste management. However, this step might have a drawback when it comes to
changing from citizens’ up to stakeholders’ behaviour and attitude towards sustain-
able waste management as this step can only be completely effectively if everyone
joins forces to do their responsibility in reducing waste.
The second step of the waste hierarchy is to reuse a product more than once.
Accumulation of the amount of waste in the landfill can be decreased by practicing
this step. This step also requires a strong commitment from the majority of the
people to succeed. Recycling is the third step in the hierarchy of waste. Similar to the
two other steps mentioned before, recycling also starts from home by segregating
waste according to its type. Recycling is a very resourceful step as it extracts the raw
material from a product to be reused or to generate new products and reduce
15 Life Cycle Assessment of Anaerobic Digestion Systems: An Approach Towards. . . 409

landfilling as well. Anaerobic digestion and composting are included in this step as it
recovers useful organic compounds from biodegradable waste.
Although composting is a traditional method that recovers and dissolves organic
material back to the land, it is considered one the most effective methods as it is
simple to carry out and the cost is minimal. However, this method may attract pests
in an area. The next level of this hierarchy is the treatment or energy recovery step,
whereby energy is recovered from waste through various types of treatment or
recovery techniques. Anaerobic digestion can also be included in this category as
it generates biogas that can be used as a source of energy to produce electricity and
fuel as discussed in this chapter. Biological anaerobic digestion gains more impor-
tance among the various conversion processes of biomass due to its economical and
efficient way of recovering carbon in the form of renewable biogas fuel (Achinas
et al. 2020). Other recovery techniques include mass burn, pyrolysis and refuse-
derived fuel (RDF). Lastly, the bottom of the hierarchy is categorised as landfilling
because it causes many environmental issues such as pollution and global warming
due to high emissions of GHGs, leachate, and toxicants.

Acknowledgement Marlia M. Hanafiah was financed by the National University of Malaysia

(GUP-2020-034 & DIP-2019-001).

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