Applied Energy 144 (2015) 73–95

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Review

A review on dark fermentative biohydrogen production from organic

biomass: Process parameters and use of by-products
Anish Ghimire a,b, Luigi Frunzo c,⇑, Francesco Pirozzi b, Eric Trably d, Renaud Escudie d, Piet N.L. Lens e,
Giovanni Esposito a
a
Department of Civil and Mechanical Engineering, University of Cassino and Southern Lazio, via Di Biasio 43, 03043 Cassino, FR, Italy
b
Department of Civil, Architectural and Environmental Engineering, University of Naples Federico II, via Claudio 21, 80125 Naples, Italy
c
Department of Mathematics and Applications Renato Caccioppoli, University of Naples Federico II, via Cintia, Monte S. Angelo, I-80126 Naples, Italy
d
INRA, UMR 050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, 11100 Narbonne, France
e
UNESCO-IHE Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands

h i g h l i g h t s

 H2 production via dark fermentation of waste biomass is promising.

 Parameters influencing dark fermentation can be optimized for higher H2 yields.
 Effluents can be utilized further for energy recovery and for other valued uses.
 Net energy gain and pH control are important issues.

a r t i c l e i n f o a b s t r a c t

Article history: Dark fermentation of organic biomass, i.e. agricultural residues, agro-industrial wastes and organic muni-
Received 18 August 2014 cipal waste is a promising technology for producing renewable biohydrogen. In spite of its potential, this
Received in revised form 10 January 2015 technology needs further research and development to improve the biohydrogen yield by optimizing
Accepted 12 January 2015
substrate utilization, microbial community enrichment and bioreactor operational parameters such as
Available online 11 February 2015
pH, temperature and H2 partial pressure. On the other hand, the technical and economic viability of
the processes need to be enhanced by the use of valuable by-products from dark fermentation, which
Keywords:
mostly includes volatile fatty acids. This paper reviews a range of different organic biomasses and their
Biofuels
Biohydrogen
biohydrogen potential from laboratory to pilot-scale systems. A review of the advances in H2 yield and
Dark fermentation production rates through different seed inocula enrichment methods, bioreactor design modifications
H2 yield and operational conditions optimization inside the dark fermentation bioreactor is presented. The pros-
Biomass valorization pects of valorizing the co-produced volatile fatty acids in photofermentation and bioelectrochemical sys-
Fermentation metabolites tems for further H2 production, methane generation and other useful applications have been highlighted.
A brief review on the simulation and modeling of the dark fermentation processes and their energy bal-
ance has been provided. Future prospects of solid state dark fermentation are discussed.
Ó 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2. Microbiology and biochemical pathways of DF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3. Potential sources of organic biomass for fermentative biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.1. Agricultural residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.1.1. Lignocellulosic waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.1.2. Livestock waste (manure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.2. Industrial waste. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

⇑ Corresponding author. Tel.: +39 081 7683 436; fax: +39 081 5938 344.
E-mail address: [email protected] (L. Frunzo).

http://dx.doi.org/10.1016/j.apenergy.2015.01.045
0306-2619/Ó 2015 Elsevier Ltd. All rights reserved.
74 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

3.3. Organic fraction of municipal waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

4. Factors affecting DF pathways and H2 yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.1. Inoculum and enrichment methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.2. Design and operation of bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.2.1. Bioreactor configuration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.2.2. Hydraulic retention time (HRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.2.3. pH and temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.2.4. H2 partial pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.3. Substrate pre-treatment for enhanced H2 yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.3.1. Addition of nutrients and trace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.3.2. Inhibition due to heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5. Use of by-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.1. Photofermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.2. Microbial electrolysis cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
5.3. Anaerobic digestion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
5.4. Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6. Pilot scale applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
7. Challenges and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
7.1. Modeling and simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
7.2. Energy balance and COD conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
7.3. pH control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
7.4. Solid state dark fermentation (SSDF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

1. Introduction alternative energy carriers, hydrogen could be the fuel of the future
because of its high energy content, environmental friendliness of
Environmental friendly energy carriers and sources are the production, and also because it can give substantial social, eco-
most highlighted topic in the energy and environmental sector. nomic and environmental credentials [2]. Hydrogen is a carbon-
The current global energy demand is mostly dependent on reserves free clean fuel, as the only final by-product of its combustion is
of fossil fuels, which are depleting, and the world is facing severe water [2]. Hydrogen can also be helpful in addressing global warm-
pollution problems from the by-products of fossil fuels uses [1]. ing and increasing pollution problems. Furthermore, it is preferred
The scientific community has widely accepted the fact that the over methane owing to its wider industrial applications, i.e. H2 is
increasing CO2 level due to the use of fossil resources is impacting used in the synthesis of ammonia and hydrogenation of edible
the greenhouse gas effect and global warming. Therefore, different oil, petroleum, coal and shale oil [3]. Hydrogen can be directly used
ways to harness the energy from clean renewable sources are either in combustion engines because of its highest energy per unit
being developed, but the search for reliable energy sources is still weight, i.e. 143 GJ per ton [2] among known gaseous biofuels or to
on. produce electricity via fuel cell technologies [4]. Thus, the creation
In the past years, the research and development interests have of a hydrogen economy which incorporates the production and use
been directed towards renewable energy technologies like the of hydrogen as an energy carrier could in the future lead to sustain-
anaerobic digestion (AD) of organic biomass and waste. For able energy systems [1,5].

Biophotolysis
12H2O → 12H2 + 6O2 (Green algae)
CO + H2O → H2 + CO2 (Photosynthetic bacteria)
Light
dependent
Photofermentation
C6H12O6 + 6H2O → 6CO2 + 12H2 (Phototrophic bacteria)
(Organic carbon)
Biohydrogen
(H2)

Dark fermentation
C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2 (heterotrophs)
(Organic carbon)
Light
independent
Microbial electrolysis cell
C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2
Anode : CH3COOH + 2H2O →2CO2 + 8e- + 8H+
Cathode : 8H+ + 8e- → 4H2

Fig. 1. Biological pathways to produce hydrogen.

 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 75

35 enhance the biohydrogen production by improving the biodegrad-

Google Scholar (Peer-reviewed articles, abstracts & book chapters)
Number of published articles

Scopus (Peer-reviewed articles in journals) ability of substrates [18–20]. Recently, there has been growing
30
interest on coupled processes to obtain a higher H2 yield by inte-
25 grating DF with processes like photofermentation (PF) [21,22] or
bioelectrochemical systems [23–25]. Because of the profitable pro-
20
duction of biomethane, a coupled DF-methanogenic stage has also
15 been a popular choice which increases the sustainability of the
coupled-process by improving the energy recovery from the DF
10
residues [26–28].
5 The aim of this paper is to provide an updated overview of
0
advancements in biohydrogen production via DF of organic bio-
Before 2006 2007 2008 2009 2010 2011 2012 2013 2014 mass. Regardless of the increasing number of research articles
2005 and reviews published, there is a need to provide an extended
Year overview of dark fermentative biohydrogen production with the
utilization of by-products and the future challenges and prospects
Fig. 2. Number of peer reviewed publications on DF published in the last decade
for its up-scaled development. This review provides an insight on
[9,10].
the factors that influence the biochemical pathways in dark fer-
mentative biohydrogen production to increase the H2 yield and
post-utilization of DF residues to realize its future sustainability.
The major challenge in the use of this promising energy carrier To summarize, this review provides an extended insight on (a) pos-
lies in its sustainable production and storage. In commercial appli- sible feedstock or substrate sources and their biohydrogen poten-
cations, hydrogen has been produced from natural gas (48%) and tial (BHP), (b) factors that influence the fermentative H2 yield: (i)
oil (30%) by steam reforming processes, and also by other indus- inoculum sources and enrichment methods, (ii) pre-treatment of
trial methods such as coal gasification (18%) and water electrolysis substrates and (iii) bioreactor operation and design (culture pH,
(4%) [6]. However, these processes are highly energy intensive and temperature and OLR, HRT, H2 partial pressure, nutrients and trace
use non-renewable sources of energy which makes them less elements addition), (c) utilization of DF residues, (d) pilot scale sys-
attractive from an environmental point of view. In order to produce tems and (e) challenges and future prospects: (i) modeling and
a cleaner and more sustainable fuel, the hydrogen should come simulation of DF process, (ii) energy balance and conversion of
from processes that avoid or minimize CO2 emissions. organic carbon, (iii) natural pH control and (iv) future prospects
Hydrogen can be produced from biological processes which are of solid state dark fermentation.
less energy intensive and more environmental friendly in terms of
global reduction of CO2. These renewable biohydrogen producing
technologies have potential to become cost competitive as they 2. Microbiology and biochemical pathways of DF
can use low value waste biomass as feedstock [2], e.g. municipal,
agricultural and industrial organic waste and wastewater. Biohy- In DF processes, carbohydrate-rich substrates are broken down
drogen can be produced by both autotrophic and heterotrophic anaerobically by hydrogen-producing microorganisms, such as fac-
microorganisms (Fig. 1) [2,7]. In autotrophic conversions (also ultative anaerobes and obligate anaerobes. Molecular hydrogen
known as direct or indirect biophotolysis), solar energy is directly (H2) is produced in the process of disposing the excess electrons
converted to hydrogen via photosynthetic reactions mediated by through the activity of the hydrogenase enzyme [8,29]. Under
photosynthetic microorganisms, i.e. microalgae, protists and pho- anaerobic environments, protons (H+) can act as electron acceptors
tosynthetic bacteria. Under heterotrophic conditions, the organic to neutralize the electrons generated by oxidation of organic sub-
substrates are transformed into simpler organic compounds with strates, consequently producing H2. In contrast with aerobic respi-
simultaneous production of molecular hydrogen [7,8]. There are ration, where oxygen is reduced and water is the final product
two types of heterotrophic conversions, photo-fermentation car- [7,30].
ried out by photosynthetic bacteria and dark fermentation (DF) In the DF of glucose as the model substrate, H2-producing bac-
carried out by anaerobic bacteria that convert carbohydrates into teria initially convert glucose to pyruvate through glycolytic path-
biohydrogen. ways producing adenosine triphosphate (ATP) from adenosine
DF is the most studied and promising technology for biohydro- diphosphate (ADP) and the reduced form of nicotinamide adenine
gen production owing to its higher production rates and treatment dinucleotide (NADH) [8]. Pyruvate is further oxidized to acetyl
capacity for organic wastes. Several substrates rich in carbohy- coenzyme A (acetyl-CoA), carbon dioxide (CO2) and H2 by pyruvate
drates are also usable, such as first generation fuel crops (e.g. sugar ferredoxin oxidoreductase and hydrogenase. Depending on the
cane, wheat, corn, and sugar beets) as well as second generation type of microorganism and environmental conditions, pyruvate
biomass like agricultural residues as well as industrial waste and may also be converted to acetyl-CoA and formate which may be
wastewater [7]. In recent years, there are increasing research activ- further converted into H2 and CO2. Also, acetyl-CoA might be con-
ities in this domain, as shown by the increasing number of peer- verted to acetate, butyrate, and ethanol [8]. DF of complex carbo-
reviewed articles with ‘‘dark fermentation’’ in the title (Fig. 2). hydrates by mixed anaerobic microbiota can result in a wide
At present, DF process development at industrial scale is limited range of intermediates and by-products depending on the opera-
by its lower hydrogen yield compared to its theoretical maximum tional parameters, such as substrate type, substrate loading rate,
yield of 4 mol of H2 per moles of hexose, as well as the estimated pH, temperature and other operating and environmental condi-
costs associated with the H2 production. There are areas for tions, as they also influence the microbial community structure
improvement to achieve higher H2 yields and production rates by in bioreactors. Fig. 3 gives a schematic representation of the differ-
optimizing the design and operation of DF bioreactors [11]. The ent steps and biochemical pathways involved in the DF of complex
H2 production cost in scaled-up systems can be minimized by organic biomass.
using low cost renewable materials such as waste biomass as feed- These biochemical pathways (Fig. 3) can be mediated by strict
stock [12,13]. Inoculum enrichment methods [8,14–17] can anaerobes (Clostridia, methylotrophs, rumen bacteria, methano-
improve the H2 yield, and pre-treatment of substrates can also genic bacteria, archea, etc.), facultative anaerobes (Escherichia coli,
76 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

Organic Biomass Inert particulate and

(Agricultural, Municipal and Industrial Waste) solubles (Lignins, etc)
Disintegration

Carbohydrates Proteins Lipids

Hydrolysis
Monosaccharides Long Chain Fatty Acids
Amino Acids

Acidogenesis

Lactate Ethanol Propionate Butyrate Valerate

Acetogenesis
Acetate H2

Fig. 3. Biodegradation steps and microbiological pathways involved in the fermentative breakdown of waste biomass (adapted and modified from [31]).

Enterobacter, Citrobacter), and even aerobes (Alcaligenes, Bacillus) gen Demand (COD)/L, pH 5.5 and 12 h hydraulic retention time
[8]. Acetate and butyrate are the most common products of DF (HRT). They also reported that a B/A ratio higher than 2.6 indicated
[32]. Common biochemical reactions during DF undertaken by fac- an efficient H2 production by anaerobic microbiota. In DF with
ultative anaerobes are: mixed cultures, when a B/A ratio of 3:2 is generally observed,
results in a H2 yield of 2.5 mol H2 per mole of hexose fermented
C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ2CO2 þ 4H2 ð1Þ
ðAcetic acidÞ as given in Eq. (7) [32]:

C6 H12 O6 ! CH3 CH2 CH2 COOH þ2CO2 þ 2H2 ð2Þ 4C6 H12 O6 þ 2H2 O ! 3CH3 CH2 CH2 COOH þ 2CH3 COOH
ðButyric acidÞ
þ 8CO2 þ 10H2 ð7Þ
When the metabolic pathway is such that it favors the produc-
In contrast, Guo et al. [37] showed in their study performed
tion of acetic acid, the stoichiometric yield of H2 is 4 mol for each
with lignocellulosic substrates that this ratio might not give a good
mole of glucose (i.e. 544 mL H2/g hexose at 25 °C) as in Eq. (1),
indication, particularly in batch tests where homoacetogenic activ-
whereas the yield of H2 is 2 mol for a mole of glucose (i.e.
ity prevails. Therefore, higher acetate concentrations cannot
272 mL H2/g hexose at 25 °C) when the final product is butyric acid
always give an indication of a higher H2 yield. Some homoaceto-
(Eq. (2)) [8]. However, the actual hydrogen yield is lower than the
gens belonging to the genus Clostridium (e.g. C. aceticum) can lower
theoretical yield as part of the substrate is utilized for biomass pro-
the H2 yield by converting H2 and CO2 to acetate or can convert
duction and the degradation of the substrates might follow other
hexose directly to acetate [32,36]. However, analysis of soluble
biochemical pathways without hydrogen production [33,34].
metabolites can give an indication of the fermentation pathways
Under some conditions, the metabolic pathways lead to ethanol
and thus the H2 production performance.
and acetate production, lowering the stoichiometric hydrogen
Clostridia have been identified as the dominant hydrogen pro-
yield to 2 mol of H2 for a mole of glucose (i.e. 272 mL H2/g hexose
ducing microorganisms in DF operated with mesophilic mixed cul-
at 25 °C) as represented in Eq. (3) [8]:
tures at a pH of 5.5 [38]. Fang and Zhang [38] identified that 64.6%
C6 H12 O6 þ 2H2 O ! CH3 CH2 OH þ CH3 COOH þ 2CO2 þ 2H2 ð3Þ of all the microorganisms were affiliated with three Clostridium
species (Clostridiaceae), 18.8% with Enterobacteriaceae, and 3.1%
A widely studied clostridia species, Clostridium butyricum, is with Streptococcus bovis (Streptococcaceae) based on the phyloge-
responsible for the production of butyric acid as the major product netic analysis of the rDNA sequences. Interestingly, Rafrafi et al.
of fermentation together with acetate and hydrogen [32]. Another [39] reported recently that sub-dominant species, in spite of their
fermentation pathway is the production of propionate by Clostrid- low abundance, can also have substantial impact on the hydrogen
ium articum which is a hydrogen consuming pathway (Eq. (4)). production performance. The presence of some species like E. coli
Similarly, metabolic pathways leading to only ethanol and lactic can aid in increasing the H2 yield by diverting the metabolic path-
acid production by Clostridium barkeri yield no hydrogen (Eqs. (5) ways to the acetate and butyrate hydrogen producing pathways
and (6)) [35]: (Eq. (7)), while other species communities such as Bacillus spp.
C6 H12 O6 þ 2H2 ! 2CH3 CH2 COOH þ 2H2 O ð4Þ and Lactobacillus spp. can lower the H2 yield by diverting the path-
way to lactate accumulation (Eq. (6)).
C6 H12 O6 ! 2CH3 CH2 OH þ 2CO2 ð5Þ Other results of the identification of the microbial diversity by
community fingerprinting techniques in the thermophilic DF of
rice straw showed that hydrolytic and fermentative bacteria such
C6 H12 O6 ! CH3 CHOHCOOH þ 2CO2 ð6Þ
as Clostridium pasteurianum, Clostridium stercorarium and Thermo-
Hawkes [32] and Kim [36] proposed the molar ratio of butyric anaerobacterium saccharolyticum dominated in the sludge of a
to acetic acid (B/A ratio) as a quantitative indicator of the biohy- repeated fed-batch reactor [40]. Shin et al. [41] detected the hydro-
drogen yield associated with microbial metabolic pathways. Kim gen producing microorganisms Thermoanaerobacterium thermosac-
et al. [36] found that B/A ratios were directly proportional to H2 charolytium and Desulfotomaculum geothermicum in a thermophilic
yields (mol H2/mol hexose) during DF of sucrose in CSTR reactors acidogenic culture, while Thermotogales strains and Bacillus species
operated at an organic loading rate (OLR) of 10–60 g Chemical Oxy- were detected in a mesophilic acidogenic culture by Polymerase
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 77

Table 1
Dark fermentative biohydrogen potential of different waste biomass under varying operating conditions.

Substrate type Microbial inoculum Reactor type Temperature pH Maximum H2 yield Maximum H2 H2 in Reference
source (°C) (mL H2/g VSadded) production rate biogas
(%)
Food waste Heat shock treated Leaching bed 37 5.5–7 310 151.25 mL H2/L/ 10–55 [60]
anaerobic sludge reactor h
Food waste Thermophilic Batch 55 4.5 46.3 3 mL H2/g VSS/h 23 [41]
acidogenic culture
Vegetable kitchen waste Kitchen waste compost Intermittent- 55 6.0 38a 1.0 L H2/L/d 40 [61]
CSTR
Food waste and sewage Anaerobic digester Batch 35 5.0–6.0 122.9a 111.2 mL H2/ – [62]
sludge sludge g VSS/h
b
OFMSW Anaerobic digestate Semi- 55 6.4 360 – 58 [45]
continuous
CSTR
OFMSW Non-anaerobic inocula Packed bed 38 5.6 99b – 47 [63]
(soil, pig excreta) reactor
Wheat straw Cow dung compost Batch 36 6.5 68.1 10.14 mL H2/ 52 [64]
g VS/h
c
Rice straw Wastewater treatment Batch CSTR 55 6.5 24.8 – – [40]
plant sludge (initial)
Corn stalk wastes with Enriched cow dung Batch CSTR 50 7 149.69 7.6 mL H2/h 45–56 [46]
acidification pre- composts (initial)
treatment
Rice slurry Anaerobic digester Batch 37 4.5 346d 2.1 L/g VSS/d 45–56 [65]
sludge (initial)
e
Cheese whey Adapted anaerobic Batch 55 7 111 3.46 mL H2/L/h – [66]
sludge (initial)
Pig slurry Mesophilic CSTR 70 6.7 3.65 – – [67]
methanogenic sludge (feed)
Untreated de-oiled algae Anaerobic digester Batch 29 6 66f 0.08 mL/h – [50]
cake sludge (initial)
Potato and pumpkin mixture BESA treated anaerobic Batch 35 7.4 171.1 – – [68]
sludge (initial)
a
mL H2/g COD.
b
mL H2/g VSremoved.
c
mL/g TS.
d
mL H2/g carbohydrate.
e
mL H2/g total sugar.
f
mL H2/g algal biomass.

Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis availability and biodegradation rate [12,13,16,43,44]. Carbohy-
(DDGE) analysis during DF of food waste. In another study, Quémé- drate rich substrates have been extensively used in DF studies, in
neur et al. [42] investigated the potential of a molecular capillary particular pure glucose, sucrose and starch mixtures [30]. But
electrophoresis-single strand conformation polymorphism (CE- renewable biohydrogen production requires the substrate or feed-
SSCP) fingerprinting method based on the hydA functional genes stock to come from renewable resources [12,32]. Second genera-
to better describe the bacterial community dynamics in a mixed tion biomass sources, such as waste biomass, are abundant and
dark fermentative culture at different pH conditions. can thus support the supply of renewable substrates for DF
Some undesirable microorganisms which lower the total H2 [13,14,43]. Besides biohydrogen and volatile fatty acids as valuable
yield might be present in mixed cultures of fermentative microor- by-products, DF also offers biological treatment of the organic
ganisms, either by consuming the H2 produced or by altering the waste.
biochemical pathways of the H2 synthesis [8]. The main H2 con- In more recent dark fermentative studies, complex substrates
sumers include methanogens, homoacetogenic bacteria and sulfate have been considered, such as the organic fraction of municipal
reducing bacteria (SRB). The activity of these hydrogen consumers solid waste (OFMSW) [40,45–48], agricultural residues like ligno-
can be controlled by inoculum pre-treatment methods or bioreac- cellulosic biomasses (e.g. rice straw, wheat straw and corn stalks),
tor operating conditions [30,43]. The activity of methanogens and agro-industrial wastes like those from food processing industries
SRB can be significantly reduced by operating at a pH below 6 (e.g. olive mill wastewater and cheese whey), effluents from live-
along with the control of the HRT and OLR. Therefore, hydrogen stock farms and aquatic plants [13,14]. With the integration of
production via a mixed dark fermentative culture is a complex DF within a biorefinery concept, the waste generated from biofuel
microbial system, influenced by a number of parameters such as production such as crude glycerol [25,49], de-oiled algal cake [50]
substrate type, substrate pretreatment, inoculum type, inoculum or cotton seed cake [51] can be utilized as a substrate, while dark
enrichment method, bioreactor design and operation. fermentative metabolites can be utilized in the production of
micro-algal biomass [52–54] and biodiesel [55], which in turn
can serve as feedstock for DF processes.
3. Potential sources of organic biomass for fermentative Based on their availability, novel low-cost substrate sources
biohydrogen production need to be explored and assessed for their biohydrogen potential
(BHP). Table 1 presents the biohydrogen production potential of
The substrate plays an important role in the H2 yield, H2 pro- different organic biomasses by dark fermentative process. The fer-
duction rate and the overall economy of the process. These are mentation pathways depend on the substrates and the microbial
mainly dependent on the substrate’s carbohydrate content, bio- metabolism [8]. It has been well established that the type of sub-
78 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

Table 2
Composition of typical agricultural waste (% of dry matter).

Component Rice strawa (%) Wheat strawb (%) Barley strawc (%) Corn stalkc (%) Corn cobd (%)
Cellulose 38.6 44.1 37.2 36.7 35.3
Hemicellulose 19.7 36.0 24.4 26.2 37.1
Lignin 13.6 6.9 16.1e 16.9e 16.4e
Ash – 6.1 6.4 4.9 1.5
a
Data obtained from [70] on wet basis.
b
Data obtained from [71].
c
Data obtained from [72].
d
Data obtained from [73].
e
Acid-insoluble lignin.

strate influences the biohydrogen yields [37,56]. Monlau et al. 3.1.2. Livestock waste (manure)
[57] and Guo et al. [37] in their studies reported that the soluble Livestock wastes include solid animal manure waste, fodder
and readily accessible sugars represent the main fraction of bio- waste (which generally contains a lignocellulosic fraction) and
mass that can be converted into hydrogen. However, the biohy- wastewater which includes urine and faeces. A large quantity of
drogen production also depends on a number of parameters livestock manure comes from cattle feedlots, poultry and swine
such as inoculum type and enrichment methods, bioreactor buildings, identified as pollution sources, which pose threats to
design and operation conditions. The latter are covered in the sec- the atmospheric and water environment [81]. The current prac-
tions below. tices of management of livestock waste include its application in
agricultural fields as well as biological stabilization or treatment
such as composting and AD. The former management practice con-
3.1. Agricultural residues tributes in uncontrolled greenhouse gas emissions (mainly CH4)
from land applications. Manure management practices can reduce
Agricultural residues, which mainly include lignocellulosic direct and indirect greenhouse gas emissions by generating energy
wastes, are an economically viable and renewable source of second in the form of biogas from the manure prior to its land application
generation carbon neutral biofuels [58]. These include plant bio- [81–83].
mass waste, which generally contains cellulose, hemicellulose However, manure substrates need physical and chemical treat-
and lignin formed by photosynthesis. Agricultural residues are pro- ment to inhibit the methanogenic activity that consumes H2
duced when economically valuable products of the crops are har- [82,84]. Another, problem that might occur during the use of this
vested and the residues such as straw, stover, peelings, cobs feedstock type is the inhibition of the biohydrogen production by
stalks, bagasse and others are left over [58]. The 2010 global annual ammonia as its high nitrogen content might cause failure of the
production of agricultural residues was around 5.1 billion dry ton- bioreactor: swine, poultry and dairy manure have a low C/N ratio
nes [59]. The waste generated by the agricultural, forestry and (C/N ratio of swine manure: 12.8) [85] and high levels of ammoni-
aquaculture sectors is increasing with the increasing population acal nitrogen (cattle slurry: 1.04–1.9 g/L and chicken manure:
and thus the waste from this sector will be increasing further in 7.0–12.8 g/L) [86]. Salerno et al. [87] reported that hydrogen
the future. Guo et al. [43] have reported the potentials and chal- production is possible at high concentrations up to 7.8 g N/L in
lenges of agricultural wastes as substrates for biohydrogen produc- continuous flow systems if the microbial culture is initially
tion. Examples of agricultural residues as a potential feedstock acclimated to a lower ammonia concentration of 0.8 g N/L total
source for DF processes and recent advancements in their applica- ammonia. However, the biohydrogen production decreases when
tion are discussed below. the total ammonia concentration increases to above 2 g N/L [88].
Also, high sulfate concentrations in swine manure can inhibit
the biohydrogen production due to the presence of hydrogen
3.1.1. Lignocellulosic waste consuming sulfate reducers [43].
Rice straw is an example of a typical agricultural residue. It Because of the high nitrogen content of animal manure, it can
is the world’s third largest agricultural residue, after maize and be used as a co-digestion substrate for nitrogen supplementation
wheat, with a reported global yearly production of approxi- of other agricultural residues to maintain a suitable carbon to
mately 916 million tons in 2009 [69]. Thus, the use of this nitrogen ratio. Wu et al. [82] reported a H2 yield between 1.18
abundant biomass as a feedstock in dark fermentative hydro- and 1.63 mol H2/mol glucose in a fermentation of swine manure
gen production might hold future potential for feedstock sup- supplemented with glucose. Xing et al. [83] achieved an enhanced
ply. Similarly, wheat straw, barley straw, corn stalk, corn H2 yield of 31.5 mL/g Volatile Solids (VS) with acidification pre-
cobs and others could be potential DF feedstock. The cellulose treated dairy manures while treating 70 g VS/L of substrate at
and hemicellulose part of these wastes can be hydrolyzed into operating pH 5.0.
carbohydrates which are further biologically converted to
organic acids and biohydrogen in DF processes (Table 2). The
composition of typical lignocellulosic crop residues in terms 3.2. Industrial waste
of cellulose, hemicellulose and lignin content is presented in
Table 2. Agro-industries waste such as palm oil mill wastewater [89–92]
The main limitation in the utilization of these valuable and olive mill wastewater (OMWW) [93,94], tapioca industries and
resources lies in the complex structure of lignocellulosic materi- food industries such as brewery and dairy industries [95–98] pro-
als: a cross-linking between polysaccharides (cellulose and hemi- duce large quantities of carbohydrate rich non-toxic waste in the
cellulose) and lignin via ester and ether linkages, which form of solid waste and wastewater. It can be potential substrates
decreases their biodegradability [58,74–76]. Therefore, prior to for dark fermentative biohydrogen production. Ren et al. [99] dem-
DF, these biomasses are often subjected to physical, chemical onstrated that waste molasses are an excellent substrate in a pilot
and biological pre-treatment to increase their digestibility scale system operated under mesophilic conditions (35 °C) where
[58,74–80]. very good results were obtained in terms of H2 production rate
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 79

(232 mL H2/L/h) and yield (26.13 mol/kg CODremoved). The produc- Bioreactor control
tion of large quantities of this type of waste biomass supports its
(pH, temperature, hydaulic &
utilization in up-scaled DF systems for continuous biohydrogen biomass retention times, partial
production. Similarly, cheese whey, a waste by-product generated pressure of H2)
by cheese manufacturing industries and characterized by high
organic loads, comprising mainly carbohydrates (lactose), protein
and lipids, is a very good potential substrate for biohydrogen pro-
duction [24,100,101]. Enhanced biohydrogen
production in DF

3.3. Organic fraction of municipal waste

Substrates Microorganisms
Organic fraction of municipal waste (OFMSW) generally consti- Types of substrates, substrate Pure and mixed culture,
tutes food waste which contains a high biodegradable carbohy- pre-treatment methods Enrichment of seed inocula
drates fraction with 85–95% volatile solids and 75–85% moisture
content making it a good substrate for DF [43]. Food waste present Fig. 4. Strategies to enhance the biohydrogen yield in DF of organic biomass.
in municipal waste is mainly responsible for methane emissions
and leachate production from landfills [102]. AD has been pro-
4.1. Inoculum and enrichment methods
posed as the most suitable treatment option for OFMSW or food
waste with energy recovery and other environmental credentials The hydrogen producing seed inoculum or culture is very
[103]. Thus, food waste has been used extensively in DF experi- important for the startup of the hydrogen production process. Sev-
ments [41,88,104–106]. De Gioannis et al. [17] have reviewed the eral studies using pure cultures have been done using a range of
studies of DF processes utilizing OFMW or food waste for dark fer- substrates (Table 3). Various species of Clostridia and Enterobacter
mentative biohydrogen production. are widely used in pure cultures (Table 3). Lee and Show [110]
Large quantities of waste biosolids or sludge are generated from and Elsharnouby et al. [111] have reviewed the studies of DF per-
municipal wastewater treatment plants which generally contain formed with pure cultures. Table 3 presents some of the dark fer-
carbohydrates or polysaccharides and proteins [107]. Several mentative biohydrogen studies done with pure cultures.
researchers have used the available carbohydrates present in these H2 synthesizing bacteria exist commonly in environments such
biosolids in fermentative hydrogen production [62,108]. However, as soil, wastewater sludge and compost. All these materials can
the sludge needs pre-treatment, such as ultrasonication, acidificat- thus be used as an inoculum for fermentative H2 production [8].
ion, sterilization, freezing–thawing or alkaline pre-treatment, to Indeed, cow dung, anaerobic sludge, municipal solid waste, soil
facilitate the fermentative process [107,108]. Besides, Kim et al. and compost are some of the common sources of mixed cultures.
[62] demonstrated the usefulness of sewage sludge as co-substrate A mixed culture of hydrogen producers is generally preferred over
in the DF of food waste. a pure culture due to its practicability for environmental engineer-
ing applications, economic benefits in operation (as it can econo-
mize asepsis costs), easiness in control based on differential
4. Factors affecting DF pathways and H2 yield kinetics of microbial subgroups and broader feedstock choice
[8,30,45]. However, enrichment of mixed cultures becomes neces-
DF via mixed cultures is a complex system where environmen- sary to enhance the biohydrogen production on the one hand and
tal factors and bioreactor operation conditions such as tempera- inhibit hydrogen consumers such as methanogens and homoacet-
ture, pH and H2 partial pressure regulate metabolic pathways of ogens, often present in these mixed inocula [30], on the other hand.
hydrogen producing microorganisms [8,30,43,109]. In addition, Table 4 summarizes the common pre-treatment measures adopted
substrate types and their pre-treatment methods, bioreactor con- for enrichment of hydrogen producers.
figurations, inoculum sources and enrichments also influence the Pre-treatment of the inoculum to obtain an enrichment of
biohydrogen production. Three categories of parameters that influ- hydrogen producers often relies on the spore forming characteris-
ence the DF pathways, and thus the yield of biohydrogen, can be tics of H2 producers such as Clostridium, which are ubiquitous in
distinguished (Fig. 4). These parameters are reviewed below and anaerobic sludge and sediments [8,95,118,119]. These organisms
compared in relation to H2 yield and production rate. have a better chance to survive the harsh conditions during the

Table 3
Biohydrogen production studies using pure cultures.

Culture Substrate Culture type Temp. pH Optimum H2 yield (mol H2/ Reference
(°C) mol glucose eqv.)a
Enterobacter cloacae IIT-BT08 Glucose Batch 36 6 2.2 [112]
Clostridium thermolacticum DSM 2910 Lactose Continuous 58 7 1.5 [113]
Enterobacter cloacae DM 11 Malt, yeast extract & Continuous 37 6 3.9 [114]
glucose
Caldicellulosiruptor saccharolyticus DSM 8903 Hydrolyzed potato steam Batch 70 6.9 3.4 [115]
peels
Thermotoga neapolitana DSM 4349 Hydrolyzed potato steam Batch 80 6.9 3.3 [115]
peels
C. thermocellum DSM 1237 and C. thermopalmarium Cellulose Batch 55 7 1.36 [116]
DSM 5974
Clostridium thermocellum 7072 Corn stalk Continuous 55 7.2 1.2 [117]
(100 L)
a
mol H2/mol glucose equivalent was calculated based on the information provided from references at Standard Temperature and Pressure (STP) (0 °C and pressure 1 atm).
80 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

Table 4
Pre-treatment methods used to enriching hydrogen producing microorganisms in anaerobic sludge.

Treatment Description Inoculum source Reference

Heat 100 °C for 15 min Anaerobic digested sludge [119]
Heat 80 °C, 90 °C and 100 °C for 15–30 min Anaerobic sludge [122]
Heat Heating in boiling water bath for 10–30 min Anaerobic granular sludge [74,75]
Heat 105 °C for 4 h Anaerobic granular sludge [123]
Heat Incubation at 90 °C for 1 h Anaerobic granular sludge [124]
Heat 100–105 °C in oven for 2 h Cow dung compost [125]
Acid pH to 2 for 24 h and increasing pH to 5.5 by adding a 2 N NaOH solution Anaerobic digested sludge [126,90]
Acid pH 3 with 2 N HCl for 24 h Anaerobic digested sludge [127]
Acid pH to 3 with 1 N HCl for 30 min Anaerobic digested sludge [120]
Acid pH 3 with 0.1 N HCl solution for 24 h and adjusting back to pH 7 Anaerobic granular sludge [128]
Base pH of the sludge to 3 with 1 mol/L of NaOH for 24 h Anaerobic digested sludge [119]
Base pH 8, 9 and 10 with 1 mol/L of NaOH for 3 h Anaerobic sludge [122]
Base pH 12 with 1 M NaOH for 24 h and adjusting back to pH 7 using 1 M HCl Anaerobic digested sludge [121]
Load shock Sludge (50 mL) spiked with 40 g of sucrose and acidification for 2 d Anaerobic granular sludge [124]
Load shock Sludge (50 mL) spiked with 500 mL of sucrose (50 g/L) and acidification for 2 d Anaerobic digested sludge [121]
Chemical inhibition 10 mmol of BESA for 30 min and gravity separation for 2 h Anaerobic digested sludge [69,70]
Chemical inhibition 0.2 g/L BESA for 24 h Anaerobic granular sludge [95]
Chemical inhibition 0.1% (v/v) chloroform for 24 h Anaerobic digested sludge [90]
Aeration Aerate with air for 24 h Anaerobic sludge [119]
Aeration Flushing with air for 30 min Anaerobic sludge [120]
Microwave irradiation Microwave radiation for 1.5 min Cow dung compost [129]

Table 5
Comparison of various inoculum pre-treatment methods for enriching hydrogen producing inocula.

Inoculum source Inoculum treatment Substrate Culture Culture Optimal pre- Maximum H2 yield Maximum H2 Reference
methods temperature pH treatment (mol H2/mol glucose production rate
(°C) method eqv.)a (mL H2/L/h)
Anaerobic digested HSb, aeration, acid, Sucrose 35 – Base treatment 3.06 – [120]
sludge base, BESAc and
iodopropane
Anaerobic granular HS, acid and base Glucose 35 – Chloroform 1.55 – [128]
sludge
d
Anaerobic sludge Acid, BESA, HS and Dairy 29 – BESA 0.0317 – [95]
(UASB) their four wastewater
combination
Anaerobic digested Acid, base, HS, Glucose 36 7 Heat shock 1.9 120.4 mL H2/h [119]
sludge aeration and treatment
chloroform
Anaerobic digested Acid, base, LSe, HS and Sucrose 60 5.5 Load shock 1.96 11.2 mmol H2/L/h [121]
sludge BESA treatment
Suspended & granular HS, chloroform and Ground 37 7 Repeated heat 25.7f – [130]
anaerobic sludge combination of both wheat shock
mixture solution treatment
Anaerobic sludge HS, acid and base Glucose 35 6.2 Heat treatment 3.84 – [122]
at 80 °C for
30 min
a
Calculated based on the information provided from references at standard temperature (0 °C and pressure 1 atm).
b
HS: heat shock.
c
BESA: 2-bromoethanesulfonic acid.
d
mmol H2/g COD.
e
LS: load shock.
f
mL H2/g cells/h.

pre-treatment of the inoculum than the non-spore forming bacte- activity of methanogens drops sharply at a pH below 6.3 or above
ria such as methanogens, as the spores can germinate again under 7.8 [8], while the activity of Clostridia sp. and other hydrogen pro-
favorable conditions [8,15]. Heat treatment of mixed cultures for ducers is not affected by an acidic pH (below pH 6).
the enrichment of H2 producers is a simple, inexpensive and effec- Other pre-treatment methods such as chemical pretreatment
tive method [8,30]. However, the effect of heat treatment might be and aeration are directed towards the selective inhibition of meth-
different depending on the inoculum source such as activated anogens present in anaerobic sludge, which are very sensitive to
sludge or anaerobic sludge [30]. Some studies [120,121] reported changes in environmental conditions. Besides being strict anaer-
a lower hydrogen yield by a heat shock treated seed inoculum than obes, methanogens are sensitive to many chemicals [8]. Thus,
obtained by other pre-treatment methods. This could be due to the oxygen can inhibit their activity during aeration [119,120]. Wang
inhibition of other non-spore forming hydrogen producing bacteria and Wan [119] aerated the inoculum sludge with air for 24 h to
which might destabilize the main hydrogen production pathways. inhibit the activity of methanogens. Likewise, chemical inhibitors
Similarly, acid or base treatment is based on the notion that the like sodium 2-bromoethasulfonic acid (BESA), iodopropane, chlo-
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 81

Table 6
Evaluation of inoculum pre-treatment methods to enhance the DF capacity of the inoculum sludge.

Pretreatment method Energy requirement Chemical requirement Economic cost Scale-up application
Heat shock treatment +++ + +++ ++
Acid treatment + +++ ++ +++
Chemical treatment + +++ +++ ++
Aeration +++ + ++ +++
Load shock treatment ++ ++ + +++

+ Less intensive; ++ Moderately intensive; +++ Very intensive.

Table 7
Examples of innovative continuous DF bioreactors.

Major substrate Biomass Reactor type Optimum Optimum organic Optimum H2 Reference
retention HRT (h) loading rate (OLR) production index
system
Glucose Granule CSTR 0.5 10 g glucose/L H2 yield [163]
1.81 mol H2/
mol glucose
Cheese whey Granule UASB reactor 6 20 g COD/L/d H2 production rate [164]
0.36–0.38 L H2/L/d
Food waste Biofilm Batch pilot scale up-flow rector (packed with coir pith) 0.50 m/ 50 g COD/L H2 production rate [165]
daya 9.67 L H2/L/h
Food waste (pre- Suspended ASBR (fill: 0.5 h; reaction: 8 h; settle: 3 h & discharge: 36 30 g COD/L H2 yield [166]
treated with alkali) 0.5 h) 0.69 mol H2/
mol hexoseadded
Tequila vinasse Suspended ASBR (fill: 3 min; reaction: 5.33 h; settle: 30 min & 12 3 g COD/L H2 production rate [167]
discharge: 7 min) with 50% volumetric exchange rate 50.5 mL H2/L/h
Kitchen waste Suspended Inclined plug-flow reactor (inclined at 20°) 168 6.5 kg VS/m3/d H2 yield 72 mL H2/ [168]
g VS
Municipal food waste Suspended Anaerobic baffled reactor (ABR) 38.4 29 g CODtotal/L/d H2 production rate [48]
& kitchen 6 L H2/d
wastewater
Glucose Suspended Anaerobic membrane bioreactor (MBR) 12 (SRTb) 5.8 g glucose/L H2 production rate [169]
640 mL H2/h
a
Up-flow velocity.
b
SRT: solid retention time.

roform and acetylene are used to inhibit methanogens or at process start-up. A high rate hydrogen producing microbial
[8,68,95,120–122]. Thus, selective inhibitors like chloroform or community can be developed in the fermentative bioreactors when
BESA selectively inhibit the activity of H2 consumer methanogens. applying appropriate reactor operating conditions [98,133–135].
In methanogens, BESA functions by inhibiting the activity of co-
enzyme M reductase, which is a key co-enzyme of methanogenesis 4.2. Design and operation of bioreactors
[95,120].
The effect of inoculum enrichment methods on H2 production is The process design for dark fermentation depends mostly on
different based on the source of inoculum (Table 5). However, in substrates which limit the operational conditions of bioreactors
order to select an inoculum pre-treatment method for scaled-up such as culture temperature (mesophilic or thermophilic), reactor
systems, several parameters needs to be considered, such as oper- configuration (reactor types, wet, semi-dry or dry conditions) and
ational costs, feasibility or complexity of the methods, time for the feeding mode (mono substrate or co-substrates) [136]. Weiland
enrichment of the hydrogen producing seed, use of the DF residues [137] reported the several types of bioreactors used for the conver-
in the post treatment processes. Table 6 gives a simple assessment sion of agricultural biomass to energy through upscaled AD sys-
of the commonly applied inoculum pre-treatment methods based tems. Although these bioreactors are designed for
on the authors’ information from the literature. The selection of a biomethanation by AD, these can be used for biohydrogen produc-
chemical treatment method such as using BESA inhibits the meth- tion after modification of some operational parameters [43].
anogens, which will give problems when the DF residues are to be
used in AD. In addition, BESA is not environmental friendly and 4.2.1. Bioreactor configuration
expensive to use at large industrial scale [8]. Likewise, heat shock Different DF bioreactor configurations have been used in labo-
treatment requires large energy inputs, which makes it less attrac- ratory studies for a wide range of substrates (see Tables 2, 7 and
tive for large scale applications. Acid and shock load pre-treatment 8). Most of the dark fermentative hydrogen production studies
can be applied at large scale to select the hydrogen producing inoc- are carried out in a batch CSTR under wet conditions (<10% total
ula without net energy concerns. solids, TS). Besides CSTR, many studies have been carried out in
There have been some dark fermentative studies done without anaerobic fluidized bed reactor (AFBR), anaerobic sequencing batch
the addition of seed inoculum, utilizing the microorganisms pres- reactors (ASBR), fixed or packed bed reactors, UASB reactors, leach-
ent in the waste itself [131,132]. The fermentative hydrogen ing bed reactors, anaerobic baffled reactors, plug flow reactors or
production took longer than in the tests with inoculum supply. membrane bioreactors (MBR) and with an objective to enhance
Nonetheless, inoculum pre-treatment is important in batch tests the biohydrogen yield and production rate. Recent research
82 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

Table 8
Effects of operational temperature and pH on fermentative hydrogen production.

Substrate type Microbial inoculum Optimum Optimum Reactor HRT Maximum H2 Major acid Reference
pH temperature type (days) yield (mL H2/ type
(°C) g VS) produced
Food waste Heat shock treated anaerobic sludge 6.3 35 Leaching 25 310 Acetate [60]
bed reactor
Cassava stillage Heat treated UASB sludge 7 60 Batch CSTR 3.5 53.8 Butyrate [152]
Cassava stillage Heat treated UASB sludge 5 60 Batch CSTR 3.5 66.3 Butyrate [152]
OFMSW Untreated anaerobic digestate 5.5 37 Semi- 21 165a Butyrate [45]
continuous
CSTR
OFMSW Untreated anaerobic digestate 6.4 55 Semi- 21 360a Acetate [45]
continuous
CSTR
Wheat straw Cow dung compost 7.0 36 Batch CSTR 6.25 68.1 Acetate and [64]
butyrate
Vegetable kitchen Enriched from kitchen waste compost 7.0 55 Batch CSTR 7.0 12.8b Butyrate and [135]
waste lactate
c
Cattle wastewater Mixed wastewater sludge, cow dung 5.5 45 Batch CSTR 1.25 319 Butyrate [155]
compost, chicken manure compost, river
sludge
Rice straw Heat treated wastewater sludge 6.5 55 Batch CSTR 6.5 24.8d Acetate [40]
a
mL H2/g VSremoved.
b
mL H2/g COD.
c
mL H2/g CODconsumed.
d
mL/g TS.

[136,138–140] has focused on the application of high solids pro- The incompatibility of the use of high organic loading rates and
cesses such as semi-dry (10–20% TS) and dry (>20% TS) DF pro- rapid microbial growth in biofilm systems makes them thus less
cesses for biohydrogen production, as the interests in the attractive than granular systems. Show et al. [142] recommended
conversion of second generation lignocellulosic biomass (mostly the column-shaped granular reactor for fermentative biohydrogen
agro-industrial residues) is growing. production from wastewater though the system is not suitable for
Studies have correlated the biohydrogen production with the digestion of substrates with a high solids content or for a longer
size of the microbial population and therefore different cell reten- retention time in which anaerobic granules may disaggregate. High
tion strategies have been investigated [11,141,142]. The latter rate bioreactors are necessary to convert complex organic biomass
include sludge granulation and biofilm systems to increase the like OFMSW and agricultural waste.
bacterial concentration in the reactor. The results of these studies
showed that the volumetric hydrogen production rate of a bioreac- 4.2.2. Hydraulic retention time (HRT)
tor depends on the ability to maintain a high microbial density. The HRT can affect substrate hydrolysis and thus the production
Gravala et al. [143] showed higher hydrogen production rates in of intermediates and products, affecting fermentative H2 produc-
a UASB (which has a granular biomass retention) than in a CSTR tion. Besides hydrolysis, the HRT can also be used as control
at low retention times (19.05 and 8.42 mmol H2/h/L, respectively parameter of the methanogenic activity. Some studies have dem-
at 2 h HRT), while the CSTR reactor gave higher hydrogen yields onstrated the effect of the HRT on the biohydrogen production in
(mmol H2/mol glucose) at all HRTs tested. This suggests a compro- DF processes [144–147]. The different growth rates of hydrogen
mise should be sought between technical efficiency (based on H2 producers and consumers make it possible to use the HRT as a con-
yields) and economic efficiency (based on H2 production rate), trolling parameter to inhibit the activity of H2 consumers in the DF.
when one of these two systems is selected. It has been reported that low HRTs favor hydrogen production as
Show et al. [142] compared the performance of a CSTR and an the methanogens are washed out, and hydrogen production
AFBR for biohydrogen production using different biomass growth increases as the HRT decreases [144–146,148]. However, the opti-
strategies with glucose as the substrate. The different bioreactor mum HRT for biohydrogen production in DF depends on the type of
configurations used in their research were: suspended sludge CSTR substrates used as the hydrolysis rate depends on the biodegrad-
system, granular sludge CSTR system, granular sludge AFBR system ability of the substrates (Tables 2, 6 and 7).
and biofilm AFBR system. The maximum H2 yield of their sus- However, the HRT alone is not sufficient to fully suppress the
pended sludge CSTR system, granule reactor and biofilm system methanogenic activity [145]. Liu [145] investigated the effects of
amounted to, respectively, 1.92 mol H2/mol glucose at a HRT of pH and HRT on hydrogen production using household solid waste
6–12 h, 1.83 ± 0.09 mol H2/mol glucose at a HRT of 0.5 h and as a substrate in a hyperthermophilic (70 °C) CSTR. The effect of the
1.81 ± 0.08 mol H2/mol glucose at a HRT of 0.5 h. HRT (1, 2, 3, 4 and 6 days) at a constant pH of 7 and the effect of pH
Besides the specific advantages of these different bioreactor (5, 5.5, 6, 6.5 and 7) at a constant HRT of 3 days was investigated.
systems, the major drawbacks are the washout of hydrogen- The results of the experiments at different HRTs and constant pH 7
producing bacteria at short HRT in CSTR systems, low conversion showed unstable H2 production with subsequent methanogenic
rates in granular reactor systems and rapid biofilm development activities at the end. However, a combination of pH 5.5 and HRT
leading to fragmentation and separation from the supporting of 3 days gave the optimum biohydrogen production conditions.
media in biofilm systems [142]. In another study by Zhang et al.
[141], the biohydrogen production potential of biofilm based and 4.2.3. pH and temperature
granule based reactors were compared. They concluded that The operational pH and temperature are the most crucial
the granule based system was advantageous as it gave better parameters that determine the optimum metabolic pathways of
results in terms of biomass retention without being subjected to hydrogen synthesis as well as the inhibition of the hydrogen con-
wash-out of the biomass support carriers. suming processes which may occur simultaneously [35,149]. An
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 83

acidic operational pH (below 6) mainly inhibits the methanogenic acetate as major end-product at mesophilic culture while butyrate
activity under both mesophilic and thermophilic conditions, but levels and hydrogen production was higher by the thermophilic
the inhibition of hydrogen consuming homoacetogenic activity culture, obtained in DF of food waste carried at pH 5.5. In another
can only be achieved under thermophilic conditions at the initial study, Wang and Wan [158] found the maximum substrate degra-
pH of 5.5 [127]. Thus, the control of the process pH and tempera- dation efficiency, maximum H2 yield and production rate at 37.8 °C
ture plays an important role in achieving high biohydrogen conver- in DF of glucose. These studies suggest temperature influences bio-
sion rates by minimizing the activity of the hydrogen consumers. chemical pathways, although other factors such as culture pH, sub-
The pH is one of the key parameters that can influence the met- strate types and loading rates are equally important.
abolic pathways as it may directly affect the hydrogenase activity, The H2 yields depend on temperature as it affects the hydrolysis
an iron containing enzyme which plays a major role in DF [150]. An rate [41,45,144]. Biomass such as agricultural residues require a
acidic pH affects the activity of the hydrogenase enzyme while it is high temperature to achieve a higher H2 yield because a better
one of the important parameters for the inhibition of methanogen- hydrolysis of lignocellulosic compounds is needed [43]. Kongjan
ic activities in a mixed culture system [8,35]. and Angelidaki [156] demonstrated biohydrogen production from
The optimum pH range for biohydrogen production varies from extreme thermophilic DF of wheat straw hydrolysate. Similarly,
pH 4.5 [35] to 9 [151] in DF of sucrose. Table 8 provides optimum thermophilic temperatures are favored in the DF of food waste
operating pH ranges in different studies. The possible explanations [41]. In contrast, easily biodegradable substrates prefer mesophilic
for the disagreements in optimum pH in the various studies can be temperatures for an optimal H2 yield. The difference between the
differences in inoculum sources, inoculum enrichment methods, optimum operational temperatures is due to the difference in the
substrate types and applied OLR [30]. fraction of easily biodegradable compounds present in the feed
The operational pH influences the metabolic by-products and substrate and the different inocula used. Table 8 reports ranges
biohydrogen yields. In most of the studies, acetate and butyrate of optimum temperatures which vary depending on the type of
are the major end products of favorable hydrogen synthesis (Eqs. substrate and inoculum used.
(1), (2) and (7)). Table 8 shows that a neutral operational pH favors There are some techno-economic studies done which compare
the acetate pathways, while acidic pH conditions favor the buty- the mesophilic and thermophilic operation of DF processes. A
rate pathways. However, Khanal et al. [35] concluded the indepen- thermophilic process seems to be more economical because of
dence of the acetate and butyrate levels from different initial pH its higher yield and lower requirement of feedstock in comparison
ranges studied (4.5–7.5). Similarly, Luo et al. [152] reported buty- to mesophilic DF processes [159]. Foglia et al. [159] reported a
rate as a major VFA in the DF of cassava stillage in both BHP tests better economic performance for thermophilic DF in comparison
carried at the initial pH 5 and 7. Luo et al. [127] found acetate as a to a two-step mesophilic process, converting sugars to hydrogen,
major metabolic product when the operational pH was 7, while CO2 and organic acids followed by a photo-heterotrophic
butyrate dominated at an initial pH 5.5 in the BHP tests carried fermentation.
under mesophilic (37 °C) conditions using an acid pre-treated inoc-
ulum. Luo et al. [127] further reported the inhibition of homo-
acetogenesis can be achieved at pH 5.5 and thermophilic 4.2.4. H2 partial pressure
temperatures (55 °C). In a recent study of the DF of cheese whey The partial pressure of hydrogen inside a biohydrogen reactor
from mozzarella production at different pH ranges (5.5–7.7) and can influence the dark fermentative biohydrogen production as a
a temperature of 39 °C, De Gioannis et al. [153] reported pH 6 as lower partial pressure in the headspace of the reactors facilitates
the optimal pH and acetate levels were higher in all the tests the mass transfer of hydrogen from the liquid to gas phase
except at pH 6.5 where butyrate and propionate levels exceeded [114,160]. During the fermentation process, the hydrogenase is
those of acetate. involved in reversibly oxidizing and reducing ferredoxin. If the
A lower pH (64.5) favors the solvent production [154]. In the DF hydrogen concentration in the liquid phase increases, the oxidation
of glucose by C. pasteurianum, a pH below 5 favors the butanol and of ferredoxin becomes less favorable and the reduction of ferre-
acetone production [150]. Selection of the operational pH is also doxin takes place [44], thus reducing the H2 production.
substrate type and OLR dependent, which determines the VFA con- Lee et al. [161] studied the effect of the reduced partial pressure
centrations and thus the pH of the solution. The optimum temper- on the hydrogen production in a CSTR reactor. Reduction in the
ature for DF processes varies with the substrate type and partial pressure during the DF could lead to an improvement in
operational pH (Table 8). The optimum pH for organic food waste H2 production. At a HRT of 6 h, they found an optimum hydrogen
varies from 4.5 to 7, for lignocellulosic waste it varies from 6.5 to yield and hydrogen production efficiency of 4.50 mol H2/mol su-
7, whereas a neutral pH is optimal for animal manure [43]. How- crose and 56.2%, respectively. Similarly, the reduced pressure of
ever, Tang et al. [155] reported an optimum pH of 5.5 at 45 °C 380 mm Hg gave a higher yield than the partial pressure of
for the DF of cattle wastewater. Thus, it is important to determine 760 mm Hg in another study done by Mandal et al. [114].
the optimum pH conditions for DF of a selected substrate type at a In the AD process, the H2 and CO2 partial pressure is reduced by
particular loading rate and operational temperature. methanogens by their conversion into CH4. Jung et al. [162]
A range of operational temperatures, i.e. mesophilic (35 °C), reported strategies to remove dissolved H2 from the mixed liquor,
thermophilic (55 °C) and extreme thermophilic (>65 °C) has been including avoiding supersaturation by strong mixing, sparging
studied to determine its effect on the biohydrogen production with N2 and CO2 and application of a H2-permeable membrane to
[41,45,156]. These studies have shown that the temperature can withdraw dissolved H2 from the mixed liquor. Similarly, the partial
affect the metabolic pathways, thus shifting the composition of pressure of H2 could be reduced directly by decreasing the operat-
the by-products of DF (Table 8). Valdez-vazquez et al. [45] reported ing pressure in the reactor using a vacuum pump [161]. Mandal
higher H2 yields for thermophilic fermentation than in the meso- et al. [114] reduced the partial pressure of H2 in a methanogenic
philic temperature range. Also acetic acid was a dominant by-prod- reactor by adjusting the saline level of the gas collector using a
uct in thermophilic digestion, whereas butyrate was formed in a peristaltic pump. However, the use of vacuum pumps increases
higher proportion during mesophilic digestion. Similarly, results the cost of the process, while the sparging with N2 and CO2 might
of the extreme thermophilic (70 °C) DF of household organic waste render the recovery of H2 difficult due to the dilution of the H2
also showed acetic acid as the dominant by-product in DF tests stream. An effective way to reduce the H2 partial pressure would
conducted at pH 7 [157]. In contrast, Shin et al. [41] showed be to continuously collect the produced gas phase from the reactor.
84 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

Table 9
Examples of different pre-treatment methods applied to complex substrates used in DF.

Substrate H2 yield (mL H2/ Pretreatment methods Reactor mode Temperature Reference
g VS) (°C)
Rice 24.8a Size reduction, <0.297 mm Bath 55 [40]
straw
Wheat 17.6 Size reduction, 1 mm Batch 35 [181]
stalks
Wheat 212b Hydrothermal (180 °C for 15 min) Continuous UASB 70 [156]
straw reactor
Corn 2.84c Steam explosion, 190.220 °C for 3–5 min Batch 35 [182]
stover
c
Corn 3.0 Acidic steam explosion (1.2% H2SO4), 180 and 200 °C for 1–3 min Batch 35 [182]
stover
d
Beet-pulp 66.7 ± 10.1 Alkaline at pH 12 using 2 M NaOH for 30 min + microwaves (170 °C for 30 min) Batch 35 ± 2 [183]
Bagasse 300 100 °C for 2 h +4% NaOH (w/v) + cellulase (20 FPU/g) Batch 55 [171]
Grass 72.2e 4% HCl (w/v), boiled 30 min Batch 35 [184]
Grass 39.5e 4% NaOH (w/v), boiled 30 min Batch 35 [184]
Corn 209.8 1.5% H2SO4, 121 °C for 60 min + 9.4 IU/g of cellulase 52 °C at pH 4.8 in 0.1 M sodium Batch 36 ± 1 [185]
stalks citrate buffer at 5% (w/v)
Corncobs 107.9 100 °C, 30 min and 1% HCl (w/w)) Batch 36 [172]
a
mL/g TS.
b
mL H2/g sugars.
c
mol H2/mol glucose.
d
mL H2/g COD.
e
mL H2/g dry grass.

4.3. Substrate pre-treatment for enhanced H2 yield cultivation pH 6.5 and temperature of 55 °C. The results of the
study showed that rice straw of a particle size <0.297 mm gave
Fermentative biohydrogen production from lignocellulosic sub- the highest cumulative H2 production (191 mL H2/L) with a H2
strates is limited by biological hydrolysis [19]. The complex yield of 6.4 mL/g TS. The substrate with a larger particle size had
organic substrates cited earlier, such as lignocellulosic biomasses, an extended lag phase and lower hydrogen production. This can
demand physical, chemical, biological or a combination of these be explained by the fact that decreasing the particle size increases
pre-treatments to enhance the degradation process, system perfor- the substrate availability for microbial hydrolysis and fermenta-
mance and biogas production [69,75,76,80]. These pre-treatment tion. Kongjan and Angelidaki [156] pretreated wheat straw at
methods reduce the crystallinity of the cellulose and increase the 180 °C for 15 min to obtain a hydrolysate which mostly contained
surface area of the materials to improve the separation of the lignin hemicellulose leaving the cellulose and lignin in solid form. The
and hemicellulose fractions [170]. There have been some studies hydrolysate was used as the substrate for fermentative hydrogen
on the effect of the pre-treatment on fermentative biohydrogen production. Similarly, Zhang et al. [46] reported the use of acid pre-
production [70,156,171,172]. These pre-treatment methods have treated corn stalks for fermentative biohydrogen production. The
in most cases a positive influence on the H2 yield, as the biohydro- biohydrogen yield from acid pretreated corn stalks was higher than
gen production depends on the soluble fraction of sugars or that of untreated waste. However, mostly physical pretreatment
carbohydrates. methods are applied in combination with chemical or biological
Physical pre-treatment methods which generally include pre-treatment methods to obtain better and rapid hydrolysis of
mechanical comminution (chopping, grinding, milling), irradiation substrates (Table 9).
with gamma-rays, electro-beam or microwaves, hydrothermal The effect of pre-treatment methods for different lignocellulosic
treatment, high pressure steaming or pyrolysis are effective in substrates have a diverse effect on the hydrolysis of soluble sugars
breaking the crystallinity, increasing the accessible surface area and release of inhibitory products [176–179]. This needs to be fur-
and decreasing the degree of polymerization [80]. Chemical meth- ther investigated for the selection of suitable pre-treatment meth-
ods such as ozonolysis, acid or alkaline hydrolysis, solvent extrac- ods. These studies have shown that the pre-treatment methods can
tion, explosion with steam ammonia fiber or CO2 are effective in enhance the system performance enhancing the biogas production.
increasing the surface area, delignification and also decreasing However, the selection of a pre-treatment process should be based
the crystallinity and rendering the partial or complete hydrolysis on effectiveness, energy balance, economic feasibility and environ-
of hemicelluloses. These physical and chemical treatment methods mental sustainability [18]. In addition, some studies have reported
can be promising for industrial applications as they are rapid. How- that during the pre-treatment of lignocellulosic biomass, various
ever, these methods demand energy and chemical inputs. More- undesirable compounds are released which exert inhibitory effects
over, lignocellulosic substrates can also be biologically treated on microorganisms [74,176–178]. The most commonly reported
with fungi and actinomycetes which provide delignification and inhibiting substances which are released during the pre-treatment
partial hydrolysis of cellulose, while some enzymes (hemicellulase processes are furfural, hydroxyl-furfural and phenolic substances.
and cellulase) can aid in the hydrolysis and degradation of the lig- Quéméneur et al. [74] and Monlau et al. [180] investigated the
nocellulosic materials [69]. inhibition and control of these inhibitors on the biohydrogen pro-
The physical pre-treatment, especially the reduction of sub- duction. Thus, the selection of pre-treatment methods for lignocel-
strate particle size, has an effect on the biogas yield and process lulosic substrates should also consider these aspects.
kinetics [173–175]. Chen et al. [40] investigated the effects of the
rice straw particle size and concentration on cumulative dark fer- 4.3.1. Addition of nutrients and trace elements
mentative biohydrogen production. They used a meshed rice straw Microorganisms in fermentation processes require nutrients for
concentration of 30 g TS/L with five particle sizes (<0.297, 0.297– bacterial activity and growth. Thus, nutrients such as nitrogen,
0.58, 0.58–1.19, 1.19–10 and >10 mm) as the substrate at an initial phosphate, metal ions and other micronutrients are needed in
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 85

fermentation processes for enzymatic activities and biomass production was enhanced by 71%. O-Thong et al. [89] obtained
growth, which affects the H2 production. Biomass rich in carbohy- the optimal hydrogen production when the substrate contained
drates such as wheat wastes and palm oil effluents may be defi- 257 mg Fe2+/L during the thermophilic DF of POME.
cient in nutrients (such as nitrogen, phosphorous) or minerals
(such as trace metals). Therefore, nutrients or micronutrients must 4.3.2. Inhibition due to heavy metals
be provided as supplement for optimum microbial activities for Toxic heavy metals such as cadmium (Cd), chromium (Cr), zinc
biohydrogen conversion from carbohydrate rich substrates [186– (Zn), copper (Cu), nickel (Ni), and lead (Pb) which may be present
189]. in industrial and municipal solid waste may lead to upset or ulti-
mately failure of anaerobic reactors [8]. Altasß et al. [195] studied
4.3.1.1. Nitrogen and phosphorous. Nitrogen has great significance the inhibitory effect of heavy metals on methane producing anaer-
for hydrogen producers as it is an important component of pro- obic granular sludge. The order of toxicity for the individual heavy
teins, nucleic acids and enzymes. Similarly, besides being an metals in decreasing order was: Zn (most toxic, 7.5 mg/L) > Cr
important nutrient, phosphate also serves in buffering the bio- (27 mg/L) > Ni (35 mg/L)  Cd (least toxic, 36 mg/L).
chemical reactions [30]. In the thermophilic DF of palm oil mill Lin and Shei [192] showed the relative toxicity of the heavy
effluents (POME), O-Thong et al. [89,92] showed that supplement- metals to fermentative hydrogen production was in the order of
ing iron (257 mg Fe2+/L), adjusting the C/N ratio from 95 to 74 Zn > Cu > Cr. The maximum concentration of these metals that
(using peptone as nitrogen source) and the C/P ratio from 650 to reduced the hydrogen producing activity by 50% was 4.5 mg Zn/L,
559 (using Na2HPO42H2O) could enhance H2 production. In these 6.5 mg Cu/L and 60 mg Cr/L [192]. However, Li and Fang [193]
studies, the hydrogen production rate increased by 60% [89] and reported the relative toxicity to H2 production in the following
COD removal efficiencies improved from 35.5 ± 9.8% to order: Cu (most toxic)  Ni  Zn > Cr > Cd > Pb (least toxic). The
62.2 ± 2.8% [92] compared to raw POME without nutrient bioactivity of the sludge was reduced to 50% of the control at
supplementation. 30 mg Cu/L, 1600 mg Ni and Zn/L, 3000 mg Cr/L, 3500 mg Cd/L
Likewise, Argun et al. [186] studied the effects of the C/N and C/ and >5000 mg Pb/L.
P ratio on the hydrogen yield and specific H2 production rate in DF
of wheat powder solution by supplementing nitrogen and phos-
5. Use of by-products
phorous. The results of the study showed the highest H2 yield of
281 N mL H2/g starch were obtained at a C/N ratio of 200 and C/P
The low process yield and the incomplete conversion of organic
ratio of 1000. However, there are some disagreements in the car-
biomass are two major bottlenecks for commercial dark fermenta-
bon to nitrogen and phosphorous ratios. Lin and Lay [189] achieved
tive biohydrogen production [12,27]. As overviewed in Section 4,
a 500% and 80% increased hydrogen yield and hydrogen production
dark fermentative biohydrogen can be enhanced by suitable sub-
rate at a C/N ratio of 47 compared with the blank. Similarly, O-
strate selection, inoculum enrichment strategies, and optimal
Thong et al. [89,92] attained an optimum hydrogen production
operation of bioreactor or substrate pre-treatment. However, a sin-
and COD removal at a C/N ratio of 74 and a C/P ratio of 559. Several
gle DF system cannot achieve beyond the highest yield of 4 mol H2
studies have used the integration of co-substrates as a strategy to
per mole hexose, as DF has a maximum yield of 33% (on sugars)
maintain an appropriate C/N ratio, examples include the use of
[27]. Besides, DF residues mainly contain volatile fatty acids, major
swine manure as a source of nitrogen in co-fermentation with veg-
by-products of the DF process, which need to be utilized to achieve
etable waste [190] and use of cassava starch in co-fermentation
complete conversion of the organic biomass. Dual systems are inte-
with the microalgae Chlorella pyrenoidosa [191].
grated by the conversion of carbohydrates to organic acids in the
first stage (DF) and the conversion of by-products in the second
4.3.1.2. Metal ions and micronutrients. Higher concentrations of stage, either to H2 (photofermentation, bioelectrochemical cells)
metal ions exert inhibitory effects on the hydrogen producers or CH4 (AD). Also, AD can be considered as the final stabilization
[8,192,193]. However, trace amounts of some metal ions enhance stage to stabilize the residues of DF, photofermentation and bio-
the reactor performance [194]. Karadag and Puhakka [194] found electrochemical cells. Fig. 5 shows an example of different possibil-
that iron and nickel improved the reactor performance and H2 ities of integrating DF to other post treatment processes.

H2 Cleaning Application of H 2

H2 Production

Agricultural and
Industrial Waste
Products of Dark Fermentation

Photofermentation
(Mainly Organic Acids)

H2 Production
Physical-chemical
pretreatment of Dark Microbial
feedstock Fermentation Electrolysis Cell
Reactor

CH4
Anaerobic Digestion
Production

II Stage: Treatment of end Application of CH 4

I Stage: Conversion of Organic
products from Dark Fermentation after cleaning
Biomass into Volatile Organic Acids

Fig. 5. Different strategies for integrating DF with post treatment processes for improved biofuel production.
 86
Table 10
Examples of operational conditions and system performances of integrated DF systems.

First stage: DF Second stage: Photofermentation Reference

Substrate type Microbial inoculum pH T HRT Max. H2 Max. H2 Dominant end Process & microbial pH T HRT Max. biogas prod. Max. H2 yield
(°C) days prod. rate yield products inoculum (°C) days rate
Sucrose Clostridium pasteurianum 7 37 – – 3.85 mol H2/ Butyrate and Rhodopseudomonas 7.1 32 96 h 25.2 mL H2/L/h 4.03 mol H2/ [213]
mol sucrose acetate palustris WP3-5 mol sucrose
Sucrose Heat treated cattle dung 6 38 Batch 360 mL H2/ 3.67 mol H2/ Butyrate and Rhodobacter sphaeroids 7 30 Batch – 4.06 mol H2/ [224]
and sludge from biogas L/h mol sucrose acetate SH2C mol sucrose
plant
Acid hydrolyzed Enterobacter aerogenes 6.8 38 Batch 1000 mL H2/ – Butyrate and Rhodopseudomonas BHU 6.8 34 Batch 755 mL/L – [22]

A. Ghimire et al. / Applied Energy 144 (2015) 73–95

sugarcane MTCC 2822 L acetate 01
bagasse
First stage: DF Second stage: AD Reference
Substrate type Microbial inoculum pH T SRT Max. H2 Max. H2 Dominant end Process & microbial pH T SRT Max. CH4 prod. rate Max. CH4
(°C) days prod. rate yield products inoculum (°C) days yield
OFMSW Heat treated sludge from 5.2 37 2 640 mL H2/ 43 mL H2/ Acetate and Sludge from biogas plant 7.5 37 15 7500 mL CH4/d 500 mL CH4/ [109]
biogas plant d g VSadded butyrate g VSadded
Food waste Indigenous microbial 5.2– 40 6.66 – 65 mL H2/ Acetate and Anaerobic granular sludge 6.8 40 26.67 – 546 mL CH4/ [238]
cultures from food waste 5.8 g VS butyrate from UASB g VS
Micro algae Clostridium thermocellum – 55 Batch – 53.4 mL H2/ Acetate and Anaerobic granular sludge – 55 Batch 22.38 mL CH4 g VS d 320.6 mL CH4/ [251]
(Chlorella g VS butyrate from ASBR g VS
vulgaris)
First stage: DF Second stage: Bioelectrochemical systems Reference
Substrate type Microbial inoculum pH T HRT Max. H2 Max. H2 Dominant end Process & microbial pH T HRT Max. H2 prod. rate Max. H2 yield
(°C) days prod. rate yield products inoculum (°C) days
Molasses – – – Batch 700 mL H2/ 0.27 mol H2/ Ethanol, acetic Domestic wastewater 6.7– 25 Batch 1410 mL H2/L/d – [252]
L/d mol COD and butyric acid 7.0
Corn stover Clostridium thermocellum 6.8 50 Batch 0.25 L H2/L/ 1.67 mol H2/ Acetic acid and Inoculum from microbial 7.3 – Batch 1 ± 0.19 L/L/d 750 ± 180 mL/ [235]
d mol glucose ethanol fuel cell wastewater g COD
Corn stalk Microwave irradiation 7.0 36 Batch 1.73 m3 H2/ 129.8 mL H2/ Acetate, Spent dark fermentation 7.0 36 Batch 3.43 ± 0.12 m H2/3
257.3 mL H2/g [234]
pre-treated cow dung m3/d g corn stalk butyrate, medium (single m3 d corn stalk
propionate, chambered cell)
ethanol
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 87

Lactate : C3 H6 O3 þ 3H2 O ! 6H2 þ 3CO2 ð10Þ

Propionate : C3 H6 O2 þ 4H2 O ! 7H2 þ 3CO2 ð11Þ

ADP
Bacterial
H2 Butyrate : C4 H8 O2 þ 6H2 O ! 10H2 þ 4CO2 ð12Þ
photosystem e-
Purple non sulfur species such as Rhodospirillum rubrum, Rhodo-
H+ pseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter capsul-
Nitrogenase ATP
atus and Rhodopseudomonas faecalis have been widely used in
photofermentation studies for H2 production [215,216], while some
studies have been done with mixed cultures isolated from waste-
Reverse
water sludge [220–222]. Yangling et al. [223] evaluated the micro-
e-
transport bial community dynamics in a mixed photofermentative culture
enriched from a digestate from the AD of pig dung and found the
prevalence of mostly R. palustris.
Organic Acids Redwood et al. [21] presented different integration strategies
for combining two step dark and photofermentation processes.
Fig. 6. Schematic presentation of photofermentation (adapted and modified from Nath et al. [202] studied the combined dark and photofermenta-
[217]). tion for biohydrogen production using glucose as the substrate.
DF was carried out by Enterobacter cloacae strain DM11, followed
by photofermentation by R. sphaeroides strain O.U.001 using the
A number of studies have been carried out combining DF either
spent medium from the DF which mainly contained acetic acid.
with photofermentation [196–203] or/and using bio-electrochem-
The combined hydrogen yield was higher than a single biohydro-
ically assisted microbial reactors [204–208] for improving the bio-
gen system, i.e. 1.86 mol H2/mol glucose in DF and 1.5–
hydrogen yield or with the AD process for improving the economic
1.72 mol H2/mol acetic acid in the photofermentation. Similarly,
viability [100,109,209–212]. Light dependent fermentative pro-
combining the two fermentation processes, Chen et al. [213,224]
cesses can be a good option for a second stage H2 production,
and Tao et al. [224] attained a total yield of 10.25 mol H2/mol
because of their higher substrate conversion efficiency, and being
sucrose and 6.63 mol H2/mol sucrose, respectively. In a study by
less energy intensive and environmental friendly [213]. On the
Su et al. [196], a yield of 4.16 mol H2 mol/mol glucose was
other hand, bio-electrochemically assisted microbial fuel cells are
obtained from photofermentation of DF effluents using glucose as
also an option to treat the effluents from DF and increase the H2
the substrate, which increased the total yield to 5.48 mol H2/
yield [214]. Likewise, the economic viability of the DF process
mol glucose.
can be enhanced by AD as a final step. Table 10 gives some exam-
Other researchers have used effluents from DF of diverse sub-
ples of integrated processes of DF combined with post treatments.
strate types in photofermentative biohydrogen production. Argun
et al. [203] used the DF effluent of a ground wheat solution with
5.1. Photofermentation a H2 yield of 781 mL/g total VFA. In another studies by Su et al.
[197,198], cassava starch and water hyacinth were used as the sub-
Under anaerobic conditions, purple non sulfur photosynthetic strates in DF and its effluent was utilized successfully for photofer-
bacteria carry out anaerobic photosynthesis using light as energy mentative biohydrogen production. The studies reported the
source for synthesizing hydrogen [215,216]. The purple non sulfur increase in total H2 yield from 240.4 mL H2/g starch to
bacteria use the captured light energy to produce ATP and high 402.3 mL H2/g starch [197] and 76.7 to 596.1 mL H2/VS [198] using
energy electrons through reverse electron flow which reduces fer- R. palustris. These studies have shown that combined dark and
redoxin (Fig. 6). Then, the ATP and reduced ferredoxin drives the photofermentation is a potential technology for biohydrogen pro-
proton reduction to hydrogen by nitrogenase [217]. The research duction using diverse substrates.
attention to these organisms is increasing because of their higher Some drawbacks of photofermentative systems include the
biohydrogen yield potential and better light utilization proficiency, inherent high energy demand associated with the nitrogenase
as they are able to absorb and utilize both visible (400–700 nm) enzyme, lower solar conversion efficiencies and economic issues
and near infrared (700–900 nm) light. Moreover, they are able to
use a wide variety of substrates [215].
The ability of purple non sulfur bacteria to convert the organic Power
supply
acids to biohydrogen makes photofermentation a good post treat- e-
ment for biohydrogen production from DF effluents. An example of Carbon dioxide - Hydrogen
e
integrated dark and photofermentative conversion of acetic acid to
biohydrogen is:

DF : C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 2CO2 þ 4H2 ð8Þ

Light energy +
Photofermentation : CH3 COOH þ 2H2 O ƒƒƒƒƒƒ! 4H2 þ 2CO2 Microorganism H
ð9Þ
Depending on the operating conditions of the bioreactors and
other parameters described earlier (see Section 4), DF might follow Proton Exchange
Membrane (PEM)
different pathways rather than only the acetic acid pathway.
Anode Cathode
Therefore, a theoretical biohydrogen potential of DF effluents con-
taining acetate, propionate and butyrate can be written as Fig. 7. Schematic diagram of two chambered MEC separated by a proton exchange
[218,219]: membrane and power supply (adapted and modified from [208]).
88 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

of anaerobic photobioreactors covering large areas [33]. However, approximately 300 mV. Thus, hydrogen can theoretically be pro-
these inefficiencies can be overcome by developing an efficient duced at the cathode by applying a circuit voltage higher than
photobioreactor [225,226]. Besides the presence of light condi- 110 mV (i.e. Vapplied = Vanode  Vcathode = 410  (300) mV),
tions, the culture medium of photofermentation should be under though it has been found that a minimum applied voltage of more
ammonia limitation and oxygen should be absent, as both inhibit than 250 mV is needed due to ohmic resistance and electrode over-
the nitrogenase activity [227–229]. Higher ammonia concentra- potential [7,208]. This applied voltage required is considerably
tions (in excess of 2–5 mmol) can be detrimental to hydrogen pro- lower than 1210 mV, the theoretical voltage needed for hydrogen
duction [227,230]. Thus, the effective removal of ammonia from DF production via electrolysis of water at neutral pH conditions [208].
residues can be a bottleneck in coupling photofermentation with Lalaurette et al. [235] tested a two stage process, combining DF
DF processes. Therefore, substrates with a high C/N ratio seem using cellulose as a substrate and MEC systems for hydrogen pro-
more suitable for H2 conversion in these systems. Nonetheless, duction. This improved the total hydrogen yield to 9.95 mol H2/
several ammonia removal strategies such as stripping, natural zeo- mol glucose from the fermentative hydrogen yield of 1.64 mol H2/
lites and selective membranes can be applied which could facilitate mol glucose using cellulose. Similarly, Liu et al. [205] used the vol-
the coupling of the two processes [231,232]. atile acids accumulated in the fermentation of waste activated
In addition to biohydrogen production, accumulation of poly- sludge as a carbon source for biohydrogen production in a MEC
hydroxybutyrate (PHB) could raise future interest, as it possesses with a H2 yield and production rate of 1.2 mL H2/mg COD and
economic value as a precursor of biodegradable polymers [233]. 120 mL H2/g VSS/d, respectively. The results from the analysis of
Thus, energy recovery and economic sustainability of the commer- the electrohydrogenesis end products showed that more than
cial development of DF also depends on the development of post- 90% of the acetate and propionate were converted to hydrogen,
treatment processes like photofermentation. but with lower conversion of n-butyrate and n-valerate (<20%).
Likewise, Moreno et al. [24] obtained 94.2 L H2/kg VS from two
5.2. Microbial electrolysis cells stage DF-MEC systems using cheese whey wastewater.
The MECs are still under research and development. One of the
Biohydrogen production from DF residues is also possible challenges of MECs is to suppress the methanogenic activity during
through an emerging technology known as electrohydrogenesis the electrohydrogenesis with mixed cultures as it negatively
or biocatalyzed electrolysis or microbial electrolysis affects the H2 production rate. Hu et al. [236] has proposed to inhi-
[24,25,27,29,205,234]. Electrochemically assisted Microbial Fuel bit the methanogenic activity by exposing the cathodes to air. They
Cells (MFCs), Microbial Electrolysis Cell (MECs) or Bioelectrochem- studied a single chambered MEC to investigate the hydrogen pro-
ical Systems (BES) use microorganisms to catalyze the biochemical duction using mixed and pure (Shewanella oneidensis MR-1) cul-
reactions at the anode and/or cathode, producing protons and elec- tures. The major objective was to reduce the potential losses
trons from the oxidation of organic matter [204,208,214]. MECs associated with the membrane and increase the energy recovery
should not be confused with MFCs, the former is an electrolysis of the process. Studies of the long term performance of MEC sys-
reactor which produces hydrogen, while a MFC is a fuel cell that tems are needed to further develop and achieve the technical
produces electricity [214]. In MECs, on oxidizing acetate under and economic edge of this technology.
standard biological conditions (25 °C, 1 bar pressure and pH 7)
hydrogen can be produced at the cathode by applying a small cir- 5.3. Anaerobic digestion
cuit voltage, theoretically 0.14 V [214] (Fig. 7). Some exoelectro-
genic microorganisms which are capable of electron transfer to Anaerobic digestion (AD) is a proven biological waste treatment
an electrode (anode) include the genera Geobacter, Shewanella method for volume reduction, waste stabilization and biogas
and Pseudomonas sp. [24,208,214], while the function and the com- recovery (CH4) from organic waste [103]. The AD process can be
munity composition of the microorganisms at the cathode are not combined with DF to achieve further conversion of end products
known [214]. The evolution of hydrogen in BESs can be repre- of DF and the residues from photofermentation and MECs systems
sented in the following reactions: (Fig. 5). Photofermentation requires a clear medium for efficient
C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 2CO2 þ 4H2 ð13Þ light utilization. Thus, the residue from the filtration of DF efflu-
ents, microbial biomass produced in photofermentation and the
Anode : CH3 COOH þ 2H2 O ! 2CO2 þ 8e þ 8Hþ ð14Þ residues from MECs (if any) can be utilized in AD for the final sta-
bilization. The two stage processes, combining biohydrogen pro-
duction in the first stage and AD in the second stage, not only
Cathode : 8Hþ þ 8e ! 4H2 ð15Þ
increase the sustainability of the process, but also guarantee the
A minimum theoretical voltage required to produce hydrogen complete treatment of the organic waste [27].
at pH 7 is 410 mV (Normal Hydrogen Electrode). However, the A number of studies have been done on dual systems
anode potential produced by the oxidation of organic matter is [100,109,209–212,237]. DF followed by AD (Fig. 8) has shown

H2 & CO2 is recovered CH4 & CO2 is recovered

H2 & CO2 CH4 & CO2

Hydrolysis
Organic waste and
acidogenesis Organic
Acids Methanogenesis

Dark fermentation Biomethanation

Fig. 8. Two-stage process for hydrogen and methane production from organic waste.
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 89

technical and economic feasibility of the integrated process up to of DF processes [88,99,105,168,253,254]. Ren et al. [99] studied a
pilot scale [88,105]. Wang and Zhao [238] ran a successful pilot 1.48 m3 continuous flow anaerobic reactor for 200 days at an OLR
scale unit consisting of hydrolysis–acetogenesis for H2 generation of 3.11–85.57 kg COD/m3/d fed with molasses. The maximum
in a rotating drum of 200 L, followed by a methanogenesis stage hydrogen yield was 26.13 mol H2/kg COD removed in the OLR
in a 800 L reactor. Likewise, Cavinato et al. [209] established suc- range of 35–55 kg COD/m3/d and a maximum production rate of
cessful two stage conversion of hydrogen and methane from 5.57 m3 H2/m3 reactor/d was reached. Jayalakshmi et al. [168]
organic waste. Similarly, Antonopoulou et al. [239] investigated worked with a plug-flow inclined DF reactor of volume 0.15 m3
two stage hydrogen and methane production using sweet sorghum with kitchen waste as the substrate. The reactor gave a H2 yield
with a H2 yield of 10.41 L H2/kg sweet sorghum and a methane of 72 mL H2/g VS added.
yield of 29 L CH4/kg sweet sorghum utilizing DF residues as a sole Another reported long term pilot-scale study was carried out at
substrate in AD, while Kvesitadze et al. [240] obtained a cumula- Fen Chia University (Taiwan), comprising of two feedstock storage
tive H2 and CH4 yield of, respectively, 104 L H2/g VS and tanks (0.75 m3 each), a nutrient storage tank (0.75 m3), a mixing
520 L CH4/g VS using OFMSW. Similarly, Antonopoulou et al. tank (0.6 m3), an agitated granular sludge bed fermenter (working
[239] showed the feasibility of a two stage hydrogen–methane volume 0.4 m3), a gas–liquid–solid separator (0.4 m3) and a control
process using cheese whey. panel. A pilot-scale high-rate reactor was operated for a period of
Jung et al. [241] showed that two stage H2–CH4 conversion from 67 days under mesophilic conditions (35 °C) at an OLR of 40–
molasses is economically feasible. Ruggeri et al. [211] used the 240 kg COD/m3/d with sucrose as the substrate. An OLR of
energy balance as a tool to determine the sustainability of inte- 240 kg COD/m3/d gave a hydrogen production rate of 15.59 m3/
grated DF and AD, which showed the positive energy gain. Simi- m3 d and a hydrogen yield of 1.04 mol H2/mol sucrose. In another
larly, Schievano et al. [242] reported 8–43% increment in energy study, Cavinato et al. [88] carried out a two-stage pilot-scale ther-
production in two stage systems in comparison to a single stage mophilic DF and AD of food waste for the production of, respec-
AD. Thus, in light of recent popularity of two stage AD processes tively, biohydrogen and methane with recirculation of AD
for treating high strength wastewater or concentrated solids, the effluents to DF to control the pH (5–6). The organic loading rate
former stage can be modified to be used for hydrogen production of 16.3 kg TVS/m3 d was maintained with a HRT of 3.3 days in
[43]. Also, the DF process can be seen as a pre-treatment stage if the DF stage, yielding 66.7 L H2/kg TVS.
the organic waste of interest is subjected to complete stabilization
[238]. Thus, in order to improve the economic competence of com-
mercial DF, AD could provide an attractive solution [243]. 7. Challenges and future prospects

7.1. Modeling and simulation

5.4. Other applications

Several researches have been proposed to integrate DF pro-

Besides the conversion of volatile fatty acids and other reduced
cesses with AD, photofermentation or bioelectrochemical systems
carbon sources to biomethane or biohydrogen in biological pro-
to utilize the VFAs produced to increase its viability. Modeling of
cesses, VFAs can be used in various applications: biological nutri-
kinetic parameters and biohydrogen production becomes impor-
ent removal from wastewater [244,245], sulfur and sulfate
tant for the design, analysis and operation of the fermentative pro-
reduction [246,247], biopolymer (such as polyhydroxybutyrate)
cesses. Also, the predictive capacity of the model for end products
production [94] and microbial lipids production [55,248]. Lim
helps to design the downstream processes. Several models have
et al. [244] studied the use of volatile fatty acids produced from
been proposed to describe the biohydrogen production, growth
food waste as carbon sources in the removal of nitrogen and phos-
of hydrogen fermenters, substrate consumption and intermediate
phorous from municipal wastewater in a sequential batch reactor
biochemical processes [255–257]. With increasing research on
(SBR) [244]. Similarly, Elefsiniotis et al. [245] studied the denitrifi-
DF, the modeling of the biohydrogen production process could be
cation process (20–200 mg NO 3 -N/L) using VFA generated from
of primary interest to achieve a better understanding of the DF
the AD of starch rich industrial and municipal wastewater as a car-
pathways and control of the process.
bon source in batch reactors.
Parameters such as substrate concentration, pH, temperature,
Similarly, a ‘‘carboxylate platform’’ or third biorefinery platform
and HRT affect the H2 yield and production rate and the nature
has been introduced to generate a mixture of carboxylates as inter-
of the end products (see Section 4). Wang and Wan [256] reviewed
mediates for the production of complex fuels utilizing waste bio-
existing mathematical models such as the Modified Gompertz
mass [249]. Ntaikou et al. [94] investigated the combined
model for product formation (H2 production), the Logistic model
production of biohydrogen and biopolymers from the DF of olive
for biomass growth [258], substrate utilization based on Monod
mill wastewater and the use of DF effluents which mostly con-
Kinetics, the Arrhenius model for temperature effects, pH inhibi-
tained VFAs in SBR using polyhydroxyalkanoates (PHAs) producing
tion models based on the IWA Anaerobic Digestion Model No. 1
bacterial culture.
(ADM1) [259] and the Modified Luedeking–Piret models for the
Tuna et al. [250] used the volatile fatty acids produced in DF
formation of by-products [258].
processes for hydrogen production by electrohydrolysis. Hydrogen
There is, however, a need to upgrade the different kinetic mod-
was generated by applying a low voltage in the range of 1–3 V DC
els, including complex biochemical processes, which involve the
current to DF effluents of wheat powder containing different VFAs
fermentative biohydrogen production such as hydrolysis, acido-
concentrations. The applied voltage of 2 V and 10.85 g/L of total
genesis and H2 production from complex substrates (Fig. 2). The
VFA gave the highest energy efficiency (56%).
IWA ADM1 has been used extensively to model AD processes
[174,175,259,260]. ADM1 is a structured mathematical model
6. Pilot scale applications based on the COD balance of composite substrates and includes a
number of biochemical processes involving disintegration of sub-
Most DF studies have been carried out at laboratory scale batch, strates such as hydrolysis, acidogenesis, acetogenesis and metha-
semi-continuous or continuous reactors. To the best of our knowl- nogenesis, biomass growth and decay processes and the physical
edge, no studies have reported the DF process at industrial or full interaction of the gas–liquid phases. Because of the similarity of
scale. Limited studies have been done on pilot-scale applications some initial biochemical and physical processes, a modified
90 A. Ghimire et al. / Applied Energy 144 (2015) 73–95

ADM1 has been proposed to model dark fermentative biohydrogen maintain the pH in the DF step around 5.5, giving a H2 yield of 51 L/
production processes [31,255,257]. Nonetheless, a model that can kg VS of food waste fed with a H2 content of 37% in the biogas.
simulate the process and predict the formation of all the major However, the major concern with the recirculation of the AD reject
intermediates and biohydrogen considering all influencing param- water is the activity of methanogens present in the reject water,
eters is a necessity. which can affect the purity of the biohydrogen produced in the
DF step. In addition, inhibition of H2 production due to higher lev-
7.2. Energy balance and COD conversion els of ammonia present in the reject water could be another con-
cern as reported in a study by Cavinato et al. [88]. Thus, long
The net energy gain in DF processes is an important issue that term studies to assess the effect of reject water recirculation from
has been addressed by few researchers [211,261,262]. The energy the AD step on the H2 content in biogas produced from DF could
balance is an important factor for the process sustainability. Higher open further doors to ensure the sustainability of DF systems.
culture temperatures have been suggested in the literature
[40,41,45,135,263] for maximizing H2 yield, without considering
7.4. Solid state dark fermentation (SSDF)
the net energy gain [261]. Some studies [211,261] suggested that
DF processes have to be operated at ambient temperature in order
Anaerobic reactors are generally categorized into wet (<10% TS),
to obtain a positive net energy. After evaluation of literature data
semi-dry (10–20% TS) and dry (>20% TS) processes [265]. However,
on DF of different substrates, Perera et al. [261] reported the net
some categorized wet digestion for low (<15% TS) and dry diges-
energy gain in dark fermentative processes is positive when the
tion for high (>15% TS) solids processes [136]. By increasing the
process temperature is below 25 °C. In another study by Ruggeri
TS content, dry fermentation processes can be operated at a high
et al. [211], the optimum working temperature of 20 °C has been
OLR with little water addition, which offers advantages such as
recommended, which offers 20% of the available energy. However,
smaller reactor volume, easy handling of the digestate residues
these studies have suggested to couple DF processes with AD,
and technical simplicity [136,265]. This could be attractive for
microbial fuel cells, bioelectrochemical systems or photofermenta-
commercialization of these processes. However, the drawbacks of
tion to obtain a more positive net energy balance from the recovery
SSDF are the low H2 yields due to mass and energy transfer limita-
of energy from the DF end-products and residues.
tions, which affect the product formation [139].
Perera et al. [261] reported that DF combined with BES or DF
Using agro-industrial wastes (70% sugarcane bagasse, 15% of
with AD can result in a positive energy yield. Similarly, Ruggeri
pineapple peelings and 15% of waste activated sludge) under mes-
et al. [211] found that the AD step after DF can deliver a positive
ophilic conditions, Robledo-Narváez et al. [139] found a decrease in
net energy with 40–90% available energy. Su et al. [197] obtained
H2 yield (3 mmol H2/g TS) at a TS content higher than 18% TS in a
a higher conversion efficiency of the heat value in DF from 13.3%
tested TS content range from 15% to 35%. Similar results were
to 46.0% when combined with photofermentation. This was due
obtained by Valdez-vazquez and Poggi-Varaldo [140], where the
to an increase of the H2 yield from 1.59 to 5.48 mol H2/mol glucose.
highest H2 productivity and yield (463.7 N mL/kg d and
Lower rates of COD reduction efficiencies are a concern if the DF
54.8 N mL/g VS removed, respectively) was obtained at a TS of
process aims to treat waste biomass. The conversion of COD to
20.9% using organic solid waste (paper (40%) and food (60%)
hydrogen is low; theoretically 16 g of COD reduction is achieved
wastes) for the tested TS range from 20.9 to 35.1% TS. Likewise,
per mole of H2 obtained. However, the COD remains in the by-
Motte et al. [138] also reported 19% TS as the limit to achieve
products as VFAs and alcohols. Mohammadi et al. [90] obtained
higher H2 production performance during the DF of wheat straw,
0.41 mmol H2/g COD from mesophilic DF of POME with a COD
as metabolic pathways shifted towards lactic acid formation at
removal efficiency of 86%. In another study, O-Thong et al. [89]
higher TS content. Further research is required on SSDF in order
obtained a COD removal efficiency of 55% with a H2 yield of
to elucidate the mechanisms involved during dark fermentation
0.142 L H2/L POME. Nonetheless, it has been suggested from the
at high TS contents. This research could provide practical solutions
studies (Table 10), that combining DF processes with AD, BES or
for biohydrogen production from organic solid waste.
PF will not only improve the energy recovery, but give higher
COD reduction efficiencies and provide complete treatment of
organic waste biomass. 8. Conclusions

7.3. pH control DF technology has an excellent future potential for biohydrogen

production as renewable biomass can be used as a feedstock and
Unlike AD processes where the production of acidity from VFAs the integration with other systems could foster a higher H2 yield
generation is balanced by alkalinity of the systems, DF processes and economic feasibility. The economic considerations and pro-
are unstable because of the continuous production of acidity (VFAs duction at industrial scale recommend a continuous bioprocess.
production). As discussed earlier (Section 4.2.3), a very low pH can Thus, more research on continuous DF processes needs to be car-
inhibit the hydrogen production, while the acidic range (5–6) ried out to demonstrate the long term operational feasibility of
favors H2 production depending on the type of substrate. The use continuous processes. The microbial community of hydrogen pro-
of an excessive amount of buffers, acids or base to maintain the ducers and innovative substrates needs to be explored. The use of
pH acidic can decrease the economics and sustainability of the pro- spent dark fermentation residues in photofermentation and, or
cess as well as increase the salt concentration of the DF effluents. electrochemical systems as a secondary step could pave the way
One of the sustainable solutions could be to explore substrates towards sustainable biohydrogen production in up-scaled systems.
with a higher pH or alkalinity to equilibrate the system. Choi et al. Finally, anaerobic digestion is required to further stabilize the res-
[56] suggested the use of substrates with a high pH to replace the idues generated from the upstream processes. The future design
use of buffers. Life Cycle Analysis (LCA) of two-step thermophilic and configuration of industrial scale dark fermentative processes
DF followed by photofermentation of potato peels, showed that is expected to be similar to anaerobic digestion processes, with
most of the impact was generated by the use of the phosphate buf- some modifications in process parameters. Existing two stage
fer during the process [264]. To provide natural buffering, Cavinato methane producing plants can be modified for dark fermentation,
et al. [263] recycled the reject water (effluent) from the AD step in while solid state dark fermentation opens new opportunities for
the two-step DF and AD. The AD reject water provided alkalinity to biohydrogen production from renewable biomass.
 A. Ghimire et al. / Applied Energy 144 (2015) 73–95 91

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