Role of Natural Deep Eutectic Solvents

(NADES) in the Pretreatment 4

of Lignocellulosic Biomass for an Integrated
Biorefinery and Bioprocessing Concept

Shaishav Sharma and Adepu Kiran Kumar

Abstract
Currently, biorefinery process from cellulosic biomass is complex and energy-
intensive where biomass fractionation, a key step for production of biomass-
derived chemicals, demands conventional organic solvents. Since there is a clear
and an unmet need for a robust and affordable biomass conversion technology,
researchers are constantly developing novel, cost-effective green solvents that are
eco-friendly, benign, renewable, and biodegradable in nature. In this perspective,
natural deep eutectic solvents, commonly referred as NADES, have been evolved
as the most advanced green solvents entirely made up of natural compounds to
combat and deconstruct the recalcitrance of lignocellulosic biomass into the
extraction of potential renewable chemicals. Although NADES are characteristi-
cally similar to deep eutectic solvents (DESs) and ionic liquids, NADES have
shown advantages to combat the challenges associated with ionic liquids and
DES by being highly specific and green in nature. Moreover, NADES could be
recovered and recycled without losing their efficiency, making these solvents
highly economical and sustainable. In this chapter, we have comprehensively
described the applicability of NADES in biorefinery and bioprocessing as an
effective tool of lignocellulosic biomass pretreatment for bioconversion
processes.

Keywords
Natural deep eutectic solvents (NADES) · Pretreatment · Lignocellulosic
biomass · Biorefinery · Bioprocessing · Bioconversion

S. Sharma · A. K. Kumar (*)

Bioconversion Technology Division, Sardar Patel Renewable Energy Research Institute,
Vallabh Vidyanagar, Gujarat, India
e-mail: [email protected]

# Springer Nature Singapore Pte Ltd. 2018 73

P. K. Sarangi et al. (eds.), Recent Advancements in Biofuels and Bioenergy
Utilization, https://doi.org/10.1007/978-981-13-1307-3_4
74 S. Sharma and A. K. Kumar

Abbreviations

Ala Alanine
BA Butyric acid
BE Betaine
CA Citric acid
CC Choline chloride
DA Decanoic acid
DEG Diethylene glycol
DOA Dodecanoic acid
EG Ethylene glycol
Fru Fructose
GA Glutaric acid
Gal Galactose
Glu Glucose
Gly Glycerol
H 2O Water
HA Hexanoic acid
LA Lactic acid
LE Levulinic acid
MA Malic acid
MAL Malonic acid
Mnl Menthol
NA Nicotinic acid
OA Oxalic acid
OCA Octanoic acid
PD Propane diol
Pro Proline
Suc Sucrose
TA Tartaric acid
TEG Triethylene glycol
Ur Urea
Xtl Xylitol
Xyl Xylose

4.1 Introduction

Bioconversion of lignocellulosic agricultural residues to biofuel and value-added

products is gaining importance globally. The development of second-generation
bioethanol from agro-residues has several advantages in terms of energy and envi-
ronment. Lignocellulosic biomass is an abundantly available sustainable and
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 75

renewable feedstock having the potential to replace fossil fuels and other fossil-
based materials and chemicals. Lignocellulose is the basic structural constituent of
all the plants (woody and non-woody) and mostly consists of three main fractions,
i.e., lignin, cellulose, and hemicellulose. The optimized utilization of these key
constituents has a major role in the viability and sustainability of biorefinery and
bioprocessing industries. In a biorefinery, along with the production of second- and
third-generation liquid biofuels such as bioethanol and biodiesel, the valorization of
other by-products generated in the process also plays a major role. The conversion of
biomass to liquid fuels (bioethanol) commonly involves five process steps:
(i) selection of suitable lignocellulosic biomass, (ii) effective pretreatment, (iii)
enzymatic saccharification of cellulose and hemicellulose, (iv) fermentation of
monomeric sugars (hexose and pentose), and (v) downstream processing for recov-
ery and reuse of solvents. Lignocellulosic biomass despite being a potential candi-
date for biofuel and other value-added products suffers from various obstacles that
are associated in their effective deconstruction.
The structure of a plant cell wall is highly recalcitrant owing to the structural
intricacy of lignocellulosic portions. In addition, the inhibitors and by-products
produced during the pretreatment process create a strong hindrance. Some other
obstacles that are needed to be taken into consideration are the selection of an
appropriate pretreatment method, understanding the physicochemical complexity
of feedstock cell walls, and degree of cell wall destruction for production of value-
added by-products. Lignocellulosic biomass pretreatment is one of the key processes
in biorefinery and bioprocessing. Within the context of production of fuels from
biomass, pretreatment is known as the process by which cellulosic biomass is made
available to the action of hydrolytic enzymes.
There are several points that need to be considered for selecting a suitable
pretreatment method: (i) size reduction of biomass particles should not be required,
(ii) deconstruction of hemicellulose fraction should be minimum, (iii) there should
be minimum energy demands, (iv) there should be no or minimum inhibitory
products formation, (v) catalyst involved should be inexpensive and recyclable,
and (vi) there should be a recovery of value-added products such as lignin
(Wyman 1999). The selected pretreatment method should defend as well as justify
its effect on the cost of subsequent downstream processing steps and the trade-off
between capital costs, operating costs, biomass costs, etc. (Lynd et al. 1996). At
present, the technologies developed and applied for pretreatment have several
drawbacks, such as (i) high cost and harmful impact of chemicals involved in the
pretreatment process; (ii) high cost of reactors developed to withstand high temper-
ature, pressure, and corrosive effect of chemicals; (iii) inhibitory effect caused due to
the formation of several by-products; (iv) incomplete destruction of lignin-
carbohydrate matrix; (v) large volumes of water required for washing, neutralization,
etc.; and (vi) an expensive process of recovery of solvents.
In order to overcome the above mentioned disadvantages, a new class of green
solvents with low melting temperatures has been developed. Developing these types
of new and advanced green solvents for a wide variety of applications has attracted
the researchers working in the field of green chemistry worldwide. Recently, such
76 S. Sharma and A. K. Kumar

group of green solvents termed as natural deep eutectic solvents (NADES) is gaining
interest rapidly. As the name suggests, these solvents are very similar to the well-
established deep eutectic solvents (DES) related to its functions and properties;
however, they differ in the chemical components involved in their synthesis. DES
involves mixtures of chemically synthesized inorganic and organic chemicals,
whereas NADES are solely produced from natural metabolites such as sugars,
amino acids, organic acids, sugar alcohols, and amines. This provides NADES an
inherent advantage over other conventional solvents. Moreover, NADES are
completely eco-friendly, nontoxic, cost-effective, easy to handle, synthesizable
and recyclable, biocompatible, and highly biodegradable. In the recent years,
NADES generated a great attention in many industrial applications ranging from
pharmaceutical, chemical, and food processing to enzyme and biofuel industries due
to their mode of action. Observing their prospective application in several industries,
NADES are considered as the solvents of the twenty-first century (Paiva et al. 2014;
Smith et al. 2014).
The current advances in NADES pretreatment have shown high specificity for
lignin solubility and extraction from lignocellulosic biomass (rice and wheat straw)
(Kumar et al. 2016) and woody biomass (Alvarez-Vasco et al. 2016). Another
important aspect is the mechanism involved in the interaction of NADES and
water, which has brought additional focus on them. It is well known that water
being the most abundant solvent plays a vital function in biological systems. Apart
from water and lipids, NADES in combination with water and biological fluids may
possibly play a major role as another type of solvent inside the cells of living
organisms (Dai et al. 2015). Supporting this phenomenon, a research study on
NADES showed that they involve in the synthesis, transportation, and storage of
various inadequately water-soluble compounds (Choi et al. 2011). Through a
biological viewpoint, the simultaneous existence of NADES and water in living
organisms is very important to understand the effect of water on NADES to further
enhance their applicability. The presence of NADES in organisms points toward a
vital phenomenon, i.e., the presence of an alternate medium for conducting various
metabolic functions that are not able to occur in water and lipids. The presence of
NADES can provide answers to the mechanisms of various reactions that could not
occur in water and lipids alone such as biosynthesis of hydrophobic metabolites, the
adaptive ability of organisms in extreme climatic conditions, etc. (Abbott et al. 2009;
Dai et al. 2013a). This chapter deals with different pretreatment methods with
emphasis on NADES pretreatment, its synthesis, physicochemical properties, and
its role in biorefinery and bioprocessing.

4.2 Lignocellulosic Feedstocks

The world is directly or indirectly dependent on agriculture for food requirements,

and this leads to the generation of million metric tons of lignocellulosic biomass and
residual wastes annually. This resource has not been fully utilized and in some cases
yet untapped and has huge potential for bioconversion into an excellent source of
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 77

Fig. 4.1 Illustration depicting the lignocellulosic framework

energy generation (Prasad et al. 2007). Lignocellulosic biomass is defined as a

biological material available in the form of vegetation, forest waste, by-products of
crops, and agro- or food industrial waste. A variety of biomass resources are
available and classified on the basis of their physicochemical nature, viz., grasses,
woody plants, fruits, vegetables, agricultural crop residues, municipal and industrial
waste, etc. (Kumar and Sharma 2017). An illustration depicting the typical lignocel-
lulosic biomass framework and its main constituents is shown in Fig. 4.1.
The organic by-products produced during the processing and harvesting of
agricultural crops are called as agricultural residues. Based on the time of yield,
the agricultural residues can further be categorized into primary and secondary
residues. Agro-residual waste materials obtained at the time of yield in the field
are termed as primary residues. Sugarcane tops and rice straw are the examples of the
primary residues, while secondary residues are the residues that are obtained during
the processing of the crops such as rice husk, sugarcane bagasse, etc. (Murali et al.
2008). The primary residues are mainly used as animal feed, fertilizers, etc. and
therefore cannot be used for energy applications. Secondary residues are obtained in
78 S. Sharma and A. K. Kumar

Agricultural wastes:
Rice and wheat husk, coconut coir,
sugarcane bagasse, cotton stalk, straws of
pulses, coatings of oil seeds

Agro-Industrial Wastes: Forest Wastes:

Pulp waste from food
Sawdust, leaves, chips, barks,
industries, molasses from sugar Lignocellulosic
feedstock for wood logs
industries, wastes from paper
mills, textile mills etc. integrated
biorefinery
approach

Municipal Solid Waste: Energy Crop Plantation:

Food and kitchen wastes like Prosopis, bamboo, Switchgrass,
vegetables and fruits, paper, Hybrid poplar, Napier grass
green wastes etc.

Fig. 4.2 Different types of biomass resources

large quantities and have potential to be developed as a raw material for energy
production. A simple classification of biomass resources based on their source is
shown in Fig. 4.2 (Kumar et al. 2015a).
Wood is primarily composed of hemicellulose, cellulose, and lignin. Based on the
varying proportions of these substances, woods are classified into softwoods and
hardwoods. Softwood hemicellulose has a higher fraction of glucose and mannose
than hardwood hemicellulose, which has a higher fraction of xylose. Moreover,
hemicellulose has been found to be more acetylated in hardwoods as compared to
softwoods. Lignin is divided into two categories: syringyl lignins and guaiacyl
lignins. The difference between the two lies in the substituents of phenyl-propanoid
skeleton. Syringyl lignin has methoxy group in three-carbon position, while guaiacyl
lignin has a methoxy group in three-carbon position. Hardwoods contain guaiacyl-
syringyl lignin, while softwoods majorly contain guaiacyl lignin (Palmqvist and
Hagerdal 2000). Hardwoods have been found to contain a lesser quantity of lignin
than softwoods (Saka 1991). The examples of hardwoods are maple, beech, teak,
oak, and walnut, while Douglas fir, cedar, pine, redwood, juniper, and spruce are
examples of softwoods. Other lignocellulosic residues include garden waste, dried
leaves, forest waste, etc. In all these lignocellulosic biomass residues, the main
constituents are cellulose, hemicellulose, and lignin, and their compositional varia-
tion in a few biomasses is shown in Table 4.1.
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 79

Table 4.1 Cellulose, hemicellulose, and lignin content in common lignocellulosic feedstocks
Lignin Cellulose Ash
Lignocellulosic fraction fraction Hemicellulose content
materials (wt %) (wt %) fraction (wt %) (wt %) References
Agro-residues 25–50 5–15 37–50 – Kumar and Sharma
(2017)
Banana peels 14 13.2 14.8 – Kumar and Sharma
(2017)
Corncobs 15 45 35 12–16 Kumar and Sharma
(2017) and Saini
et al. (2015)
Corn stover 19 38 26 3.6 Kumar and Sharma
(2017) and Saini
et al. (2015)
Cotton seed 0 80–95 5–20 – Kumar and Sharma
hairs (2017)
Grasses 10–30 25–40 25–50 – Kumar and Sharma
(2017)
Hardwood 18–25 40–55 24–40 0.6 Kumar and Sharma
(2017) and Saini
et al. (2015)
Hardwood 25–40 20–25 45–47 0.8 Kumar and Sharma
barks (2017) and Saini
et al. (2015)
Leaves 0 15–20 80–85 – Kumar and Sharma
(2017)
Newspaper high 18–30 40–55 25–40 – Kumar and Sharma
grade (2017)
Newspaper low 25–40 12 40–55 – Kumar and Sharma
grade (2017)
Nutshells 30–40 25–30 25–30 – Kumar and Sharma
(2017)
Rice straw 18 32 24 14–20 Kumar and Sharma
(2017) and Saini
et al. (2015)
Softwood 25–35 45–50 25–35 0.5 Kumar and Sharma
(2017) and Saini
et al. (2015)
Softwood barks 25–29 30–60 40–45 0.8 Kumar and Sharma
(2017) and Saini
et al. (2015)
Sorted refuse 20 60 20 – Kumar and Sharma
(2017)
Sugarcane 20 42 25 1.5–5 Kumar and Sharma
bagasse (2017) and Saini
et al. (2015)
Sweet sorghum 21 45 27 – Kumar and Sharma
2017
(continued)
80 S. Sharma and A. K. Kumar

Table 4.1 (continued)

Lignin Cellulose Ash
Lignocellulosic fraction fraction Hemicellulose content
materials (wt %) (wt %) fraction (wt %) (wt %) References
Switchgrass 12 45 31.4 – Kumar and Sharma
(2017)
Waste paper 12–20 6–10 50–70 – Kumar and Sharma
pulps (2017)
Wheat straw 16–21 29–35 26–32 6–8 Kumar and Sharma
(2017)
White paper 0–15 85–99 0 – Kumar and Sharma
(2017)

4.3 Biomass Pretreatment Processes

Lignocellulosic feedstock represents an extremely large quantity of renewable

bioresource available in excess on earth and is an appropriate raw matter for a
variety of applications for human sustainability. However, effective utilization of
lignocellulosic biomass is a hard nut to crack due to several obstacles associated with
it. Some of the major factors are the recalcitrance of the plant cell wall due to the
integral structural complexity of lignocellulosic fractions and strong obstruction
from the by-products and inhibitors formed during pretreatment. Moreover, other
challenges such as understanding the physicochemical structure of their cell walls,
appropriate pretreatment method, and extent of cell wall decomposition for produc-
tion of value-added by-products.
Lignocellulosic biomass pretreatment is an indispensable step required for
modifying the strongly bound lignocellulosic structure, which exposes the
constituents for effortless accessibility to an enzyme for hydrolysis, which in turn
increases the pace and amount of reducing sugars (Alvira et al. 2010). Based on the
type of agent involved, pretreatment methods are classified into two categories:
biological and non-biological. Non-biological pretreatment methods which do not
involve any microbial agent are broadly classified into physical, chemical, and
physicochemical methods. A list of promising and most commonly used
pretreatment methods is shown in Fig. 4.3.

4.4 Biological Pretreatment

Biological pretreatment methods are more efficient, environmentally safe, and low
energy consuming as compared to conventional chemical pretreatment methods.
Nature supplies an extensive array of hemicellulolytic and cellulolytic
microorganisms, which can be exclusively applied for efficient biomass pretreatment
(Vats et al. 2013). Among these microorganisms, white rot, soft rot, and brown rot
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 81

Fig. 4.3 Overview of different pretreatment processes

fungi possess the capacity to denature hemicellulose and lignin (Sanchez 2009).
White rot fungi have the ability to degrade lignin due to the presence of laccases
(lignin-degrading enzymes) and peroxidases (Kumar et al. 2009). Normally
employed white rot fungi species are Pleurotus ostreatus, Phanerochaete
chrysosporium, Cyathus stercoreus, Pycnoporus cinnabarinus, Ceriporiopsis
subvermispora, and Ceriporia lacerata. The microorganisms that are reported to
have shown high delignification efficiency are Fomes fomentarius, Bjerkandera
adusta, Irpex lacteus, Ganoderma resinaceum, Lepista nuda, Trametes versicolor,
and Phanerochaete chrysosporium (Kumar et al. 2009; Shi et al. 2008). Different
classes of microbes used for pretreatment along with their effects on different
lignocellulosic biomasses are summarized in Table 4.2. Although biological
pretreatment is captivating, hydrolysis rate has been found to be slow which
obstructs the application of biological agents for pretreatment at commercial scale.
Other robust fungal species having the capability to delignify the lignocellulosic
biomass need to be tested for further applications.
82 S. Sharma and A. K. Kumar

Table 4.2 Different biological pretreatment strategies involved for pretreatment of lignocellulosic
biomass and its advantages
Significant alterations after
Agro-residues Biological agent pretreatment References
Bamboo Punctularia sp. >50% removal of lignin Suhara et al.
culms (2012)
Cornstalks Irpex lacteus 82% reducing sugar yield Du et al.
(2011)
Corn stover Fungal consortium >44% lignin removed, hydrolysis Song et al.
efficiency improved by sevenfold (2013)
Corn stover Irpex lacteus >66% saccharification efficiency Xu et al.
(2010)
Corn stover Ceriporiopsis Reducing sugars enhanced by 2–3 Wan and Li
subvermispora times (2011)
Eucalyptus Pleurotus ostreatus/ Hydrolysis efficiency increased by Castoldi
grandis Pleurotus pulmonarius 20-fold et al. (2014)
sawdust
Plant Fungal consortium Toxic chemicals completely Dhiman
feedstock removed et al. (2015)
Rice husk Phanerochaete – Potumarthi
chrysosporium et al. (2013)
Rice straw Dichomitus squalens >55% enzymatic hydrolysis Bak et al.
(2010)
Straw Fungal consortium Hydrolysis efficiency increased by Taha et al.
20-fold (2015)
Wheat straw Ceriporiopsis Negligible loss of cellulose Cianchetta
subvermispora et al. (2014)

4.5 Physical Pretreatment

The non-biological pretreatment includes various physical, chemical, and physico-

chemical methods. Various pretreatment methods summarised here as the detailed
analysis of these methods is beyond the scope of this chapter. The readers are
suggested to refer the recent review by Kumar and Sharma (2017) for further details
on various pretreatment methods.
The physical pretreatment methods involve the use of different physical forces for
altering the tightly bound lignocellulosic structure for exposure to the enzyme.
Commonly applied physical pretreatment methods are:

1. Mechanical extrusion: It is the oldest and most common method used for the
lignocellulosic biomass pretreatment. Under this process, the biomass is
subjected to high temperature (>300
C) along with shear mixing. However,
this method is highly energy-intensive thereby increasing the cost and unfeasible
for industrial-scale applications (Zhu and Pan 2010).
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 83

2. Milling: This technique is majorly applied to reduce the crystallinity of cellulose.

This process commonly includes grinding, chipping, and milling techniques.
Chipping reduces the size of biomass up to 10–30 mm, while grinding and
milling reduce it further up to 0.2–0.4 mm. Chipping process decreases the
limitations of heat and mass transfer, whereas grinding and milling efficiently
decrease the cellulose crystallinity and particle size owing to the shear forces
produced during milling (Kumar and Sharma 2017). There are various milling
methods, viz., hammer milling, two-roll milling, vibratory milling, and colloid
milling, which are used to increase the degradability of the lignocellulosic
biomass (Taherzadeh and Karimi 2008).
3. Microwave: It is an extensively used method for pretreatment of lignocellulosic
agro-residues. This method is useful because of (i) low energy requirement,
(ii) simple operating conditions, (iii) minimum inhibitor formation, (iv) high
heating capacity in short time duration, and (v) the ability to degrade cellulose
structural organization (Kumar and Sharma 2017).
4. Sonication: Ultrasound waves produce chemical and physical effects which alter
the morphology of the lignocellulosic biomass. This results in the formation of
cavitation bubbles, which disrupts the cellulosic and hemicellulosic portions
resulting in the increased availability of cellulose-degrading enzymes for efficient
breakdown into simpler sugars. However, the duration and power of sonication
need to be optimized further for maximum pretreatment efficiency.
5. Pulsed electric field: This treatment subjects the biomass to a rapid burst of high
voltage in the range of 5–20 kV/cm for small durations (nano- to milliseconds).
The pulsed electric field has certain advantages, viz., low energy requirement,
ambient conditions required for pretreatment, and simplicity in design due to lack
of moving parts (Kumar et al. 2009).

4.6 Physicochemical Pretreatment

These methods involve a blend of chemical effects and mechanical forces for the
pretreatment of lignocellulosic biomass. A few of such methods have been described
here:

1. Steam explosion: This method involves autohydrolysis of acetyl groups of hemi-

cellulose and pressure drop as chemical and mechanical effects, respectively, for
the effective pretreatment of biomass. High-pressure (0.7–4.8 MPa) saturated
steam at elevated temperatures (between 160 and 260
C) for few seconds to
minutes is applied to biomass, which leads to hydrolysis and release of hemicel-
lulose. Steam penetrates the biomass leading to expansion of fibers thereby
increasing the accessibility of enzymes for cellulose. Steam explosion has several
disadvantages such as partial digestion of lignin-carbohydrate matrix, creation of
fermentation inhibitors at high temperature, and the hydrolysate washing which
reduces the yield of sugar by 20% (Agbor et al. 2011).
84 S. Sharma and A. K. Kumar

2. Liquid hot water: This method applies high temperature (170–230
C) pressure
(up to 5 MPa) leading to hydrolysis of hemicellulose and lignin removal increas-
ing cellulose accessibility. Low cost of the solvents, low temperature require-
ment, and minimum formation of inhibitory compounds are advantages of liquid
hot water. However, liquid hot water becomes energy-intensive due to the
requirement of large amount of water for downstream processing (Agbor et al.
2011).
3. Wet oxidation: This method is most suited for lignin-rich biomass residues. This
method works on the principle that when water reaches above 170
C, it starts
behaving like an acid and catalyzes hydrolytic reactions. Hemicelluloses are
converted to simpler pentose monomers along with oxidation, but the cellulose
remains unaltered (Kumar and Sharma 2017).
4. SPORL pretreatment: SPORL is the abbreviated form of sulfite pretreatment to
overcome recalcitrance of lignocellulose. It is commonly carried out in two steps.
In the first step, lignin is removed from lignocellulosic biomass by treating with
magnesium or calcium sulfite. In the second step, disk milling is used to reduce
the size of the biomass. The advantages of SPORL pretreatment are rapid
conversion of cellulose to glucose, less energy consuming, ability to process
diverse types of biomass, and easy to scale up the existing mills for commercial
production of biofuels, while certain drawbacks of this method are expensive
recovery of pretreatment chemicals, degradation of sugars, and large amount of
water needed for post-pretreatment washing (Bajpai 2016).
5. Pretreatment using ammonia: Various methods that are known to use ammonia
for lignocellulosic biomass pretreatment are ammonia fiber explosion (AFEX),
ammonia recycle percolation (ARP), and soaking aqueous ammonia (SAA).
Under AFEX, the biomass is heated with ammonia in 1:1 ratio in a closed reactor
at 60–90
C and >3 MPa pressure for 30–60 min. After the biomass has
undergone desired conditions for 5 min, the valve is opened to release the
pressure explosively resulting in evaporation of ammonia and drop in temperature
(Alizadeh et al. 2005). The difference between AFEX and steam explosion lies in
the use of ammonia instead of water in AFEX (Rabemanolontsoa and Saka 2016).
In ARP, 5–15 wt% aqueous ammonia is passed through biomass in a reactor.
Optimum temperature for this process is between 140 and 210
C, while the
percolation rate is 5 mL/min with 90 min reaction time. After the pretreatment is
completed, the ammonia is recycled and reused (Sun and Cheng 2002; Kim et al.
2008).The high energy requirement in ARP to maintain the process temperature
is a major drawback. SAA is a modification of AFEX in which the biomass
undergoes treatment with aqueous ammonia in a reactor at 30–60
C. The
advantage of this method is the reduction of liquid throughput in pretreatment
(Kim and Lee 2005).
6. CO2 explosion: Supercritical CO2 is used in this method, which implies that the
gas behaves like a solvent. Biomass is subjected to supercritical CO2 in a high-
pressure vessel (Kim and Hong 2001). Biomass under the effect of CO2 at high
pressure forms carbonic acid, which leads to hydrolysis of hemicellulose. The
biomass organization is easily disrupted when the pressurized gas is released
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 85

(Zheng et al. 1995). The biomass having low or zero moisture content cannot be
treated through this method. Advantages of this method are low cost of CO2, high
solid loading, minimum inhibitor generation, and low temperature requirement.
However, the expensive reactors capable of withstanding high pressure create
hindrance in its scaling up for commercial applications (Agbor et al. 2011).
7. Oxidative pretreatment: Various oxidizing agents, viz., hydrogen peroxide,
ozone, oxygen, or air, are used for the treatment of lignocellulosic biomass in
this method (Nakamura et al. 2004). During this process, several chemical
reactions such as side-chain displacement, cleavage of aromatic ether linkages,
and electrophilic substitution may occur. This method leads to delignification due
to the conversion of lignin to acids, which may act as inhibitors. These acids must
be removed for carrying out further steps (Alvira et al. 2010). In addition,
hemicellulose is damaged to an extent making it unavailable for fermentation
(Lucas et al. 2012).

4.7 Chemical Pretreatment

These methods involve the use of different chemicals and their characteristic
reactions for disrupting the complex lignocellulosic structure. A few commonly
used chemical pretreatment methods have been briefed here:

1. Dilute acid: The low cost of acids makes it the most commonly used pretreatment
method. The formation of a high amount of inhibitory products such as phenolic
acids, furfurals, aldehydes, and 5-hydroxymethylfurfural is a major disadvantage
of this method. In addition, the reaction vessel should be constructed from a
suitable material, which has the capacity to handle extreme experimental
conditions and corrosive property of acids (Saha et al. 2005).
2. Mild alkali treatment: In this method, hydroxyl derivatives of potassium, cal-
cium, ammonium, and sodium salts are used for the lignocellulosic biomass
pretreatment. Unlike acid treatment, this method can be carried out at ambient
pressure and temperature. The side chains of esters and glycosides are degraded
by alkali reagents resulting in modification of lignin, decrystallization and
swelling of cellulose, and hemicellulose solvation. However, the high down-
stream processing cost is a major drawback of this method. In addition, the
method requires a large volume of water for removal of salt from the biomass
and is an exhaustive process to remove them.
3. Ozonolysis: Ozone is mainly applied for decreasing the lignin portion of ligno-
cellulosic biomass residue as it majorly degrades lignin (Kumar et al. 2009).
Although ozonolysis is an effective method, the large amount of ozone required
makes it a difficult option for commercial applications.
4. Organosolv: This method uses various organic and aqueous solvents, viz., glycol,
ethanol, methanol, acetone, etc. for the pretreatment of lignocellulose biomass
under specific pressure and temperature (Ichwan and Son 2011; Alriols et al.
2009). This method is commonly carried out in the presence of a base, acid, or salt
86 S. Sharma and A. K. Kumar

catalyst (Bajpai 2016).The drawbacks with this process are high acid concentra-
tion, long reaction time, and formation of inhibitors.
5. Ionic liquids (IL): These solvents are synthesized from cations and anions. Major
characteristics of these solvents are low melting points (<100
C), high polarity,
high thermal stabilities, and negligible vapor pressure (Behera et al. 2014; Zavrel
et al. 2009). Ionic liquids disrupt the lignocellulosic network by competing with
its components or hydrogen bonding (Moultrop et al. 2005). However, drawbacks
such as difficulty in recycling and reuse, high cost, and formation of inhibitors
obstruct the commercial application of ionic liquids (Kumar and Sharma 2017).
6. Deep eutectic solvents (DES): These eutectic mixtures are commonly synthesized
by mixing two or three components, which have the capability of self-association
(usually hydrogen bonds). A major characteristic feature of these solvents is that
the melting point of the mixture is lower than that of each individual component
(Zhang et al. 2012a, b). DESs are very similar to ionic liquids, but the difference
lies in their composition. Unlike ionic liquids, DESs are not composed of ionic
species and can be synthesized from nonionic species. In the last few years, a
number of natural products, viz., urea, choline, sugars, amino acids, and many
other organic acids, have been considered in the DES category (Dai et al. 2013a).
These solvents have been categorized separately into a new class of solvents
termed as natural deep eutectic solvents (NADES). In contrast to ionic liquids,
NADES are easier to synthesize, cost-effective, nontoxic, recyclable, reusable,
biocompatible, and highly biodegradable. NADES synthesis, components, char-
acteristic properties, and applications have been described in the next sections.

4.8 Natural Deep Eutectic Solvents (NADES): Synthesis

and Mechanism

NADES have proved to be an alternative to ionic liquids. The ionic liquids’ green
nature has been strongly challenged owing to their poor biocompatibility,
sustainability, and biodegradability. DESs are similar to ionic liquids in having an
ionic character but comprise a mixture of organic compounds having a melting point
remarkably lower than that of individual components (Paiva et al. 2014). A sche-
matic representation of the lowered melting temperature of the so formed NADES is
shown in Fig. 4.4. A timeline of various processes and various solvents developed in
chronological order is shown in Fig. 4.5.
NADES are synthesized by a complex formation between a hydrogen-bond donor
(HBD) and hydrogen-bond acceptor (HBA). Table 4.3 shows various HBA and
HBD components that can be combined to form NADES. The decrease in the
melting point of the mixture as compared to its constituents is mainly due to the
delocalization of charge. Another advantage of NADES is the availability of a wide
variety of solvents with the possibility of a large number of combinations, which
give rise to tailored solvents with desired properties for various applications.
NADES are synthesized in two ways. It can either be prepared from the aqueous
solutions consisting of each compound in which a component is in the molten state
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 87

Fig. 4.4 Schematic design

showing the lowering of the
melting point of the mixture
than individual components

Fig. 4.5 A road map of reported developments, both on applications and fundamental studies on
deep eutectic solvents
88 S. Sharma and A. K. Kumar

Table 4.3 Commonly used HBA and HBD for synthesis of NADES
Hydrogen-bond donors (HBD) Hydrogen-bond acceptors (HBA)
Oxalic acid Ethylammonium chloride
Malonic acid Nicotinic acid
Xylitol Trimetylammonium chloride
Urea Betaine
Benzoic acid Choline nitrate
Citric acid Tetraethylammonium chloride
Imidazole Choline chloride
Cinnamic acid Histidine
Ethylene glycol Alanine
D-sorbitol Acetylcholine chloride
Lactic acid Glycine
Levulinic acid Choline fluoride
Glycerol Proline
Succinic acid Lidocaine
Hexanoic acid Methyltriphenylphosphonium chloride
Stearic acid Tetrabutylammonium chloride
Oleic acid
Linoleic acid
Adipic acid
Suberic acid

and the other component is dissolved or by mixing the two solid components heated
to a predetermined value (Francisco et al. 2013; Paiva et al. 2014).
NADES are most commonly synthesized from carboxylic acids, choline chloride,
and hydrogen-bond donors such as succinic acid, glycerol, urea, citric acid, etc.
NADES despite being similar to ionic liquids have an upper hand over ionic liquids
due to their low cost, less toxic, and biodegradable nature. In the recent past, a large
variety of stable NADES have been developed from natural compounds such as
organic acids, amino acids, and sugars (Dai et al. 2013a; Choi et al. 2011).
The composition of a eutectic mixture is commonly determined through differ-
ential scanning calorimeter (Morisson et al. 2009; Kareem et al. 2010). This process
determines the freezing point of the eutectic mixture. The eutectic point refers to the
lower melting point of the mixture, which is significantly lower than the individual
components, thereby allowing the mixture to be used at room temperature (Durand
et al. 2013). A possible reason behind the depression in freezing point of the mixture
is that HBD acts as an agent for complex formation with the anionic species leading
to an increase in its effective size, thereby decreasing the interaction with a cation
(Abbott et al. 2006; D’Agostino et al. 2011). Choline chloride (CC) is the most
widely used cationic constituent for NADES. The major reasons behind the popu-
larity of CC are its nontoxic biodegradable nature and low cost (Hayyan et al. 2012).
In addition, its combination with other HBD components such as glycerol, urea, etc.
provides a sustainable mixture. Moreover, Dai et al. (2015) have extensively studied
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 89

the supermolecular structure of synthesized NADES, i.e., malic acid and proline
(MA-Pro) mixture at a molar ratio of 1:1 using Fourier-transform infrared spectros-
copy (FTIR), and have observed that an extensive network of hydrogen bonds exists
between the components of the NADES, making these green solvents as
supermolecule with coherent hydrogen bonding network system.

4.9 Types of NADES Reagents

4.9.1 Hydrophilic

The most commonly synthesized NADES have shown high miscibility in water and
are regarded as hydrophilic NADES reagents. Since these form monophasic
solvents, they could perform similar functions as aqueous solvents and have signifi-
cant applications in broad areas of research. Bosiljkov et al. (2017) studies revealed
that extraction of wine lees anthocyanins was enhanced using hydrophilic NADES
mixture containing choline chloride and malic acid when coupled with ultrasonic
waves. Unlike conventional phenolic extraction technique, green solvent approach
opened a new branch of study for aromatic compound extraction. Similarly, studies
performed by Kumar et al. (2016) showed that hydrophilic NADES could replace
other harsh chemical agents such as hydrochloric acid and sodium hydroxide for
pretreatment of lignocellulosic agro-residues. Moreover, based on the pH, they have
grouped hydrophilic NADES into acidic or neutral NADES reagents (Kumar et al.
2016). The high stability of Cellic CTec2, a commercial cellulose-degrading enzyme
cocktail, was also recorded in neutral NADES reagent. Hydrophilic NADES
synthesized along with their components and molar ratios are described in
Table 4.4. Besides, the applicability of hydrophilic NADES has also been found in
the improvement of chondroitinase ABCI (cABCI) stability from Proteus vulgaris
(Daneshjou et al. 2017), CO2 capture (Mulia et al. 2017), microalgal lipid extraction
(Lu et al. 2016), and bioavailability of nutraceutical products (Faggian et al. 2016).

4.9.2 Hydrophobic

Synthesis of hydrophobic DES is relatively a new concept, and studies are recently
initiated exploring novel environmentally friendly green solvents that could replace
conventional organic solvents. Since the majority of complex organic compounds
are either sparingly or completely insoluble in aqueous solvents, the dependency is
entirely on the common organic reagents such as hexane, dimethyl sulfoxide,
acetone, phenols, and chloroform for extraction processes. Multiple cost-intensive
downstream processing is needed to recover these chemicals. Recently, for the first
time, Van Osch et al. (2015) synthesized hydrophobic DES using a mixture of
decanoic acid and quaternary ammonium salts. The hydrophobicity was maintained
from the long alkyl chain of fatty acids and a two-phase system was generated. The
efficiency of recovery of volatile fatty acids from diluted aqueous solutions was
90 S. Sharma and A. K. Kumar

Table 4.4 Hydrophilic NADES tested in different studies

NADES Molar
reagenta ratio References
CC-MA 1:1 Kumar et al. (2016) and Zhang et al. (2016)
CC-MAL 1:1, 1:2 Kumar et al. (2016) and Rengstl et al. (2014)
CC-Gly 1:1, 1:2 Kumar et al. (2016), Alvarez-Vasco et al. (2016), Xia et al. (2014),
and Zhang et al. (2016)
CC-Ur 1:1, 1:2 Kumar et al. (2016), Jablonsky et al. (2015), and Xia et al. (2014)
CC-TA 1:1 Kumar et al. (2016)
CC-OA 1:1 Kumar et al. (2016), Jablonsky et al. (2015), and Zhang et al. (2016)
CC-EG 1:1, 1:2 Kumar et al. (2016) and Zhang et al. (2016)
CC-LA 1:5, 1:9, Kumar et al. (2016), Alvarez-Vasco et al. (2016), and Jablonsky
1:10 et al. (2015)
CC-GA 1:1 Zhang et al. (2016)
BE-LA 1:1, 1:2 Kumar et al. (2015b)
Suc-MA 1:1 Yiin et al. (2015)
a
NADES reagents used in different studies: CC-MA choline chloride-malic acid, CC-MAL choline
chloride-malonic acid, CC-Gly choline chloride-glycerol, CC-Ur choline chloride-urea, CC-TA
choline chloride-tartaric acid, CC-OA choline chloride-oxalic acid, CC-EG choline chloride-
ethylene glycol, CC-LA choline chloride-lactic acid, CC-GA choline chloride-glutaric acid,
BE-LA betaine-lactic acid, Suc-MA sucrose-malic acid

Table 4.5 Hydrophobic DES tested in different studies

Component 1a Component 2b Molar ratio References
Mnl AA 1:1 Ribeiro et al. (2015)
Mnl BA 1:1 Florindo et al. (2017)
Mnl DA 1:1 Florindo et al. (2017)
Mnl DOA 2:1 Florindo et al.(2017)
Mnl HA 1:1 Florindo et al. (2017)
Mnl LA 1:2 Ribeiro et al. (2015)
Mnl OCA 1:1 Florindo et al. (2017)
Mnl PA 1:2 Ribeiro et al. (2015)
MTAC Bnl 1:4 Cao et al. (2017)
a
Component 1: Mnl menthol, MTAC methyltrioctyl ammonium chloride
b
Component 2: AA acetic acid, BA butyric acid, Bnl butanol, DA decanoic acid, DOA dodecanoic
acid, HA hexanoic acid, LA lactic acid, OCA octanoic acid, PA pyruvic acid

studied by altering the alkyl chain length of HBD. Similarly, Florindo et al. (2017)
also studied the applicability of hydrophobic DES in the extraction of pesticides, a
major micro-pollutant in wastewater treatment plants (Table 4.5).
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 91

4.10 Physicochemical Properties of NADES

4.10.1 Structure

Various spectroscopy techniques, viz., nuclear magnetic resonance (NMR) spectros-

copy and Fourier-transform infrared (FTIR) spectroscopy, have been applied by the
researchers to study the structure of NADES. NMR studies found the presence of
hydrogen bond in the NADES. Abbott and coworkers performed heteronuclear
Overhauser effect spectroscopy (HOESY) and found a cross-correlation between
protons from urea and fluoride from choline fluoride (Abbott et al. 2003). Mele et al.
(2003) also observed a clear intramolecular as well as intermolecular bonding
between 1-n-butyl-3-methylimidazolium tetrafluoroborate molecules through 1H–1
H-nuclear Overhauser effect spectroscopy (NOESY). Dai et al. (2013b) conducted
HOESY spectrum studies on 1,2-propanediol, choline chloride-water (PD-CC-H)
and proline-malic acid-water (Pro-MA-H). A signal was revealed corresponding to a
proton present on the methyl group of 1,2-propanediol which was found to have
interactions with both methylene carbon and methyl carbon and of CC.
The spectrum revealed that there was strong interaction between protons on
hydroxyl groups from 1,2-propanediol, choline chloride, and water thereby
confirming the presence of hydrogen bond between hydroxyl groups. Similar
observations were observed in Pro-MA-H. Also, the stability of NADES is affected
by the varying ratio of different components. For example, glucose/CC is stable at
2:5, but solid precipitation takes place at 2:1, 1:1, and 1:4 molar ratios. This confirms
that a chloride ion of CC forms two hydrogen bonds with two hydroxyl groups from
sugars. Similar results were also observed in a mixture of CC and carboxylic acid
(Abbott et al. 2004). An equal proportion of the two components (1:1) have been
found suitable for most other combinations. Sugars, organic acids, amino acids, and
sugar alcohols belong to both HBA and HBD which is considered to be a possible
reason for the formation of a complex by the components forming liquids with
supramolecular structure. This brings to the conclusion that NADES can be consid-
ered as liquid crystals where the arrangement of molecules takes place through
hydrogen bonding along with other intermolecular binding forces. Dai et al.
(2013a) also observed the effect of the structure on the formation and stability of
NADES and found that a number of groups of HBA and HBD, the position of the
bonds, and the spatial structure of those groups influenced the formation and stability
of NADES. It has been observed that organic acids, malic acids, citric acid, and
tartaric acids form a mixture in a liquid state with choline salts, while such phenom-
enon is not observed in succinic acid. Based on the structure of these acids, it can be
assumed that the presence of an extra hydroxyl or carboxyl group leads to the
formation of more hydrogen bonds, which in turn increases the stability of liquids.
Similarly, the mixture of organic acids and sugars having more number of
carboxylic groups (citric acid) forms a stable mixture with more number of sugars
than those with less number of carboxylic groups (malic acid).The hydrogen bond
formation and its stability also depend on the spatial structure of NADES. It has been
observed that NADES formed by CC and galactose is not stable and precipitates,
92 S. Sharma and A. K. Kumar

while the combination of glucose and CC forms a stable liquid mixture. Other sugars
and sugar alcohols, viz., sorbitol and mannitol, have also shown similar results.
Disaccharides such as trehalose have been found to form liquid mixtures with
choline salts, while cellobiose, which has different glycosidic bond than trehalose,
does not form a liquid mixture (Dai et al. 2013a). Aissaoui et al. (2016) used TmoleX
for studying the molecular structure and charge density of NADES synthesized from
triethylene glycol and CC, diethylene glycol and CC, ethylene glycol and CC, and
glycerol and CC.

4.10.2 Viscosity

The high viscosity of NADES is a major obstacle in their wide-scale application. The
viscosity of NADES lies in the range of 200–500 mm2/s at 40
C which leads to slow
mass transfer in extractions/dissolutions leading to time-consuming solvent transfer
operations (Dai et al. 2013b, 2015). Researchers have found that diluting the
solvents with a small amount of water led to a major drop in viscosity and modulates
the function of the solvents (Kohno and Ohno 2012). This property is useful in
designing appropriate NADES for specific applications. There is an interesting
aspect regarding the mechanism of interaction between NADES and water. Water
plays a very vital role in the biological systems. Besides water and lipids, NADES
can have an important role as a solvent in biological systems. NADES can provide
an alternate medium for carrying out various metabolic activities that could not occur
in water and lipids. Their presence can explain metabolic activities such as biosyn-
thesis of hydrophobic metabolites, an adaption of organisms in extreme climatic
conditions (Abbott et al. 2009). Figure 4.6 shows the changing behavior of NADES
viscosity with increasing water content.
Dai et al. (2013b) added 25% water to glucose-choline and 1,2-propanediol-
choline chloride (PD-CC) which reduced the viscosity of mixtures from 397 to
7.2 mm2/s and 33 to 6.1 mm2/s, respectively. Dai et al. (2015) explained the
mechanism of effect of the addition of water to NADES with the help of 1H NMR
and FTIR. It was observed that the strong hydrogen bonds between the NADES
constituents weakened gradually with the addition of water and ended completely
upon addition of 50% (v/v) water.
A small amount of water addition decreases the viscosity of NADES and brings it
down to the range of water, while the conductivity of some NADES increases by up
to 100 times. Therefore, dilution of NADES with water not only reduces NADES
viscosity but also alters the structure of NADES facilitating their application for
specific purposes.

4.10.3 Density

Density is an important criterion for the purpose of process designing. The effect of
pressure and temperature on density is needed for the optimization of
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 93

Fig. 4.6 Change in viscosity with temperature for aqueous NADES reagents. (a) LA-CC (9:1), (b)
LA-CC (5:1), and (c) LA-CC (2:1). (♦) 5%, blue; (■) 10%, red; (~) 20%, green; (x) 30%, purple;
(*) 40%, magenta; (●) 50%, orange; (+) 60%, light blue; () 70%, pink; () 80%, green; (♦) 90%,
purple; and (■) 100%, light blue

thermodynamic equations necessary for the development of industrial processes

(Valderrama 2003). The majority of NADES that has been synthesized and tested
has been found to have densities in the range of 1.0–1.35 g/cm3 at 298.15 K. Density
of NADES is largely affected by the molar ratio of the constituents of NADES and
molecular characteristics of HBD (Garcia et al. 2015).
The group reported higher density for 1:3 NADES synthesized from ethanol-
amine and 2,2,2-trifluoroacetamide. In addition, the density of NADES increases
with the number of hydroxyl (OH) groups present in HBD, e.g., higher density was
obtained for glycerol than ethylene glycol. The group also reported that the density
of NADES decreased with the introduction of aromatic group in the HBD, e.g.,
phenol was found to have the lowest density among the group of NADES evaluated.
In addition, it was observed that in diacid NADES, density decreases with the
increase in chain length. The order of density in such condition would be oxalic
acid followed by malonic and glutaric acid. Hence, it has been proven that the
density of NADES is determined by the stearic effects and ionic strength of HBD.
The type of salt present in the NADES is also a major factor behind the density of
NADES. It has been observed that for a specific HBD, i.e., ethylene glycol, NADES
based on ammonium have less density in comparison to phosphonium-based
NADES. The density of NADES has been found to decrease with the increase in
the length of cation alkyl chains. Increase in the length of the alkyl chain in NADES
enhances the compressibility, which leads to increase in the free volume.
94 S. Sharma and A. K. Kumar

Similarly, the anion has an effect on the density, as in the case of halogen; the
bromide salts have a higher density than chloride ones. This is because the NADES
containing bromide are more compressible as compared to the chloride ones which
are probably due to larger free volumes of NADES that contain bulkier anions and
weaker anion-HBD and anion-cation interactions for larger anions (Garcia et al.
2015).

4.10.4 Conductivity

Temperature-dependent specific conductivity (k) of NADES reagents was measured

in a custom-designed apparatus between 25 and 85
C (Rengstl et al. 2014) and
dielectric relaxation spectroscopy (Craveiro et al. 2016). Conductivity data can
clarify the mechanism of charge transport of the NADES. The conductivity of tested
NADES was nearly similar to common ionic liquids but higher than imidazolium
ionic liquids. With respect to hydrogenbond donor (HBD), dicarboxylic acid-based
NADES showed higher conductivity than urea-based NADES and varies with
variation in temperature. Furthermore, the calculated activation energy (EA) using
simulation studies through Vogel-Fulcher-Tammann (VFT) model predicted that the
smaller the size of the HBD, the lower the activation energy. One such example is EA
of choline chloride with the succinic acid mixture is 54.3 kJ/mol whereas with oxalic
and malic acid is 34.6 kJ/mol and 29.0 kJ/mol, respectively. The VFT equation
typically explains the dependence of temperature on the relaxation time linked with
the dynamic glass transition. Another study by Kumar et al. (2016) investigated the
electrical conductivity of choline chloride-based acidic and neutral NADES
reagents. Diols as HBD showed marginally higher conductivity than dicarboxylic
acids. Similarly, Craveiro et al. (2016) studied conductivity of NADES synthesized
from organic acids, choline chloride, amino acids, sugars, and amino acids and had
demonstrated that high polar organic acid-based NADES have higher conductivities
as compared to NADES based on sugars and amino acids.

4.10.5 Thermal Properties

Thermal properties are commonly studied by differential scanning calorimetry

(DSC) and thermogravimetric analysis (TGA). Dai et al. (2013a) reported that
heating a variety of NADES up to 100
C for 1 h did not show any sign of
decomposition. They also observed that NADES formed from sugars have a low
decomposition temperature (~135
C), while other NADES have significantly higher
decomposition temperature (>200
C). The glass transition temperature
(Tg) corresponds to the temperature at which the structure of the material transforms
from a glassy state to rubbery state (Craveiro et al. 2016). The glass transition
temperatures (Tg) of different NADES evaluated were found to be below 50
C,
but their melting point was not present which concludes that NADES are stable
supramolecular liquid complexes over a large range of temperature. In addition, the
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 95

liquid state of NADES supports the theory that NADES play a critical role in plants
for providing resistance against cold. Moreover, it also implies that NADES can be
used as solvents in the range of 0–100
C (Dai et al. 2013b). Different thermal
properties of various NADES have been presented in Table 4.6 (Craveiro et al.
2016).
Craveiro et al. (2016) applied DSC for evaluating the thermal stability of NADES
up to 250
C. The NADES samples evaluated were found to have only one
degradation peak signifying decomposition temperature above 120
C. Tg helps in
classifying all the NADES studied as glass formers and found it true for all the pure
components except CC. The reason behind the decease of Tg upon hydration is the
plasticizing effect of water (Carvalho et al. 2012). The plasticizing effect of water on
NADES was confirmed by adding water to CC-Xyl (2:1) and was inferred that upon
addition of 5% water, Tg decreased by 4
C.

4.10.6 Polarity

The polarity of NADES is an important property as it affects their solubilizing

capacity. NADES consisting of organic acids are found to be most polar
(~45 kcal/mol) followed by NADES synthesized from sugars and amino acids
(polarity equivalent to water ¼ ~48 kcal/mol). NADES consisting of sugars and
polyalcohols have the least polarity almost equivalent to that of methanol (~52 kcal/
mol). Addition of water to NADES also affects their polarity largely. The polarity of
Pro-CC-H (1,2-propanediol-CC-water) and LA-Glu-H (lactic acid-glucose-water)
was affected by adding 50% (v/v) water signifying a major change in the structure of
Pro-CC-H and LA-Glu-H. This structural modification possibly occurs owing to the
breakdown of hydrogen bonds between the components. Similar results were
observed with urea-CC and glycerol-CC (Dai et al. 2013b). The physical
characteristics of NADES are summarized in Table 4.7 as reported by Dai et al.
(2013a).

4.11 Functional Properties of NADES

4.11.1 Specificity

Although vast studies were performed on DES specificity, there were very limited
studies on NADES which are confined mainly toward solubilization of individual
components of lignocellulosic biomass. Kroon et al. (2014) have synthesized a
group of NADES reagents and tested for solubility of commercial products, viz.,
lignin, starch, and cellulose in a mixture. In their study, a specific solubility of lignin
was observed in NADES mixtures of lactic acid-proline (2:1), lactic acid-betaine
(2:1), and lactic acid-choline chloride (3:1, 2:2, and 5:1), respectively. Among these
lactic acid-betaine (2:1) showed a maximum solubility of 12.03 wt% with no
solubility of starch and cellulose. Similarly, a specific solubility of starch was
 96

Table 4.6 Thermal properties of different NADES, viz., degradation temperature (Td), melt and cold crystallization temperatures (Tcmelt and Tccold), melting
temperature (Tm), and glass transition temperature (Tg)
NADES#
Component 1 Component 2 Molar ratio Td (oC) Tg (oC) Tc(melt) (oC) Tc(cold) (oC) Tm (oC) References
CA Glu 1:1 130.1 9.8/48.7 – – – Craveiro et al. (2016)
CA Suc 1:1 121.2 14.0 – – – Craveiro et al. (2016)
CC CA 1:1 154.49 – – – – Haz et al. (2016)
CC CA 1:1 171.3 21.4 9.7 – 76.0 Craveiro et al. (2016)
CC EG 1:2 84 – – – – Dietz et al. (2017)
CC Glu 1:1 129.8 28.4 – – – Craveiro et al. (2016)
CC LA 1:1 212 – – – – Skulcova et al. (2017)
CC LA 1:1 196.83 – – – – Haz et al. (2016)
CC MAL 1:1 126 – – – – Skulcova et al. (2017)
CC OA 1:1 134.81 – – – – Haz et al. (2016)
CC Suc 4:1 141.7 42.0 33.9 51.8 79.2 Craveiro et al. (2016)
CC Suc 1:1 126.8 15.8 – – – Craveiro et al. (2016)
CC TA 1:1 197.84 – – – – Haz et al. (2016)
CC TA 1:1 194 – – – – Skulcova et al. (2017)
CC TA 2:1 130.8 41.6 – – – Craveiro et al. (2016)
CC Xyl 3:1 165.2 46.4 20.1 59.9 78.5 Craveiro et al. (2016)
CC Xyl 2:1 172.7 51.2 – 56.9 78.3 Craveiro et al. (2016)
CC UR 1:2 186 – – – – Dietz et al. (2017)
Glu TA 1:1 117.5 18.3 – – – Craveiro et al. (2016)
LE BE 2:1 166 – – – – Dietz et al. (2017)
#
NADES solvents tested: CA-Glu citric acid-glucose, CA-Suc citric acid-sucrose, CC-CA choline chloride-citric acid, CC-EG choline chloride-ethylene glycol,
CC-Glu choline chloride-glucose, CC-LA choline chloride-lactic acid, CC-MAL choline chloride-malonic acid, CC-OA choline chloride-oxalic acid, CC-Suc
choline chloride-sucrose, CC-TA choline chloride-tartaric acid, CC-Xyl choline chloride-xylose, Glu-TA glucose-tartaric acid, LE-BE levulinic acid-betaine
S. Sharma and A. K. Kumar
 4

Table 4.7 Physical properties of NADES using water and methanol as references
NADES Water Water activity Density (40
C) Viscosity Tdecom ENR (kcal/
reagenta Molar ratio (wt %) (40
C) g/cm3 (40
C) mm2/s (
C) Tg (
C) mol) Reference
MA-CC- (1:1:2) 11.62 0.195 1.230 445.9 201 71.32 44.81 Dai et al.
H2O (2013a, b)
Gly-CC- (2:1:1) 5.26 0.126 1.174 51.3 187 101.59 49.55 Dai et al.
H2O (2013a, b)
LA-CC (9:1) – – 1.1785 31.27 – – – This study
LA-CC (5:1) – – 1.1729 35.62 – – – This study
LA-CC (2:1) – – 1.1546 79.44 – – – This study
LA-CC (1:1) – – 1.1453 157.55 – – – This study
LA-CC-- (9:1:3.3) 30 – 1.1292 1.83 – – – This study
H2O
LA-CC- (5:1:3.3) 30 – 1.1257 5.31 – – – This study
H2O
LA-CC- (2:1:3.3) 30 – 1.1207 6.10 – – – This study
H2O
LA-CC- (1:1:3.3) 30 – 1.0417 9.43 – – – This study
H2O
MA-Ala- (1:1:3) 19.48 0.573 1.352 174.6 164 70.88 48.05 Dai et al.
H2O (2013a, b)
Pro-MA- (1:1:3) 17.81 0.591 1.318 251.0 156 61.29 48.30 Dai et al.
Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . .

H2O (2013a, b)
Fru-CC- (2:5:5) 7.84 0.151 1.209 280.8 160 84.58 49.81 Dai et al.
H2O (2013a, b)
Xyl-CC- (1:2:2) 7.74 0.141 1.209 308.3 178 81.80 49.81 Dai et al.
H2O (2013a, b)
(continued)
97
Table 4.7 (continued)
98

NADES Water Water activity Density (40
C) Viscosity Tdecom ENR (kcal/
reagenta Molar ratio (wt %) (40
C) g/cm3 (40
C) mm2/s (
C) Tg (
C) mol) Reference
Suc-CC- (1:4:4) 7.40 0.182 1.227 581.0 >200 82.96 49.72 Dai et al.
H2O (2013a, b)
Fru-Glu- (1:1:1:11) 22.0 0.662 1.366 720.0 138 50.77 48.21 Dai et al.
Suc- H2O (2013a, b)
Glu-CC- (2:5:5) 7.84 0.162 1.207 397.4 170 83.86 49.72 Dai et al.
H2O (2013a, b)
LA-Glu- (5:1:3) 7.89 0.496 1.250 37.0 135 77.06 44.81 Dai et al.
H2O (2013a, b)
Xyl-CC- (1:2:3) 11.17 0.116 1.178 86.1 >200 93.33 59.72 Dai et al.
H2O (2013a, b)
H2O Water 100 1 0.992 1 – – 58.21 Dai et al.
(2013a, b)
MeOH Methanol – – 0.791 – – – 51.89 Dai et al.
(2013a, b)
a
NADES solvents tested: MA-CC-H2O malic acid-choline chloride-water, Gly-CC-H2O glycerol-choline chloride-water, LA-CC lactic acid-choline chloride,
LA-CC-H2O lactic acid-choline chloride-water, MA-Ala-H2O malic acid-alanine-water, Pro-MA-H2O proline-malic acid-water, Fru-CC-H2O fructose-choline
chloride-water, Xyl-CC-H2O xylose-choline chloride-water, Suc-CC-H2O sucrose-choline chloride-water, Fru-Glu-Suc-H2O fructose-glucose-sucrose-water,
LA-Glu-H2O lactic acid-glucose-water, Xyl-CC-H2O xylose-choline chloride-water, MeOH-H2O methanol-water
S. Sharma and A. K. Kumar
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 99

observed in NADES mixtures of malic acid-betaine (1:1) and oxalic acid-nicotinic

acid (9:1). When oxalic acid was mixed with histidine at a molar ratio of 9:1, the
specificity of NADES was shifted toward cellulose solubility. In some comparable
studies by Kumar et al. (2015b, 2016), the specificity of NADES remained similar
when lignocellulosic biomass residues were pretreated with selected NADES
reagents. They have found that NADES mixtures of lactic acid-CC (5:1 and 9:1)
effectively removed lignin fraction from rice straw without any effect on cellulose
and hemicelluloses. The primary reason behind the non-dissolution of cellulose in
choline chloride mixtures was that the hydroxyl groups of choline are linked to
cellulose by strong hydrogen-bond interactions and hence stabilizes the cellulose
system protecting from solubilization (Rengstl et al. 2014).
Besides solubility, recently, Martinez et al. (2016) studied enantioselective L-
proline-catalyzed intermolecular aldol reaction in biorenewable NADES formed by
glucose and malic acid. The products generated from aldol reaction are specifically
diastereomers. Moreover, NADES had proven to be a clean media to carry out
volatile organic compound-free selective process.

4.11.2 Toxicity and Biodegradability

An advantage of NADES is the fact that these solvents are synthesized by natural
components thereby having significantly lower toxicity than ionic liquids. Hayyan
et al. (2013) evaluated the toxicity of phosphonium-based NADES on brine shrimp
and gram-positive and gram-negative bacteria. This study observed that NADES
toxicity depends on viscosity, composition, and concentration of NADES. The
researchers also concluded that the eutectic mixture of the two components is
more toxic as compared to the aqueous solutions of the individual components.
The delocalization of the charges in NADES led to the disruption of cell walls of the
bacteria. This property might help in using NADES as antibacterial agents.
Another study evaluated the effect of NADES on acetyl cholinesterase (AChE)
which is an enzyme found in the nervous system of higher organisms. Choline
chloride- and amino acid (CC-AA)-based NADES have been found to have lower
toxicity than imidazolium-based ionic liquids. However, the toxic effect varies based
on amino acid present in NADES. In addition, the CC-AA NADES showed less
toxicity toward the bacteria. The biodegradability of CC-AA with different amino
acids was tested, and all the combinations were found readily biodegradable. The
extra amide or carboxyl group on amino acid side chain makes NADES more
vulnerable to microbial breakdown. Most of the combinations of CC-AA NADES
synthesized and tested were found to have low toxicity and high biodegradability
(Hou et al. 2013). This makes NADES a promising candidate for application as
green solvents in several industries. Paiva et al. (2014) studied the impact of more
than ten NADES on model cell line (L929 fibroblast-like cells) and observed that
NADES containing tartaric acid has negative impact on cellular metabolic activity.
However, choline-based NADES were found to be non-cytotoxic.
100 S. Sharma and A. K. Kumar

4.11.3 Recyclability

Sustainability of chemical processing depends on recycling the process inputs for

maximizing the benefits. Recycling of conventional chemical solvents demands
high-energy input and capital expenditure. Hence, simple recovery mechanisms
are preferred for recycling. Unlike multiple downstream recovery processing of
organic solvents, NADES recovery is a comparatively easy process. Synthesis of
NADES involves weak intermolecular hydrogen bonding between the HBA and
HBD molecules, and the mixture formed does not form a complex. Hydrogen bond
could easily be broken and involve any chemical reaction. NADES are nonvolatile in
nature due to their high boiling points, and hence no major losses are observed.

4.11.4 Biocatalysis

Despite NADES being synthesized from denaturing agents, viz., citric acid, urea,
etc., several enzymes have been found to show high activity and stability in NADES.
Durand et al. (2013) observed Candida antarctica lipase B (CALB) showing very
high activity and stability in choline chloride-based NADES. Gorke et al. (2010)
performed the transesterification of ethyl valerate with butanol by hydrolase
enzymes. The enzymatic stability of CALB showed an improvement of 20–35-
folds when choline chloride-urea was used as cosolvent as compared to the aqueous
solution. Choline chloride-based NADES have been used as cosolvents for hydroly-
sis of methylstyrene oxide by epoxide hydrolases (Lindberg et al. 2010). Reetz et al.
(2003) had proposed a two-phase separation system using ionic liquids and super-
critical fluids (supercritical carbon dioxide). However, the drawback of this tech-
nique is the high cost of ionic liquids and poor biodegradability. With the advent of
NADES, which are cheaper and greener and have excellent biodegradability, this
opens new possibilities for biocatalysis in biphasic systems. Gunny et al. (2015)
studied the applicability of NADES on cellulose-degrading enzymes. Their study
has shown that >90% of the cellulase activity was retained in the presence of 10% (v/
v) for glycerol-based DES (GLY) and ethylene glycol-based DES (EG).

4.11.5 Biorefinery and Bioprocessing

A biorefinery is an integrated facility that includes biomass fractionation and bio-

conversion processes for production of various separated products. These products
are of high importance in generation of transport fuels, power, and other value-added
chemicals. For production of transport fuels, pretreatment is considered as one of the
key steps of bioprocessing. An efficient pretreatment process improves overall
downstream processing steps in a biorefinery. For example, in bioethanol produc-
tion, the conversion efficiency of biomass residues into its constituent reducing
sugars and monomers from a pretreated biomass is significantly higher than the
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 101

untreated biomass. This further improves the overall cellulosic ethanol production
yields during fermentation.

4.11.6 Production of Reducing Sugars

The sole purpose of pretreatment is to expose the cellulose for enzymatic saccharifi-
cation leading to the formation of reducing sugars. The production of reducing
sugars from cellulose by enzymes is a key connecting step between lignocellulosic
biomass and ethanol production from biorefinery point of view. Researchers have
reported that application of NADES for the pretreatment process does not have an
inhibitory effect on the enzyme, which in turn increases the overall sugar yield.
Kumar et al. (2015b) tested several different concentrations of lactic acid-betaine
(LA-BE) (2:1 and 5:1) and lactic acid-choline chloride (LA-CC) (2:1, 5:1, and 9:1).
Among these reagents, lactic acid-choline chloride (LA-CC) (5:1)-treated biomass
produced maximum reducing sugars (333 mg/g) with 36% saccharification effi-
ciency in 24 h at a solid loading of 10%. Kumar et al. (2016) studied the effect of
CC-glycerol NADES on cellulase enzyme Cellic CTec2 and obtained maximum
reducing sugars of 226.7 g/L with saccharification efficiency of 87.1% at 20% solids
loading and 12 filter paper units (FPU) Cellic CTec2. Zhang et al. (2016) studied
three kinds of choline chloride-based NADES reagents mixed with monocarboxylic
acid, dicarboxylic acid, and polyalcohols for saccharification of corncobs. Among
these, maximum glucose yields of 96.4% were observed with CC-glycerol (CC-Gly)
(2:1) reagent, followed by monocarboxylic acid mixtures. However, dicarboxylic
acid-based NADES reagents produced a lower amount of reducing sugars. This was
possibly due to the high acidic strength of dicarboxylic NADES solvents, which had
limited the lignin extractability hindering enzyme interaction with the cellulosic
fraction. Gunny et al. (2015) studied saccharification of NADES-pretreated rice husk
using polyalcohol-based NADES reagents.

4.11.7 Bioethanol Production

Studies on bioethanol production using NADES-pretreated biomass are very limited.

Since applicability of NADES is still in nascent stage, research needs to be explored
further in cellulosic ethanol production from a large variety of lignocellulosic
biomasses and agro-residual waste materials. Following saccharification, the formed
reducing sugars are fermented by yeast to produce bioethanol. Cellulosic ethanol
production from NADES-pretreated rice straw was evaluated by Kumar et al.
(2016). Studies showed that choline chloride-glycerol (CC-Gly)-treated rice straw
produced maximum ethanol of 36.7 g/L with a conversion efficiency of 90.1%.
Besides, in this study, the tolerance level of Clavispora NRRL Y-50464, a
β-glucosidase-producing ethanol-fermenting yeast, was evaluated in acidic and
neutral NADES reagents. Fermentation of glucose in the presence of CC-glycerol
and CC-propane diol (CC-PD) at 10% (v/v) has not shown any effect on the rate of
102 S. Sharma and A. K. Kumar

yeast growth, sugar consumption, and ethanol production from Clavispora NRRL
Y-50464, while 10% (v/v) CC-ethylene glycol repressed and delayed the cell growth
of the microbe.

4.11.8 Carbon Dioxide Sequestration

Ionic liquids were found to have a high solubility for CO2, but the high price and low
biodegradability are major obstacles in the commercial application of ionic liquids.
NADES can prove to be a viable substitute for ionic liquids as they have very low
vapor pressure like that of ionic liquids and are biodegradable, biocompatible, and
inexpensive (Wu et al. 2012). Chen et al. (2014) measured CO2 solubility in NADES
synthesized from CC and dihydric alcohols, viz.,1,4-butanediol, 2,3-butanediol, and
1,2-propanediol in a molar ratio of 1:3 and 1:4 at a temperature difference of 10 K
ranging from 293.15 to 323.15 K at 6 bar pressure with the help of isochoric
saturation method. The solubility of CO2 in the mixtures increased linearly with
the decrease in temperature or increase in pressure. Paiva et al. (2014) suggested the
application of amine-based NADES for the sequestration of CO2 in order to provide
a cheaper and cleaner alternative to the conventional processes applied today.

4.11.9 Chemical Extraction

The dissolution property is mainly responsible for the efficiency of an extraction

agent. As mentioned earlier, NADES possess the capacity to donate and accept
protons and electrons which makes them suitable to form hydrogen bonds which in
turn increases their dissolution capacity (Zhang et al. 2012b). Dai et al. (2013b) used
NADES (lactic acid-glucose, CC-glucose, and fructose-glucose-sucrose) for the
removal of phenolic compounds from safflower. This study established the hypoth-
esis of the formation of hydrogen bond between the NADES and phenolic
compounds by showing a high ability of NADES for the removal of phenolic
compounds. Authors have also reported higher phenolic compound extraction as
compared to conventional solvents like water and ethanol.
Molecules having poor solubility in water, viz., griseofulvin, benzoic acid,
itraconazole, and danazol, were reported to have higher solubility in CC-urea and
CC-malic acid (Morrison et al. 2009). NADES have also been found to successfully
dissolve transition metal oxides from minerals (Abbott et al. 2004). NADES being
green and safe solvents are employed in extracting natural products in several
industries such as pharmaceutical and food industries. Since NADES have been
found to dissolve both polar and nonpolar metabolites (Biswas et al. 2006), specific
NADES can be synthesized and applied for the extraction of desired natural
components. Paiva et al. (2014) used CC-xylose (3:1), CC-tartaric acid (1:1), and
CC-citric acid (1:1) for extracting phenolic compounds from coffee beans. The
amounts of phenolic compounds were found to be greater than the conventional
solvents such as acetone and citric acid.
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 103

4.12 Technical Assessment of NADES

Comprehensive techno-economical evaluation using NADES was not reported till

date. Since study on NADES is relevantly a new topic of research, a majority of the
studies are still in nascent stage and are exploring the potentiality and applicability of
NADES in diverse applications. In continuation to our earlier studies on the appli-
cability of NADES in lignocellulosic biomass pretreatment, we have further studied
the technical assessment of NADES in an integrated process for biorefinery
applications. All the details with respect to the mass balance of the integrated
technology given in Fig. 4.7 are based on an experimental evaluation performed in
our laboratory. In brief, the integrated process includes cellulosic production from
NADES pretreated rice straw (Kumar et al. 2016), and recovery, recycle NADES
reagent, recovery of high purity lignin and xylan as the value-added products under
four different conditions (Kumar et al. 2018). The conditions maintained were
(I) biomass pretreatment at 5% solids loading and enzymatic saccharification at
10% solids loading, (II) biomass pretreatment at 5% solids loading and enzymatic
saccharification at 25% solids loading, (III) biomass pretreatment at 10% solids
loading and enzymatic saccharification at 10% solids loading biomass, and

Fig. 4.7 Process flow steps of an integrated biorefinery approach for lignocellulosic feedstock
pretreatment, cellulosic ethanol production and recovery and reuse of solvents, and recovery of
value-added products
104 S. Sharma and A. K. Kumar

(IV) biomass pretreatment at 10% solids loading and enzymatic saccharification at

25% solids loading pretreatment.
A detailed flow diagram of the process is given in Fig. 4.7. Based on the mass
balance analysis, it is evident from the conditions I and II, pretreatment at 5% solids
loading demands a 20-fold higher NADES solvent, which increases the overall cost
of the chemical reagents and makes the entire process cost-intensive. Besides, the
requirement of acetonitrile and water was also twofold high as compared to the
process conditions III and IV. Although no significant losses were observed during
recovery of acetonitrile and water, high capacity distillation units are required for
process conditions I and II, thus increasing the capital and operational expenditures.
Besides, irrespective of pretreatment conditions, hydrolysis of pretreatment biomass
also plays an important role in reducing the cost of cellulosic ethanol production.
Enzymatic saccharification at 10% solids loading (conditions I and III) yielded the
low concentration of reducing sugars (3–4%); thus an additional pre-concentration
process was required in order to obtain 10% glucose, which was found to be
optimum for Clavispora NRRL Y-50464 to produce ethanol yields up to 3.7%
ethanol. Whereas, hydrolysis at 25% solids loading (conditions II and IV), yielded
high concentrations of glucose (9–11%) and the pre-concentration process could be
avoided, thus making the entire process more cost-effective and commercially
feasible for production of cellulosic ethanol. Overall, condition IV was found to be
optimum for an integrated process for the production of cellulosic ethanol and value-
added products and recovery and reuse of solvents.

4.13 Future Perspective

Research on NADES toward unravelling the bottleneck applications in biorefinery

and bioprocessing is still in nascent stage. Applicability of NADES is needed
primarily to replace the harsh synthetic chemical solvents with eco-friendly natural
solvents. Moreover, being benign, recovery of NADES seems to be significantly
higher, and without losing the efficiency, these are preferred for industrial
applications. There are several examples of future direction of NADES and its
uses. Recently, it was reported that NADES extracts high-purity unique low-
molecular-weight lignin. The new sources of lignin extract may provide a break-
through toward truly realizing the high value potential of lignin (Kumar et al. 2016).
In another study, pretreatment of microalga enhances the lipid recovery for biodiesel
production (Lu et al. 2016). In addition, it is reported that some of the NADES could
capture carbon dioxide efficiently (Mulia et al. 2017). The hydrophilic and hydro-
phobic NADES reagents have already shown their great potential in specific solubi-
lization of macromolecules that seemed to be limited earlier. The rapidly increasing
reports clearly suggest that NADES are being explored for the discovery of new
areas of applications. Above all, preparation of NADES involves only physical
mixing of natural compounds, numerous designer NADES reagents could be
synthesized with specific usage. Thus, NADES are regarded as the next-generation
green solvents.
4 Role of Natural Deep Eutectic Solvents (NADES) in the Pretreatment of. . . 105

4.14 Conclusions

The historical developments on NADES and its versatile applications in biorefinery

and bioprocessing have shown clear advantages over ionic liquids or other chemical
solvents. Having much lower cost and greenness, applications of NADES in the
fields of biocatalysis, extraction, electrochemistry, etc. are expected to be boosted
with the in-depth understanding of the mechanism of NADES action. NADES show
a great possibility as the next generation of solvents, and though many efforts are
needed to be executed, these novel solvents will certainly make a massive impact for
the clean, green, and sustainable industrial development.

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