2.

16 Ionic Liquids for Pretreatment of Biomassq

A Vijaya Bhaskar Reddy and M Moniruzzaman, Universiti Teknologi Petronas, Perak, Malaysia
M Goto, Kyushu University, Fukuoka, Japan
© 2019 Elsevier B.V. All rights reserved.

2.16.1 Introduction 190

2.16.2 Biomass Dissolution in Ionic Liquids 191
2.16.2.1 Lignocellulosic Biomass Pretreatment 191
2.16.2.2 Microalgae Biomass 193
2.16.3 Factors Affecting Biomass Pretreatment 194
2.16.3.1 Biomass Loading 194
2.16.3.2 Pretreatment Temperature 195
2.16.3.3 Pretreatment Time 195
2.16.3.4 Water Content 195
2.16.3.5 Combined Techniques 196
2.16.4 Recovery of Ionic Liquids 196
2.16.5 Environmental Impacts of Ionic Liquids 197
2.16.6 Conclusions 197
References 197
Relevant Websites 198

Glossary
Biomass A renewable source of energy that comes from plants and animals. Biomass contains stored energy from the sun.
When biomass is burned, the chemical energy in biomass is released as heat.
Ionic liquid A salt in which the ions are poorly coordinated, which results in these solvents being liquid below 100
C.
Life-cycle analysis (LCA) To judge the environmental impact of a compound, solvent, or whole process, LCA of each single
component has to be done. Sometimes the LCA is also called eco-efficiency analysis.
Pretreatment The treatment of something before another process, stage, or chemical modification to improve the effectiveness
of the main process.
Toxicity To test the environmental impact, the toxicity of a compound toward microorganisms and different animals has to be
tested. The result is given in inhibition of growth, lethality, or other appropriate figures.

2.16.1 Introduction

Ionic liquids (ILs) can be defined as salts with a melting point below 100
C. They generally consist of a large asymmetrical organic
cation combined with an anion. ILs have been exploited as solvents and reagents in various applications owing to their “green”
properties, including negligible vapor pressure and their tunable physicochemical and biological properties. The strong electrostatic
forces between the ions in the ILs impart low volatility/flammability and high chemical and electrochemical stability. ILs have been
classified as “green solvents” owing to their extremely low vapor pressure and high thermal stability, which offer advantages such as
ease of containment, product recovery, and recycling ability. Furthermore, ILs have distinct features as “designer solvents” because
their properties can be tuned by the combination of appropriate cations and anions.1 In recent years, ILs have been extensively used
in a variety of applications such as catalysis, biocatalysis, analytical and electrochemical applications, spectroscopy, and material
science.
Besides the application of ILs in synthesis, researchers have also investigated the use of ILs for the pretreatment of biomass, and
they have found that ILs could effectively dissolve biomass and represent a remarkable platform for biomass pretreatment. It has
been demonstrated that some ILs are effective alternatives for the pretreatment of biomass, as their use can reduce by-product gener-
ation, solvent losses, energy use, and carbon dioxide generation. Compared with various physical (mechanical), chemical, and bio-
logical methods, the pretreatment of biomass using ILs is a new technology that provides better results. The use of ILs as processing
solvents for biomass treatment started with the discovery of cellulose-dissolving ILs; subsequently, they were used as alternative
solvents for the spinning of man-made cellulose fibers.2 Lignocellulosic biomass has been highlighted as a promising resource

q
Change History: February 2018. A. Vijaya Bhaskar Reddy, M. Moniruzzaman, and M. Goto wrote a completely new chapter for this edition.

190 Comprehensive Biotechnology, 3rd edition, Volume 2 https://doi.org/10.1016/B978-0-444-64046-8.00121-X

 Ionic Liquids for Pretreatment of Biomass 191

owing to its abundance and sustainability. It contains complicated and rigid structures mainly composed of cellulose, hemicellu-
lose, and lignin. These structural characteristics of biomass are involved in the biomass recalcitrance; therefore, lignocellulosic
biomass needs careful processes. Furthermore, several specific applications of ILs for the processing of algal biomass have emerged
in recent years.3
Several studies have described that ILs can effectively dissolve the plant cell walls and selectively remove hemicellulose and lignin
under mild reaction conditions in many biomass-related applications, including pretreatment and characterization. The increasing
number of publications on biomass processing shows that there is a great interest in this research area.4 However, toxicity of ILs is an
important variable to be considered in the selection of ILs. For instance, the toxicity is directly linked to the chain size in amine-
based ILs, and corrosion potential should be considered for acid-based ILs. Furthermore, the high cost of aprotic ILs (AILs) is
also a challenge for their use in biomass pretreatment. An option to reduce the cost of pretreatment process is to use protic ILs
(PILs), whose production is easier and less expensive than the production of AILs. Nevertheless, the majority of studies in the liter-
ature have evaluated AILs for lignocellulose pretreatment5; there are very few reports that have considered the pretreatment with
PILs for further enzymatic hydrolysis.6,7 The authors reported that potentially low-cost PILs produced by acetic acid and amines
were able to extract more than 70% of lignin from lignocellulosic biomass. The extraction of lignin by pretreatment has been shown
to increase enzymatic hydrolysis yields.
A large number of review articles have summarized the use of ILs for the pretreatment of biomass. Therefore, the aim of this
chapter is to discuss the pretreatment processes followed by the factors that affect the efficiency of the pretreatment as well as
the environmental impact of ILs. Finally, the recovery of ILs is discussed, followed by a summary and conclusions, including the
economic viability, scale-up, and sustainability. Furthermore, the recent progress on the pretreatment of biomass, lignocellulosic
biomass and microalgae biomass, using ILs is reviewed, with a particular focus on the advancement during the past few years.

2.16.2 Biomass Dissolution in Ionic Liquids

ILs have been introduced as an effective medium for chemical modification and dissolution of biomass and its components. The
dissolution is a physical solvent–solute interaction that mainly depends on the nature of both components of ILs and physical
factors, including the viscosity of the IL and processing temperature. Partial or complete dissolution of biomass is necessary for
the successful pretreatment of biomass. However, the dissolution of biomass is not very easy because of its firm and complicated
structure. Cellulose is the most abundant carbohydrate fraction in biomass and an important resource for many biomass-related
applications such as fiber-, paper-, membrane-, ethanol-, and furan-based products. It is composed of linear glucose polymer chains
and is insoluble in water and most common organic solvents. Cellulose can be dissolved in solvents by disrupting its inter- and
intramolecular hydrogen bondings. ILs provide several attractive features in addition to the ability to dissolve lignin and carbohy-
drates.8 The reactions can be carried out under mild conditions, and the ILs can be reused after the process. The process provides easy
recovery of the cellulose and the ILs, with no toxicity or odor emission. The enzyme-catalyzed hydrolysis of cellulose can be
controlled by two crucial stages, i.e., the adsorption of enzymes on cellulosic particles and the formation of enzyme–substrate
complexes. These two stages are further associated with several enzyme- and substrate-related factors. Enzyme-related factors
include inhibition of by-products (cellobiose and D-glucose), thermal stability, synergism, and adsorption. Substrate-related factors
include the presence of hemicellulose and lignin, cellulose crystallinity, and degree of polymerization, as well as the amount of
accessible external and internal surfaces of cellulose. Because celluloses regenerated from ILs are much less crystalline than untreated
ones and are susceptible to faster enzymatic hydrolysis, it is reasonable to speculate that regenerated celluloses are more accessible
by cellulases. In the valorization processes of lignocellulosic biomass, cellulose and hemicellulose are mostly hydrolyzed to sugar
monomers and subsequently converted into alcohols, hydrogen, or methane by fermentation processes.

2.16.2.1 Lignocellulosic Biomass Pretreatment

Lignocellulosic biomass is the most abundant basic renewable energy source present on the earth, with a worldwide production of
approximately 1.1  1011 tons/year. It is constituted of three major biopolymeric subcomponents: (1) cellulose (a semicrystalline
polysaccharide present in approximately 40%–55%); (2) hemicellulose (amorphous multicomponent polysaccharide present in
approximately 20%–30%); and (3) lignin (amorphous phenylpropanoid polymer present in approximately 15%–20%). The lignin
component is one of the richest sources of important platform organic moieties, whereas cellulose is an abundant, nontoxic, inex-
pensive, biocompatible, ecofriendly, and virtually inexhaustible source of lignocellulosic biomass. Therefore, researchers are paying
more attention to the use of lignocellulosic biomass as a major raw source to produce platform chemicals and bioenergy. However,
lignocellulose needs to convert into lower-molecular-weight fragments in primary stages, which contributes to the high cost of the
utilization process owing to its well-protected structure.
There are few aqueous and nonaqueous cellulose solvents that can dissolve cellulose, including N,N-dimethyl acetamide/
lithium chloride; dimethyl sulfoxide/tetra-N-butyl ammonium fluoride; N,N-dimethyl formamide/nitrous tetraoxide. These
solvents can dissolve cellulose but all of them have drawbacks such as high flammability, high-cost, poisonous gas production, diffi-
culty in solvent recovery, difficulty in down-streaming process, recyclability, and insufficient solvating ability. However, the above-
listed problems can be solved using nonconventional neoteric solvents known as room temperature ILs. There are different methods
that have been adopted for the treatment of lignocellulosic biomass, including physical, chemical, and physicochemical treatments
192 Ionic Liquids for Pretreatment of Biomass

such as grinding and milling, hot water, alkali and acidic treatment, and biological treatment. However, there are several drawbacks
associated with these techniques, such as severe reaction conditions, high energy consumption, toxicity, expensive operation,
disposal recovery cost, and slow reaction rate. In this case, the utilization of so-called “green solvents,” i.e., ILs, provides several
attractive features in addition to their ability to dissolve lignin and carbohydrates. There are several reports of multistep processes
that apply the pretreatment of different types of lignocellulosic biomass with a variety of ILs followed by enzymatic hydrolysis. The
research on the combination of IL pretreatment of lignocellulose and enzymatic hydrolysis in a single-step process for bioethanol
production (enzyme-ILs) is growing rapidly. The findings from these studies have stimulated interest in the use of ILs for biomass
pretreatment in a biorefinery. Imidazolium ILs have been the most popular ILs for experimental investigations of lignocellulosic
biomass treatments. The majority of the ILs of interest contain 1-ethyl-3-methylimidazolium or [EMIM], 1-allyl-3-
methylimidazolium or [AMIM], and 1-butyl-3-methylimidazolium or [BMIM] cations. The latest attempts reported for lignocellu-
losic biomass pretreatment are listed with the substrates, findings, and references in Table 1.
A study compared the use of dilute acid and 1-ethyl-3-methylimidazolium acetate [EMIM]Ac for the pretreatment of lignocellu-
lose; the authors reported that switchgrass exhibited reduced cellulose crystallinity, increased surface area, and more reduction in the
lignin content with [EMIM]Ac pretreatment compared with dilute acid pretreatment. Furthermore, subsequent hydrolysis of the
sample pretreated with [EMIM]Ac for 12 h was sufficient to obtain 90% yield compared with pretreatment with dilute acid, which
resulted in 80% saccharification after 72 h.9 In another study, six ILs, [BMIM]Cl, [EMIM]Ac, [AMIM]Cl, [MMIM]DMP, [BMIM]
[NTf2], and 1-ethyl-3-(hydroxymethyl)pyridine ethyl sulfate, were investigated to find the best one for dissolving sugarcane
bagasse.10 The results revealed that [EMIM]Ac significantly enhanced the sugarcane bagasse enzymatic saccharification and yielded
99.7% of glucose after 60 min of pretreatment at 120
C. Therefore, [EMIM]Ac is considered as an efficient IL for biomass treatment.
Next, a study used [C2mim]OAc to dissolve a wooden substance due to its enzyme compatible nature. Delignification of wood chips
was performed with the IL followed by laccase enzymatic hydrolysis, which offered the separation of cellulose fibers from wood
biomass in an IL-aqueous buffering system.11 Very recently, few studies have been reported for the conversion of cellulose and
a variety of agro wastes such as rubber wood, palm oil frond, bamboo, and rice husk into levulinic acid (LA). Among the used
ILs, [C4(Mim)2][(2HSO4)(H2SO4)4] showed a higher percent yield of LA up to 47.52% from bamboo biomass at 110
C for
60 min and up to 55% LA for cellulose without using any additional catalyst or solvent at 100
C for 3 h, which were the best results
compared with those in previous reports. The highest yield of LA was obtained by the treatment of straw with [C3SO3Hmim]HSO4
(96.6 mol% (21.6 wt%)).12–14 In another study, three fermentation strategies, viz., the increase of inoculum size, two-step fermen-
tation, and fed-batch fermentation, were investigated to alleviate the negative effect of [EMIM]Ac on lignocellulosic ethanol

Table 1 List of recent methods reported for the lignocellulosic biomass pretreatment and hydrolysis with different ionic liquids (ILs), substrates,
and main findings

Ionic liquid Substrate Purpose Main findings References

[C2mim][OAc], acetic acid, NaOAc, Switchgrass Pretreatment IL pretreatment was proved better for switchgrass than 9
DNS, TFA Saccharification acid treatment
Recovered biomass has high surface area, and resulted
three times more delignification
[EMIM][Ac] Sugarcane Pretreatment Resulted 80% glucose within 6 h and more than 90% 10
Bagasse Saccharification within 24 h
SSA of the treated bagasse was found 100 times that of
untreated bagasse
[C2mim][OAc] Wood Pretreatment IL-aqueous system offered separation of cellulose from 11
Delignification wood biomass
[C4(Mim)2] Rubber wood Pretreatment Agro waste has been converted into useful levulinic acid 12
[(2HSO4) (H2SO4)0] Rice husk Conversion (LA)
[(2HSO4) (H2SO4)2] Bamboo Higher percent yield of 47.52% was obtained from
[(2HSO4) (H2SO4)4] bamboo at 110
C for 60 min
[C4(Mim)2] Cellulose Pretreatment Selected IL showed higher catalytic activity for conversion 13
[(2HSO4) (H2SO4)2] Conversion of cellulose to LA (55%) without any additional catalyst
or solvent at 100
C for 3 h
[C3SO3Hmim]HSO4 Straw Conversion High yield of LA produced from straw, i.e., 96.6 mol% 14
(21.6 wt%)
LA production depends on acidity and hydrogen bonding
of anions
[EMIM][Ac] Wheat straw Fermentation All the tested fermentation strategies improved the ethanol 15
production of the IL [EMIM][Ac] treated with wheat
straw
[AMIM]Cl Rice straw Pretreatment The IL 1% rhamnolipid-pretreated rice straw showed 16
Delignification significant lignin removal of 26.14% and high cellulose
conversion of 36.21% than the untreated (16.16%)
 Ionic Liquids for Pretreatment of Biomass 193

production. All three fermentation processes improved the ethanol productivity at an IL concentration of 10 g/L.15 Further in a study,
a surfactant-assisted [AMIM]Cl was tested for the pretreatment of rice straw. The biosurfactant þ IL-pretreated rice straw showed
significant lignin removal (26.14%) and exhibited higher cellulose conversion (36.21%) than the untreated (16.16%) rice straw.16

2.16.2.2 Microalgae Biomass

Microalgae are a large and diverse group of aquatic organisms that lack the complex cell structures usually found in higher plants.
Most species are photoautotrophic and convert solar energy into chemical forms through photosynthesis. They have received
considerable attention in recent years as one of the most promising sources of sustainable biomass for the production of fuels
and chemicals. However, some challenges must be addressed to improve the commercial outlook for the production of commodity
chemicals. Microalgae present several advantages over terrestrial crops for sustainable biomass production. First, microalgae do not
require soil and are capable of photosynthetic growth utilizing sunlight, carbon dioxide, water, and inorganic salts as their major
nutrients, allowing a potentially carbon-negative growth process.17 Algae are also capable of producing an assortment of desirable
biochemicals for a variety of industries, but the prime importance is the production of biodiesel with accumulation of lipids by
microalgae. However, the biggest challenge remains the energy intensive and consequently costly process of drying microalgae.
Fortunately, ILs have provided the opportunity for improvements in this area by allowing the extraction of lipids from wet biomass
at low temperatures in less time than traditional lipid extraction methods. ILs have been recently shown to facilitate the extraction of
lipids from microalgae, primarily by disrupting the microalgal cell structure, allowing either autopartitioning of the lipids or
presumably improving access to the intracellular lipids for the cosolvents.
Several species of microalgae exhibit the ability to accumulate large portions of their dry weight as intracellular lipids (up to
75 wt%). Owing to their small size, single-celled algae must first be harvested from their liquid growth medium using solid–liquid
separation techniques. Traditionally, microalgae are then dried and subjected to organic solvent–based extraction. Cell disruption
can greatly aid in facilitating the recovery of lipids by improving the mass transfer properties of the system, as solvent extraction is
primarily diffusion based. However, owing to their small size, cell disruption processes such as enzymatic degradation, ultrasoni-
cation (SN), or microwave (MW) irradiation can be either material- or energy-intensive processes. Most solvent-based extraction
processes are also incompatible with wet biomass, requiring extensive drying of the algae prior to extraction, which can add signif-
icant costs to the overall process. Dewatering and drying is thought to be responsible for up to 70% of the biodiesel production cost
from microbial lipids.18
A number of studies have recently reported the extraction of microalgae using ILs and elucidated some of the limitations of this
process. The majority of ILs studied to date have focused on imidazolium-based ILs mostly owing to their commercial availability,
with only few publications describing the use of pyridinium-, ammonium-, and phosphonium-based ILs. A summary of the latest
microalgae extraction reports and the used ILs can be found in Table 2. Most common ILs assessed so far have been hydrophilic and
water-soluble ILs with the exception of [C2mim][NTf2]. Dialkylimidazolium cations combined with alkyl ester sulfates or phos-
phates have been used extensively. [C2mim] paired with either [EtSO4], [MeSO4], or [Et2PO4] has generally resulted in high yields
(>70%) of lipids when compared with traditional solvent extraction methods. [C2mim]Cl, one of the most commonly known ILs,
has also exhibited high lipid extraction efficiencies, although other dialkylimidazolium chlorides worked poorly. Interestingly,
1-hexylimidazolium exhibited moderate ability (53.4%) to extract lipids at room temperature even with 30% water content present
in the IL.19 Acidic ILs composed of carboxylic acid anions have also exhibited some lipid extraction ability, which increased with
increasing processing temperature and decreased with increasing carboxylic acid chain length. Few reports of pyridinium-,
ammonium-, and phosphonium-based ILs for lipid extraction exist. However, ammonium- and phosphonium-based ILs may be
significantly cheaper to synthesize than their imidazolium-based counterparts.
In one study, wet and saliferous microalgae (WSM) was successfully dissolved in polar ILs such as [C2mim][MeO(H)PO2] under
mild conditions within 30 min at room temperature with gentle stirring. The IL successfully recycled and maintained its ability to
dissolve WSM regardless of the number of times it was recycled.20 Furthermore, there are several studies that have reported the disso-
lution and lipid extraction of Chlorella vulgaris microalgae using different ILs, in which the lipid extraction yield using a single IL was
compared with the yield obtained with organic solvents and IL mixtures. Overall, the synergistic effects of the IL mixtures with
different anions improved the lipid extraction yield of C. vulgaris.21–24 The lipid extraction from C. vulgaris was improved by the
synergistic effects of molten salts such as Zn(NO3)2$6H2O, Mg(ClO4)2$6H2O, and FeCl3$6H2O and IL combination. A study re-
ported an IL blend system consisting of a solvent IL for biomass dissolution and an acidic IL for acid-catalyzed hydrolysis of the
released polysaccharides into simple sugars for the pretreatment of Gelidium amansii microalgae.25 Interestingly, the macroalgae
were more efficiently dissolved in the selected IL blend than in the most effective pure solvent IL. Pretreatments can be accomplished
under an atmospheric environment, but temperature control and timely reaction termination were both important to maximize the
sugar yields and minimize the subsequent sugar degradation. Another study explored the possibility of using ILs as a medium for
efficient extraction of agarose through the dissolution of red algae under varying conditions of heat or MW irradiation. Compared
with conventional methods, a very high extraction yield of good quality agarose (as high as 39 wt%) was achieved.26 Furthermore,
mixtures of ILs and methanol successfully dissolved the biomass leaving lipids insoluble; hence this IL–MeOH combination over-
comes the drawbacks of imidazolium-based ILs.27 A low-cost IL–water mixture for effective extraction of carbohydrates and lipids
from algae has been reported. The biomass derived from C. vulgaris and Spirulina platensis was pretreated with low-cost choline-
amino-acid-based ILs to effectively yield lipids (30.6% and 51.0% total lipids) and sugars (71.0% and 26.0% total sugars). The
ILs dissolved the lipids, leaving behind a carbohydrate-rich solid.28
194 Ionic Liquids for Pretreatment of Biomass

Table 2 List of advanced methods reported for microalgae extraction using ionic liquids (ILs)

Ionic liquid Substrate Purpose Main findings References

[C2mim][MeO(H)PO2] Wet and saliferous Dissolution Wet and saliferous microalgae (WSM) were well dissolved 20
microalgae in polar ILs
IL maintained its chemical structure and suggested
recyclable use
[Emim]OAc Chlorella vulgaris Lipid extraction ILs with different anions improved the lipid extraction of 21
[Emim]Cl C. vulgaris
[Emim]DEP among all [Emim]OAc and [Emim]BF4 showed good lipid extraction
[Emim]DEP performance
[Emim]Cl
[Emim]OAc C. vulgaris Lipid extraction The lipid extraction of C. vulgaris was increased adding 22
FeCl3$6H2O to IL
FeCl3$6H2O showed a high lipid extraction yield of
113.0 mg g1 cell
EMIM C. vulgaris Lipid extraction The IL-PCM cosolvent mixture supports extraction of 23
Yeast lipids in the presence of moderate water contents
[C2mim][EtSO4] C. vulgaris Wet extraction The IL is found effective over a range of water containing 24
dewatered algae
[BMIM]Cl Gelidium amansii Lipid extraction G. amansii was dissolved faster (2 h) in blend (1:1) than in 25
[BMIM]HSO4 pure [BMIM]Cl (24 h)
Temperature control and timely reaction termination were
important to maximize the sugar yields
[Emim]OAc Gracilaria dura Lipid extraction High purity agarose was extracted from algae-IL solution 26
[Emim]DEP through precipitation
[Emim][OAc] was found highly efficient, microwave
heating increased the extraction of agarose
[Bmim][CF3SO3] C. vulgaris Lipid extraction Mixtures of ILs and methanol fully dissolved biomass 27
leaving lipids insoluble
This IL–MeOH combination overcomes the drawbacks of
imidazolium-based ILs
[Emim][OAc] Spirulina platensis Extraction Low-cost choline-amino-acid-based ILs effectively 28
C. vulgaris produced lipids and sugars
Along with lipids, ash was also removed during the
pretreatment

2.16.3 Factors Affecting Biomass Pretreatment

2.16.3.1 Biomass Loading
Biomass loading largely depends on the solubility of biomass in a particular IL at the operating temperature and the biomass
dissolution rate, which influences the pretreatment time. Because the time requirement can be excessively long, the thermody-
namic solubility limit may not be reached during the given pretreatment time. Thus, the IL-to-biomass mass ratio should be
substantially higher than the solubility limit. A smaller biomass particle size typically means a faster biomass dissolution rate.
Typically, researchers use a nominal biomass loading ratio of 20:1 to 30:1 (g ionic liquid:g biomass) for IL pretreatment of
various types of biomass under different conditions of temperature and time. In comparison, a ratio of 10:1 (g ionic liquid:g
biomass) is common for aqueous ammonia, acid hydrolysis, supercritical CO2, and hydrothermal pretreatment methods. It
should be noted that if the biomass concentration reaches 10% (w/w), the IL solution is almost always very viscous, which makes
it difficult to process. Thus, the viscosity could be a limiting factor rather than the biomass solubility in a practical pretreatment
process.
The ratio of biomass to IL (% biomass loading) is an important aspect to improve the process economy, recyclability, disso-
lution, and regeneration efficiency. Studies have reported that chemical equilibrium of a reaction might have shifted to favor
more cellulose dissolution only at the appropriate biomass loading and temperature. In one study, higher dissolution rates
were obtained at 12 wt% rather than 8 wt% of biomass loading because of more frequent contact or collision between the IL
and biomass particles, which inadvertently accelerated dissolution. Conversely, a reduced dissolution rate was reported as the
biomass loading increased from 4 to 10 wt%, whereas the optimal biomass loading was 5.0 wt%. Similarly, a reduction in the
dissolution rate from 35% to 26% was observed in another study when the biomass loading increased from 1.0 to 5.0 wt%.
A higher dissolution rate at a lower biomass loading may be attributed to efficient dispersion and diffusion of the IL inside the pores
of the biomass sample. The existing literature shows that the biomass loading is an important processing parameter,
whereby researchers selected 5.0–8.0 wt% of biomass loading. However, the biomass loading may depend on the particle size
and biomass type.
 Ionic Liquids for Pretreatment of Biomass 195

2.16.3.2 Pretreatment Temperature

Besides selecting a suitable IL for the desired biomass pretreatment outcome, the pretreatment temperature is the most important
operating parameter. A higher temperature typically achieves higher biomass solubility and shortens the pretreatment time. It also
reduces the biomass recalcitrance. However, it also requires a higher energy input. Compared with steam explosion and hot water
pretreatment methods, the required pretreatment temperature for IL pretreatment is relatively mild, with a typical temperature range
of 80–180
C. This is comparable to the typical temperature range used in supercritical CO2 explosion pretreatment, but IL pretreat-
ment does not require pressurization, which is needed for supercritical CO2. Compared with the typical 140–250
C temperature
range used in steam explosion pretreatment and hot water pretreatment, ILs have a major advantage. Typically, the higher temper-
ature in the temperature range is required for the desired pretreatment outcome. Thus, IL pretreatment requires a much lower
optimal temperature. This also means that the pyrolysis side reaction is easier to avoid when IL pretreatment is used instead of steam
or hot water pretreatment.
There are several factors to be considered when choosing the optimal temperature: (1) the lowest temperature at which the IL is
in the liquid state; (2) the solubilization efficiency; (3) maintaining the enzyme activity over extended periods; and (4) the decom-
position temperature of the IL to avoid IL decomposition, which may result in unwanted cellulose derivatization at higher temper-
ature. As reported, ILs dissolve more celluloses at a higher temperature, but this could depend on the IL type. For example, in
a [BMIM]Cl system, the reduced sugar (RS) concentration was higher at 120
C after 5.0 h of pretreatment, whereas a lower concen-
tration was observed at 80
C. In contrast, for [EMIM]Ac and [BMIM]Ac systems, the RS yield was two times higher in a two-step
process. The increase in temperature reduces the viscosity of the acetate/formate-based ILs more quickly than the chloride-based ILs,
as the chloride-based ILs are more viscous. In one study, various temperatures were used to test the efficiency of the enzymatic
hydrolysis of biomass in the presence of [EMIM]DEP. When the temperature is ranged between 20 and 60
C, the highest hydrolysis
yield was obtained between 50 and 60
C after 1.0 h of pretreatment. However, to reduce the energy consumption, the pretreatment
was conducted at 50
C, which has provided a hydrolysis yield of 54.94%. Longer pretreatment (>3 h) slightly reduced the hydro-
lysis yield. This study suggested that time and temperature affected the hydrolysis rather than the composition of ILs.29
A solid evidence was also provided by researchers, whereby the molecular weight of the cellulose was investigated at 40
C in
[EMIM]Ac after 48 h. The molecular weight did not significantly deviate from the initial value at 17 h, whereas cellulose was strongly
degraded at 110
C. In one study, cellulose was treated at 130
C for 1.0 h with [EMIM]Ac, [AMIM]Cl, [BMIM]Cl, [BMIM]Ac, or
[BMIM]Cl combined with 0.5% H2SO4, and [BMIM]Cl combined with MgCl2. In general, the dissolution of cellulose was carried
out in a temperature range of 40–160
C. However, organic moieties (cation/anion) of ILs may undergo decomposition at certain
temperatures; therefore, it is critical to determine the optimal temperature. Elevated temperatures at short retention times (5.0–
15 min) were sufficient to dissolve most of the biomass. For example, 97% of the added bagasse was dissolved by pretreatment
at 165
C for 10 min, but a complete dissolution of the biomass sample was achieved at 175
C for 10 min or at 185
C for
5.0 min. The percent glucose recovery increased from 26.2% to 96.7% as the pretreatment temperature increased from 60 to
100
C for 1.5 h.

2.16.3.3 Pretreatment Time

Generally, increasing the dissolution time has a similar effect to increasing the temperature. For instance, increasing the dissolution
time from 5.0 min to 15 min leads to a lower carbohydrate yield and lower lignin content. Longer dissolution times result in more
carbohydrate degradation but facilitate the separation of lignin. More dealkylation might occur on heating to higher temperatures
even for shorter periods of time. Moreover, the use of higher temperatures, even at shorter times, appears to lead to more IL decom-
position. This suggests that care must be taken for the selection of the temperature and treatment time.
The dissolution of lignocellulosic biomass in an IL is far from instantaneous. Typically, several hours are required for biomass
dissolution, as reported in the literature. The biomass solubility limit in a particular IL may not be reached within the given pretreat-
ment time frame. The solubility of pulp cellulose in different ILs was investigated by researchers. The highest solubility was obtained
using [C4mim]Cl. Various types of biomass were pretreated with [C2mim]Cl at 150
C to study how lignin removal made the
residual biomass easier to hydrolyze. Apart from lignin dissolution, some cellulose and hemicellulose were also dissolved. Thus,
it was important to control the pretreatment time. They found that for corn stover, wheat straw, and eucalyptus, 1.0 h of pretreat-
ment time led to significantly higher glucose and xylose yields compared with 0.5 h. However, the opposite trend was observed for
lenga (Nothofagus pumilio). The authors argued that the lenga biomass pieces were smaller in size and thus faster for IL pretreatment.
More cellulose and hemicellulose were lost owing to dissolution in [C2mim]Cl when the pretreatment time was excessive.

2.16.3.4 Water Content

The deactivation effect of the ILs on the cellulases is not that significant, which provided promising basics for more investigations.
An IL–water mixture can be used to reduce the cost as well as the viscosity of the ILs, thus reducing the impact of the ILs on the
cellulases. Among the tested compositions of IL–water mixture, 1:4 (IL/water v/v) was selected as the best for conversion of cellulose
biomass. A total of 70% of cellulose was successfully converted after 24 h of hydrolysis with cellulase; increasing the IL/water ratio
to 2:3 deactivated the enzyme. A study investigated the solubility of dissolving grade pulp, hardwood pulp and softwood pulp, and
microcrystalline cellulose in a mixture of [EMIM]Ac and water, which showed that a low water content (2.0%–5.0%) gave the
196 Ionic Liquids for Pretreatment of Biomass

maximum conversion. Dissolution and conversion was significantly inhibited at water content above 10%, and a nondissolved
material was obtained at a water content of 15%.
Conversely, an IL solution with a water content of up to 15 wt% was reported to dissolve cellulose at lower concentration, i.e.,
1.0 wt%. According to one study, only low water concentrations below 25 wt% (or 0.7 in mole fraction) were studied because cellu-
lose was not completely dissolved in [EMIM]Ac/water mixtures with water content higher than 20 wt%. The presence of 1.0% water
obstructed the dissolution process as the water molecules compete with the IL anion for the H-bond formation process and conse-
quently interrupt the dissolution process. In contrast to this, a study reported that acetate anions can tolerate up to 10% water for the
biomass dissolution process. Furthermore, a study confirmed that the presence of a trace amount of water was necessary to endorse
swelling and dissolution of lignocellulose.30

2.16.3.5 Combined Techniques

Combined processes, if feasible, reveal synergetic benefit on biomass treatment and widen the process design freedom for specific
biomass applications. ILs are polar in nature and have good absorption ability of MW radiation, which promotes the dissolution by
internal molecular heating of the materials through molecular vibrations, whereas conventional heating involves the conduction of
heat flow only. Thus, only 10 wt% solubility of cellulose was observed in [Bmim][Cl] with conventional heating, whereas the solu-
bility increased to 25 wt% with MW heating. A similar type of superior solubility was also achieved in a short time by high-intensity
ultrasound (acoustic cavitation) treatment.31 In comparison with conventional heating, the dissolution time decreases profoundly
with SN; e.g., complete dissolution of 5.0 wt% cellulose solution in [Amim][Cl] IL was achieved in 2.0 min after SN, whereas
conventional heating required more than 1.0 h. The sonication promotes the dissolution process at a molecular level through
acoustic cavitation caused by the input of ultrasonic waves. It has also been reported that sonication and MW pretreatment enhances
the dissolution of cellulose in ILs. Experimentally, it was proved that the time required for complete dissolution of pine sawdust was
reduced to less than half with MW or SN pretreatment. However, there is a need to control the operating temperature and MW or SN
energy source because excess heating by MW or SN may cause rapid deprivation of ILs and cellulose. Furthermore, these MW and SN
techniques are mostly useful in lab-scale applications.
To enhance the dissolution of cellulose or lignocellulosic biomass in IL, several studies that combine IL pretreatment with heat,
MW radiation, and ultrasound technologies have been conducted. In one study, the effect of ultrasound-IL pretreatment on the
enzymatic and acid hydrolysis of the sugarcane bagasse and wheat straw was investigated. The lignocellulosic biomass was disso-
ciated in ILs ([Bmim]Cl and [Bmim]AOC) aided by ultrasound waves. The enzymatic and acid hydrolysis of the biomass samples
pretreated with ultrasound-IL result in higher yields of the reducing sugars compared with the IL-pretreated sample. The combina-
tion of ultrasound with [Bmim]OAc was more effective than [Bmim]Cl in terms of the yield of the reducing sugar.32 In another
study, MW irradiation was employed to rapidly break the glycosidic bonds in cellulose and enhance the glucose yield to 78.7%
at 160
C in only 46 min. The water concentration and reaction temperature were identified as the process parameters for regulating
the product distribution, and the optimum product yields were 78.7% for glucose at 160
C and 40% water concentration, 26.8%
for 5-hydroxymethylfurfural (HMF) at 180
C and 25% water, and 44.9% and 17.8% for LA and FA, respectively, at 200
C and 40%
water.33 Next, glucose, cellulose, and biomass were successfully converted into HMF using the probe SN with [C4C1im]Cl and CrCl3
as solvent and catalyst, respectively. A high glucose conversion and HMF yield were obtained within 3.0 min of ultrasonic
treatment.34

2.16.4 Recovery of Ionic Liquids

Owing to the high cost and potential toxicity of ILs, they must be recycled after biomass pretreatment. Various methods have been
reported to recover ILs after biomass pretreatment. Each method has its advantages and limitations. The ILs can be recovered from
the pretreated mixture by adding antisolvents such as ethanol or H2O. The addition of potassium phosphate to an aqueous solution
can salt out hydrophilic ILs from solutions. However, these processes would generate recovered ILs with impurities. The reverse
micelle method can be used to extract almost pure [Mmim][DMP] from a pretreated mixture. However, further investigation on
its economy is needed.
A method reported for the total recovery rate of renewable cholinium ILs was 75% after treating rice straw for eight cycles, sug-
gesting almost 25% of the IL was lost. Because the PILs synthesized from acetic acid and base through proton exchange can decom-
pose into the corresponding acid and base at high temperatures, the three PILs can be recovered by decomposition and resynthesis.
Almost full recovery of PILs was achieved with this process, suggesting that the thermal degradation of PILs into amide by-products
did not occur under these conditions. A hybrid membrane–based methodology of electrodialysis (ED) with ultrafiltration to recover
IL [Bmim]Br (1-butyl-3-methylimidazolium bromide) after biomass fractionation was developed to avoid the energy-extensive
distilling process. The result showed that the highest overall IL recovery ratio reached 75.2% with the current efficiency of the
ED process approaching 79.1%. The specific energy consumption in this process was approximately 514.1 g/kwh. Another study
proposed the use of membrane distillation to separate ILs and water from the rinse liquor from pretreated biomass.35 By passing
the hot ILs ([Emin][OAc] or [Emin][O2CH]) over a hydrophobic mesoporous membrane with cold water flowing over the opposite
side, the water in the IL stream was evaporated and condensed on the cold stream side. These authors found a 10-fold concentration
for the diluted ILs in the rinsed liquor. This technique can be an economically feasible option at an intermediate treatment capacity.
 Ionic Liquids for Pretreatment of Biomass 197

Membrane filtration is another method for the separation/recovery of ILs. For example, nanofiltration membranes have been
used for IL recycling by rejecting ILs while allowing smaller molecules to pass through. Membrane filtration has the advantage
that room temperature operation may be possible. However, high viscosity is often a hurdle. Ion-exclusion chromatography, which
is already in use for carbohydrate processing, can also be used for IL recovery because it can exclude charged species such as IL
species, thus separating them from nonelectrolytes such as sugar molecules. For continuous chromatographic separation, simulated
moving bed (SMB) chromatography has been used. SMB utilizes a series of ion-exchange chromatography columns with synchro-
nized moving ports to simulate resin movement. Although chromatography separation has the advantage of high resolution and the
separation of multiple fractions in the output, it is far more expensive than other separation methods.36

2.16.5 Environmental Impacts of Ionic Liquids

Owing to the increasing use of ILs in many areas of chemistry and engineering, researchers in academia and industry have started to
investigate their impact on the environment. A number of studies investigating the toxicity of ILs towards different species have been
published. However, the early work should be judged with care, as it is not always clear whether impurities resulting from the
synthesis caused the observed effect. Investigations of the toxicity and biodegradability of ILs discovered a massive influence of
several ILs on aquatic organism or bacteria. The toxicity of imidazolium- and pyridinium-based ILs was found to be dependent
on the length of the alkyl chain: the longer the side chain, the higher the toxicity of an IL. For example, 1-octyl-3-methyl imidazo-
lium (OMIM) bromide shows toxicity towards Vibrio fischeri exceeding the EC50 value of toluene and benzene, whereas 1-butyl-3-
methyl imidazolium (BMIM) shows almost no toxicity at all.37
ILs are safe to handle because they are nonvolatile and nonflammable. However, they possess toxicity. The cellulose dissolving
ILs are relatively hydrophilic and have low octanol–water partition coefficients, which cause less bioaccumulation. However, they
still need to be biodegradable, which will render nontoxic products after biodegradation of the IL. Therefore, toxic effects on
humans or the environment should be assessed for implementation of IL as solvents for scale-up. To reduce the toxicity of the
ILs themselves, the application of ions found in nature or obtained from natural substances is beneficial. Several publications
have reported the successful synthesis of ILs in which either the cations or anions are represented by amino acids. Additionally, there
are attempts to increase the biodegradability of ILs. In the area of environmental sustainability, a lot of research has yet to be done,
especially with respect to the unknown toxicity of numerous compounds.

2.16.6 Conclusions

ILs have emerged as potential solvents and/or reagents in the field of biomass pretreatment under “green” conditions, which has
been confirmed during the last decade. IL pretreatment provides a new powerful platform to improve the enzymatic hydrolysis
of biomass. The most important challenge in the use of ILs for lignocellulosic biomass pretreatment for the production of bioenergy
and biomaterials from biomass is the development of economical solutions for the recycling of ILs. The recent applications of ILs for
the utilization and characterization of lignocellulosic biomass and microalgae have been reviewed in this chapter. The outstanding
features and many varieties of ILs make it a powerful solvent/media in many biomass-related applications, including characteriza-
tion of the materials and valorization of biomass for fuel, chemicals, and materials. Current approaches suggest solutions for the
economic and environmental challenges of biomass utilization using ILs. More innovative methodologies are needed for the reuse
and recycling of ILs and cellulases in an effective way. The research in this direction is still at an initial stage.
Overall, there are many opportunities to improve enzymatic hydrolysis of biomass after IL pretreatment. In the future, the aim of
IL-based processing of algal biomass is to recover the carbohydrate fraction and recover high-value products such as carotenoids. If
these processes can be combined using a single IL-based extraction/fractionation process, this could significantly advance the
commercial outlook of microalgae-based production of renewable chemicals.

References

1. Priya, A. T.; Bassy, B. M. Room Temperature Ionic Liquids as Green Solvent Alternatives in the Metathesis of Oleochemical Feedstocks. Molecules 2016, 21 (2), E184.
2. Elgharbawy, A. A.; Md Zahangir, A.; Moniruzzaman, M.; et al. Ionic Liquid Pretreatment as Emerging Approaches for Enhanced Enzymatic Hydrolysis of Lignocellulosic Biomass.
Biochem. Eng. J. 2016, 109, 252–267.
3. Florence, J. V. G.; Agnieszka, B. T.; Clementine, L. C.; et al. Ultra-low Cost Ionic Liquids for the Delignification of Biomass, Ionic Liquids: Current State and Future Directions.
ACS Symp. Ser. 2017, 1250, 209–223 (Chapter 9).
4. Phillips, D.; Mitchell, E. J. S.; Langton, A. R. L.; et al. The Use of Conservation Biomass Feedstocks as Potential Bioenergy Resources in the United Kingdom. Bioresour.
Technol. 2016, 212, 271–279.
5. Azevedo, E. G. R.; Carvalho, A. C.; Martín, A. Use of Protic Ionic Liquids as Biomass Pretreatment for Lignocellulosic Ethanol Production. Chem. Eng. Transac. 2014, 37,
397–402.
6. Lisa, W.; Shahrokh, M.; Brandt, A. T.; et al. Effect of Pretreatment Severity on the Cellulose and Lignin Isolated from Salix Using IonoSolv Pretreatment. Faraday Discuss 2017,
202, 331–349.
7. Saha, K.; Dasgupta, J.; Sudip, C.; et al. Optimization of Lignin Recovery from Sugarcane Bagasse Using Ionic Liquid Aided Pretreatment. Cellulose 2017, 24, 3191–3207.
198 Ionic Liquids for Pretreatment of Biomass

8. Mahmood, H.; Moniruzzaman, M.; Suzana, Y.; et al. Ionic Liquids Assisted Processing of Renewable Resources for the Fabrication of Biodegradable Composite Materials. Green
Chem. 2017, 19, 2051–2075.
9. Li, C.; Knierim, B.; Manisseri, C.; et al. Comparison of Dilute Acid and Ionic Liquid Pretreatment of Switchgrass: Biomass Recalcitrance, Delignification and Enzymatic
Saccharification. Bioresour. Technol. 2010, 101, 4900–4906.
10. Da Silva, A. S. A.; Lee, S. H.; Endo, T.; et al. Major Improvement in the Rate and Yield of Enzymatic Saccharification of Sugarcane Bagasse via Pretreatment with the Ionic Liquid
1-ethyl-3-methylimidazolium Acetate ([Emim][Ac]). Bioresour. Technol. 2011, 102, 10505–10509.
11. Moniruzzaman, M.; Ono, T. Ionic Liquid Assisted Enzymatic Delignification of Wood Biomass: A New ‘green’ and Efficient Approach for Isolating of Cellulose Fibers. Biochem.
Eng. J. 2012, 60, 156–160.
12. Amir, S. K.; Zakaria, M.; Mohamad, A. B.; et al. Efficient Conversion of Lignocellulosic Biomass to Levulinic Acid Using Acidic Ionic Liquids. Carbohydr. Polym. 2018, 181,
208–214.
13. Khan, A. S.; Zakaria, M.; Azmi, M. B.; et al. Dicationic Ionic Liquids as Sustainable Approach for Direct Conversion of Cellulose to Levulinic Acid. J. Clean. Prod. 2018, 170,
591–600.
14. Liu, L.; Zhenrui, L.; Wuxin, H.; et al. Direct Conversion of Lignocellulose to Levulinic Acid Catalyzed by Ionic Liquid. Carbohydr. Polym. 2018, 181, 778–784.
15. Shengdong, Z.; Fang L,Wenjing, H.; et al. Comparison of Three Fermentation Strategies for Alleviating the Negative Effect of the Ionic Liquid 1-ethyl-3-methylimidazolium
Acetate on Lignocellulosic Ethanol Production. Appl. Energy 2017, 197, 124–131.
16. Ken, C. L.; Chen, X.; Wang, X. Q.; et al. Impact of Surfactant Type for Ionic Liquid Pretreatment on Enhancing Delignification of Rice Straw. Bioresour. Technol. 2017, 227,
388–392.
17. Pierobon, S. C.; Cheng, X.; Graham, P. J.; et al. Emerging Microalgae Technology: A Review. Sustain. Energy Fuels 2018, 2, 13–38.
18. Moniruzzaman, M.; Noriho, K.; Kazunori, N.; et al. Water-in-ionic Liquid Microemulsions as a New Medium for Enzymatic Reactions. Green Chem. 2008, 10, 497–500.
19. Ramanathan, R. K.; Polur, H. R.; Muthu, A. Lipid Extraction Methods from Microalgae: a Comprehensive Review. Front. Energy Res. 2015, 2, 1–9.
20. Kyoko, F.; Daigo, K.; Nobuhumi, N.; et al. Direct Dissolution of Wet and Saliferous Marine Microalgae by Polar Ionic Liquids without Heating. Enzym. Microb. Technol. 2013, 52,
199–202.
21. Choi, S. A.; Oh, Y. K.; Jeong, M. J.; et al. Effects of Ionic Liquid Mixtures on Lipid Extraction from Chlorella vulgaris. Renew. Energy 2014, 65, 169–174.
22. Sun, A. C.; Lee, J. S.; Oh, Y. K.; et al. Lipid Extraction from Chlorella vulgaris by Molten-salt/ionic-liquid Mixtures. Algal Res. 2014, 3, 44–48.
23. Gregory, Y.; Franz, N.; Sebastian, T.; et al. Lipid Extraction from Biomass Using Co-solvent Mixtures of Ionic Liquids and Polar Covalent Molecules. Separ. Purif. Technol. 2010,
72, 118–121.
24. Valerie, C. A. O.; Natalia, V. P.; Kenneth, R. S.; et al. Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room-temperature Ionic Liquids. ACS Sustain.
Chem. Eng. 2016, 4, 591–600.
25. Lenny, B. M.; Grace, M. N.; Neha, M.; et al. Blended Ionic Liquid Systems for Macroalgae Pretreatment. Renew. Energy 2014, 66, 596–604.
26. Tushar, J. T.; Arvind, K. Efficient Extraction of Agarose from Red Algae Using Ionic Liquids. Green Sustain. Chem. 2014, 4, 190–201.
27. Kim, Y. H.; Choi, Y. K.; Jungsu, P.; et al. Ionic Liquid-mediated Extraction of Lipids from Algal Biomass. Bioresour. Technol. 2012, 109, 312–315.
28. Trang, Q. T.; Kerryn, P.; Blake, A. S.; et al. Low Cost Ionic Liquid-water Mixtures for Effective Extraction of Carbohydrate and Lipid from Algae. Faraday Discuss 2018, 206,
93–112.
29. Kiran, A. K.; Shaishav, S. Recent Updates on Different Methods of Pretreatment of Lignocellulosic Feedstocks: A Review. Bioresources Bioprocess. 2017, 4, 1–19.
30. Elgharbawy, A. A.; Fatimah, A. R.; Alam, M. Z.; et al. Ionic Liquids as a Potential Solvent for Lipase-catalysed Reactions: A Review. J. Mol. Liq. 2018, 251, 150–166.
31. Naveena, B.; Patricia, A.; Tony, J. P.; et al. Ultrasonic Intensification as a Tool for Enhanced Microbial Biofuel Yields. Biotechnol. Biofuels 2015, 140, 1–13.
32. Xiaojie, Y.; Xinjie, B.; Cunshan, Z.; et al. Ultrasound-ionic Liquid Enhanced Enzymatic and Acid Hydrolysis of Biomass Cellulose. Ultrason. Sonochem. 2018, 41, 410–418.
33. Paula, S. K.; Saikat, C. Microwave-assisted Ionic Liquid-mediated Rapid Catalytic Conversion of Non-edible Lignocellulosic Sunn Hemp Fibres to Biofuels. Bioresour. Technol.
2018, 253, 85–93.
34. Ariyanti, S.; Zakaria, M.; Nawshad, M.; et al. A New Approach of Probe Sonication Assisted Ionic Liquid Conversion of Glucose, Cellulose and Biomass into
5-hydroxymethylfurfural. Ultrason. Sonochem. 2017, 37, 310–319.
35. Lynam, J. G.; Chow, G. I.; Coronella, C. J.; et al. Ionic Liquid and Water Separation by Membrane Distillation. Chem. Eng. J. 2016, 288, 557–561.
36. Brown, L.; Earle, M. J.; Gilea, M. A.; et al. Ionic Liquid–Liquid Chromatography: A New General Purpose Separation Methodology. Top Curr. Chem. (Cham) 2017, 375, 74.
37. Khana, M. I.; Zainia, D.; Azmi, M. S.; et al. Framework for Ecotoxicological Risk Assessment of Ionic Liquids. Procedia Eng. 2016, 148, 1141–1148.

Relevant Websites

http://ilthermo.boulder.nist.gov/ILThermo/mainmenu.uixMerck.
http://ilthermo.boulder.nist.gov/ILThermo/mainmenu.uixMerck – Ionic Liquids Database – ILThermo.
www.iolitec.com – Ionic Liquids Technologies.
http://biomasspower.gov.in/ – Biomass Knowledge Portal.
www.basionics.com – BASF – The Chemical Company.
http://www.il-eco.uft.uni-bremen.de/ – The UFT.