Chapter 5

Compatibility of DES with Enzymes

and Microorganisms

“Enzymatic processes are green and eco-friendly and thus are desired in the industrial
applications. The addition of DESs and NADESs (Natural deep eutectic solvents)
to the media could speed up the reactions and increase the yields as seen in many
studies. Enzymes maybe stabilized or activated in DES or NADES mixtures due
to the presence of the hydrogen bonds between the salt and HBD, which results
in a significant alteration in the chemical nature of the resulting DES. This may
expand the hydrogen bond interactions, lessen the thermodynamic water activity in
the reaction media, and consequently enhance the enzyme activity” (Elgharbawy
et al. 2020; Zhao et al. 2011). Enzymatic-assisted reactions in DES and NADES
have been reported (Elgharbawy et al. 2018; Juneidi et al. 2017; Tian et al. 2016;
Kim et al. 2016; Huang et al. 2014; Xu et al. 2018; Wu et al. 2014; Khodaverdian
et al. 2018).
Satlewal et al. (2018) have discussed current status of bioconversion of different
types of biomass via pretreatment with DESs and enzymatic hydrolysis. Enzymatic
hydrolysis efficiency after pretreatment with DES is presented in Table 5.1.
Enzymatic hydrolysis yields vary substantially depending upon the biomass,
temperature of pretreatment, DES, molar ratio of hydrogen bond donor and acceptor.
Although lipase and cellulase enzymes show good stabilization and activation in DES
and NADES (natural deep eutectic solvents), the effect cannot be generalized for all
types of enzymes. Aqueous solution of DES affects the enzyme by DES virtue rather
than by the synergistic use of its individual constituents. Peroxidase is different
from lipases. In this case, stabilization occurred, but no activation was found. There-
fore, several factors should be considered during designing of DES-enzyme systems.
Mechanism of enzyme action, substrates and products’ biodegradability and toxicity
should be cautiously examined (Wu et al. 2014).
Jablonsky et al. (2015) have reported that increase in hydrolysis yields after
pretreatment with DESs is mainly due to the disruption of crystalline cellulose and
delignification.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 55
P. Bajpai, Deep Eutectic Solvents for Pretreatment of Lignocellulosic Biomass,
SpringerBriefs in Applied Sciences and Technology,
https://doi.org/10.1007/978-981-16-4013-1_5
56 5 Compatibility of DES with Enzymes and Microorganisms

Table 5.1 Enzymatic hydrolysis efficiency after DES pretreatment

Substrate/Biomass DES Pretreatment Enzymatic References
conditions hydrolysis
efficiency
Rice husk Nil 50 °C, 0.5 h 0.2 mM Gunny et al.
(2015)
Rice husk ChCI ethylene 115 °C, 3 h 0.7 mM Gunny et al.
glycol (2015)
Corncob Untreated – 33% Procentese
et al. (2015)
Corncob ChCI glycerol 80 °C, 15 h 40% Procentese
et al. (2015)
Corncob ChCI glycerol 115 °C, 15 h 79% Procentese
et al. (2015)
Corncob ChCI glycerol 150 °C, 15 h 92% Procentese
et al. (2015)
Corncob ChCI urea 80 °C, 15 h 51% Procentese
et al. (2015)
Corncob ChCI urea 115 °C, 15 h 59% Procentese
et al. (2015)
Corncob ChCI imidazole 80 °C, 15 h 92% Procentese
et al. (2015)
Corncob ChCI imidazole 115 °C, 15 h 94% Procentese
et al. (2015)
Corncob ChCI imidazole 150 °C, 15 h 95 Procentese
et al. (2015)
Corncob Untreated – 22 Zhang et al.
(2016)
Corncob ChCI lactic acid 90 °C, 24 h 84% Zhang et al.
(2016)
Corncob ChCI glycolic acid 90 °C, 24 h 67% Zhang et al.
(2016)
Corncob ChCI levulinic acid 90 °C, 24 h 62% Zhang et al.
(2016)
Corncob ChCI malonic acid 90 °C, 24 h 62% Zhang et al.
(2016)
Corncob ChCI glutaric acid 90’C, 24 h 41% Zhang et al.
(2016)
Corncob ChCI oxalic acid 90 °C, 24 h 45% Zhang et al.
(2016)
Corncob ChCI malic acid 90 °C, 24 h 37% Zhang et al.
(2016)
Corncob ChCI ethylene 90 24 h 85% Zhang et al.
glycol (2016)
(continued)
5 Compatibility of DES with Enzymes and Microorganisms 57

Table 5.1 (continued)

Substrate/Biomass DES Pretreatment Enzymatic References
conditions hydrolysis
efficiency
Corncob ChCI glycerol 90 24 h 96% Zhang et al.
(2016)
Corn stover ChCI formic acid 130 °C, 3 h 99% Zhang et al.
(2016)
Rice straw ChCI lactic acid 60 °C, 12 h 36% Kumar et al.
(2016)
Oil palm trunk Nil 25% Zulkefli et al.
(2017)
Oil palm trunk Ethylammonium 100 °C, 48 h 74% Zulkefli et al.
chloride ethylene (2017)
glycol
Pine Untreated 10% Lynam et al.
(2017)
Pine ChCI formic acid 155 °C, 2 h 70% Lynam et al.
(2017)
Microcrystalline Untreated 49% Wahlstrom
cellulose et al. (2016)
Microcrystalline ChCI betaine 80 °C, 24 h 49% Wahlstrom
cellulose et al. (2016)
Microcrystalline ChCI glycerol 80 °C, 24 h −65% Wahlstrom
cellulose et al. (2016)
Microcrystalline Betaine glycerol 80 °C, 24 h −65% Wahlstrom
cellulose et al. (2016)
Eucalyptus dissolving Untreated 62% Wahlstrom
pulp et al. (2016)
Eucalyptus dissolving ChCI glycerol 8 °C, 24 h −100% Wahlstrom
pulp et al. (2016)
Eucalyptus dissolving ChCI betaine 80 °C, 24 h −100% Wahlstrom
pulp et al. (2016)
Eucalyptus dissolving Betaine glycerol 80 °C, 24 h −100% Wahlstrom
pulp et al. (2016)
Wheat straw Untreated 18% Wahlstrom
et al. (2016)
Wheat straw ChCI betaine 80 °C, 24 h 33% Wahlstrom
et al. (2016)
Wheat straw ChCI glycerol 80 °C, 24 h <20% Wahlstrom
et al. (2016)
Wheat straw Betaine glycerol 80 °C, 24 h <20% Wahlstrom
et al. (2016)
(continued)
58 5 Compatibility of DES with Enzymes and Microorganisms

Table 5.1 (continued)

Substrate/Biomass DES Pretreatment Enzymatic References
conditions hydrolysis
efficiency
Spruce saw dust Untreated 8% Wahlstrom
et al. (2016)
Spruce saw dust ChCI glycerol 80 °C, 24 h <20% Wahlstrom
et al. (2016)
Spruce saw dust ChCI betaine 80 °C, 24 h <20% Wahlstrom
et al. (2016)
Spruce saw dust Betaine glycerol 80 °C, 24 h <20% Wahlstrom
et al. (2016)
Switchgrass ChCI 160 °C, 3 h 32% Kim et al.
4-hydroxybenzyl (2018)
alcohol
Switchgrass ChCI catechol 160 °C, 3 h 77% Kim et al.
(2018)
Switchgrass ChCI vanillin 160 °C, 3 h 80% Kim et al.
(2018)
Switchgrass ChCI p°Coumaric 160 °C, 3 h 86% Kim et al.
acid (2018)
Satlewal et al. (2018). Reproduced with permission

The enzymatic hydrolysis of wheat straw, spruce saw dust, dissolving Kraft
eucalyptus pulp and cellulose before and after pretreatment with three DESs—
ChCl:boric acid (5:2), ChCl:glycerol (1:1) and betaine:glycerol (1:1)—were
compared (Wahlstrom et al. 2016). Before pretreatment, maximum hydrolysis yields
obtained were 62, 49, 18 and 8% with kraft dissolving eucalyptus pulp, cellulose
(MCC), native wheat straw and spruce saw dust respectively (Table 5.1). Pretreat-
ment with DES was found to improve the enzymatic hydrolysis yields of all the
raw materials. The maximum hydrolysis yields were ~ 100, ~ 65%, 33% and only
marginal with dissolving pulp, cellulose (MCC), wheat straw and spruce saw dust,
respectively (Table 5.1). With agro-residues such as wheat straw, corncob, switch-
grass and other low recalcitrant substrates such as amorphous cellulose, kraft pulp,
mild DESs’ pretreatment was found to be effective. More research is required for
developing and demonstrating the use of DESs for other highly recalcitrant woody
biomass like spruce saw dust, date palm, etc. Acidic DES (ChCl:boric acid (5:2))
showed better efficiency as compared to glycerol-based DESs (i.e., ChCl:glycerol
(1:1) and betaine glycerol (1:1)).
DESs having strong acidity were more effective in solubilizing lignin and
improving the enzymatic hydrolysis. Different DESs on pine residues for delignifica-
tion and enzymatic hydrolysis yields were compared by Lynam et al. (2017). These
researchers used ChCl:acid based DESs—formic acid (high acidic strength with pKa
3.75), lactic acid (pKa 3.86), acetic acid (least acidic strength pKa 4.75)). Maximum
5 Compatibility of DES with Enzymes and Microorganisms 59

lignin solubility of 14% w/w and hydrolysis yields of 70% were achieved with
ChCl:formic acid as compared to others. With wheat straw, 57.9% delignification
was obtained by highly acidic ChCl:oxalic (pKa 1.2) in comparison to ChCl:lactic
acid (Jablonsky et al. 2015).
Procentese et al. (2015) studied the impact of temperature on pretreatment and
enzymatic hydrolysis. Enzyme Cellic CTec2 from Novozyme was used. When the
pretreatment temperature was increased from 80 to 150 °C, the hydrolysis yields
of corncob increased from 39.9 to 91.5% with ChCl:glycerol. Likewise, hydrol-
ysis yields increased from 51 to 58.6% with an increase in pretreatment tempera-
ture with ChCl:urea, but no appreciable increase with temperature was noted with
ChCl:imidazole as it performed in the same way (92.3%) even at reduced temperature
of 80 °C.
In another study, Procentese et al. (2017) performed hydrolysis with the same
enzyme after treatment of waste lettuce leaves with DESs. The time required for the
completion of hydrolysis of the pretreated biomass was 9 h. Conversely, when the
pretreatment temperature was higher, the higher fraction of monomers was obtained
after enzymatic hydrolysis. These researchers also used the enzyme hydrolysate of
the pretreated lettuce in the batch culture of Clostridium acetobutylicum DSMZ 792.
Complete consumption of the sugars was found after 60 h.
Kumar et al. (2016) studied NADES-lactic acid/choline chloride at molar ratio of
5:1. Maximum lignin of 68 ± 4 mg g−1 from the rice straw biomass was obtained.
Subsequent enzymatic hydrolysis of the residual holocellulose enriched biomass
showed maximum reducing sugars of 333 ± 11 mg g−1 with a saccharification
efficiency of 36.0 ± 3.2% in 24 h at 10% solid loading.
Liu et al. (2019) used cellulases and β-glucosidases for the hydrolysis of wheat
straw pretreated with DES. About 89% of cellulose and 71% of xylan were
hydrolyzed.
Fang et al. (2017) suggested a prior treatment using hydrothermal processing
for the date palm residues before treatment with DES. Hydrothermal pretreatment
showed more efficient hydrolysis using Cellic CTec2. Glucan conversion to glucose
increased by 1.7 times as a result of the pretreatment. But cellulose crystallinity was
not affected by the pretreatment. The operating and the investment cost, however,
remain a challenge for the process.
Chen et al. (2018) treated the pretreated switchgrass with CTec2 and HTec2
enzymes. They obtained 206.5 g/L glucose and 34.7 g/L xylose with 86.2% glucose
yield within 48 h. About 90.2 g/L 2,3-butanediol without extra sugar addition was
obtained.
Wan and Mun (2018) conducted a subsequent hydrolysis step after treating the
sago waste with DESs. The hydrolysis was performed at 50 °C and 100 rpm for 48 h.
Maximum glucose yield was achieved using ChCl-urea as 5.2 mg/mL.
Higher delignification and enzymatic hydrolysis yields were observed by Zhang
et al. (2016) when the pretreatment temperature was increased from 70 to 110 °C,
but the increase in hydrolysis yields was insignificant after reaching 90 °C. It was
77.8–79.7%. Usually, enzymatic digestibility showed an improvement when the
temperature was increased from 80 to 150 °C.
60 5 Compatibility of DES with Enzymes and Microorganisms

Acidity of DESs is found to play an important role in pretreatment of biomass

and also the efficiency of delignification. The yield is generally found to improve
with increasing acidity. When a synthetic blend of cellulose and lignin was used as
a substrate, the solubility of lignin improved when the acid content of ChCl:lactic
acid was increased from a molar ratio of 1:1 to 1:9 (Francisco et al. 2012). With rice
straw, Kumar et al. (2016) obtained higher lignin solubilization of 51 to 60% with
higher acid ratio from 1:2 to 1:5, respectively.
When the molar ratio of ChCl:lactic acid was increased from 2:1 to 15:1, the
lignin extraction (64.7–93.1%) of corncob improved (Zhang et al. 2016). However, no
appreciable improvement in enzymatic hydrolysis was seen (79.1–83.5%). Further-
more, 70% removal of lignin from corncob was enough for obtaining the optimum
hydrolysis yield. This value of 70% for lignin removal is in agreement with the
finding that about 65 to 70% of lignin in biomass can be easily removed if redeposi-
tion of lignin is avoided (Bhagia et al. 2016). Furthermore, earlier research showed
that complete removal of lignin is not essential for obtaining improved enzyme
hydrolysis (Fu et al. 2010; Hou et al. 2012).
Pretreatment with liquid hot water followed by pretreatment with ChCl:glycerol
at 70 °C for 6 h of date palm residues showed 1.7 times higher enzymatic digestibility
in comparison to liquid hot water pretreatment only (Fang et al. 2017). No substantial
increase in hydrolysis with ChCl:glycerol pretreatment of date palm residues was
observed. Removal of lignin and xylan by DES increased the enzymatic digestibility
rather than reducing the crystallinity of cellulose.
Kim et al. (2018) showed that novel DESs prepared from lignin phenolics obtained
from biomass were as efficient as other DESs produced with malonic acid, oxalic acid,
levulinic acid, ethylene glycol, glycerol, urea in improving the enzymatic hydrolysis
of switchgrass. The maximum glucose yields of 85.7% were obtained with ChCl:p-
coumaric acid and 79.8% with ChCl:vanillin, whereas the lowest glucose yield (32%)
was obtained with ChCl:4- hydroxybenzyl alcohol as no extensive delignification
was achieved after pretreatment with it. Therefore, enzyme accessibility reduced
considerably.
“The two most popular process designs for biomass to ethanol production are
pretreatment followed by separate hydrolysis and fermentation (SHF), or simul-
taneous saccharification (hydrolysis) and fermentation (SSF). If the pretreatment
solvent is toxic to enzymes and microbes, it will be needed to be removed from
pretreated solids by extensive washing or by other methods for obtaining high
yields by SHF or SSF. In contrast, some studies that used ionic liquid pretreat-
ment performed a one-pot process in which the three stages are carried out in the
same vessel. Thus, biocompatibility is critical concern for one-pot processes where
pretreatment slurry undergoes hydrolysis and fermentation” (Satlewal et al. 2018).
As several ionic liquids cause high enzyme inhibition and cytotoxicity, Liszka
et al. (2016) developed IL systems for addressing these issues. By the use of aqueous
choline-based ILs, a single-pot process design consisting of pretreatment, enzymatic
hydrolysis and fermentation was made possible. Almost 75% ethanol yield and more
than 40 g/l ethanol from fed batch process at high 30 to 34% solid loading of corn
stover but at higher enzyme loading of 20 mg/g glucan was obtained (Xu et al. 2016).
5 Compatibility of DES with Enzymes and Microorganisms 61

The constituents of DES, like choline chloride and urea, are found to inactivate
proteins (Gorke et al. 2008). But when combined to form a DES, inactivation can
be reduced by many-fold. This was seen in the case of transesterification catalyzed
by lipase enzymes where conversions with certain types of DESs such as choline
chloride:(glycerol or urea) were found to be comparable to that in toluene showing
very good stability of certain lipase enzymes in DES.
The effect of DES on cellulase activity has been studied. Gunny et al. (2015)
treated cellulases from Aspergillus sp. with DESs based on choline chloride
containing glycerol or ethylene glycol or malonic acid as the hydrogen bond donors
(HBD) in ratio of 1:2 for 48 h. About10% reduction in filter paper activity after 48 h
in control was seen, while 60% loss of filter paper activity was seen within 24 h in the
presence of DES containing malonic acid (Fig. 5.1). On the other hand, only slight
reduction in cellulase activity was seen with glycerol or ethylene glycol containing
DES. Hydrolysis of Avicel with cellulases resulted in only slight reduction of ~ 1 mM
in the glucose concentration with 10% v/v choline chloride DES having glycerol or
ethylene glycol. Malonic acid is very much inhibitory to cellulases in comparison to
the other two HBDs (glycerol or ethylene glycol).
Wahlstrom et al. (2016) examined the feasibility of a single-pot biomass decon-
struction process. The effect of high concentrations of DESs on enzyme activity and
hydrolysis yields was studied. Cellobiohydrolase Cel7A, endoglucanases Cel5A and
Cel7B, and xylanase Xyn11 from T. reesei were purified and examined individually
in three DESs—choline chloride:boric acid (5:2), choline chloride:glycerol (1:1) and
betaine:glycerol (1:1) at 85% concentration at pH 5.0 in citrate buffer and at 50 °C.
1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) which is a cellulose solubilizing
ionic liquid was also used. This ionic liquid was found to be extremely inhibitory
as the activity of all enzymes reduced to almost zero in about 4 h, whereas choline
chloride:glycerol (1:1) slightly inhibited the enzymes. This might be expected as
glycerol stabilizes the enzyme (Agrawal et al. 2017). The betaine:glycerol (1:1) stabi-
lized the enzymes after an initial (24 h to 72 h) reduction in activity. The remaining
enzyme activity of the two endoglucanases was substantially higher after 144 h in
betaine:glycerol than in buffered solution without using any DES. On the other hand,
choline chloride: boric acid (5:2) was found to be extremely inhibitory among all
of the DESs studied. Enzymatic activity was completely lost within 48 h. Both the
glycerol component and the hydrogen bond acceptor played an important role in
affecting the enzyme stability. Betaine was found to be more enzyme-compatible in
comparison to choline chloride (Wahlstrom et al. 2016). For increasing the tolerance
of such unconventional solvents, enzymes can be engineered.
A high-throughput assay which was based on a fluorescent cellobiose substrate
for directed evolution of DES tolerant endoglucanase (CelA2) was developed by
Lehmann et al. (2012). Cellulase variants which showed 23-fold more cellulolytic
activity in high ionic strength mediums like DES, ionic liquid or NaCl because of
activation of a salt bridge were found (Lehmann et al. 2014).
Only few studies have been conducted on compatibility of DESs with microor-
ganisms. Hayyan et al. (2013c) conducted filter paper diffusion assay for 24 h on
Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus
62 5 Compatibility of DES with Enzymes and Microorganisms

Fig. 5.1 Cellulase activity in the presence of different DESs (HBA as choline chloride and HBD
as ethylene glycol, glycerol or malonic acid at various concentrations after a 24 h, and b 48 h
incubation times. Gunny et al (2015). Reproduced with permission

aureus for toxicity caused by choline chloride based DESs containing HBDs as
glycerol, ethylene glycol, triethylene glycol and urea, and also the pure components
forming these DES. None of the bacteria were found to be inhibited by the four DES
or their pure components.
Hayyan et al. (2013b) also studied phosphonium-based DES (methyltriph-
enylphosphonium bromide) with glycerol, ethylene glycol and triethylene glycol for
5 Compatibility of DES with Enzymes and Microorganisms 63

inhibition using the filter paper diffusion assay for 24 h on Escherichia coli, Bacillus
subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. DESs containing
ethylene glycol and triethylene glycol HBDs inhibited all the four bacteria. Only
with Pseudomonas aeruginosa, zone of inhibition was observed with DES containing
glycerol HBD.
Whole-cell biocatalysis with the use of baker’s yeast was demonstrated in different
mixtures of water with DESs—choline chloride/glycerol, 1:2 mol/mol (Maugeri
and Dominguez De Maria 2014). Enantioselective ketone reduction was seen for
longer reaction times (more than 200 h). This suggests that the whole cells remain
stable in these neoteric solvents. By varying the amount of the DES, a complete
inversion of enantioselectivity was seen, from about 95% enantiomeric excess (S) in
pure water to about 95% enantiomeric excess (R) in the pure DES. Probably, some
(S)-oxidoreductases present in baker’s yeast are found to be inhibited by DESs.
These studies show that certain types of DESs are compatible with enzymes,
bacteria and yeast. But, there are several reports which show that higher concentration
of DESs are toxic to enzymes, bacteria and yeast. For single-pot processes, it may be
advantageous to dilute the pretreatment slurry to a point at which the DESs do not
significantly affect sugar yields. More research work is required in this area (Satlewal
et al. 2018).

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