Renewable Energy 177 (2021) 259e267

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Process optimization for deep eutectic solvent pretreatment and

enzymatic hydrolysis of sugar cane bagasse for cellulosic ethanol
fermentation
Yao Liu a, Xiaojie Zheng a, Shunhui Tao a, Lei Hu a, Xiaodong Zhang a, Xiaoqing Lin a, b, c, *
a
School of Chemical Engineering and Light Industry, Guangdong University of Technology, No. 100 Waihuan Xi Road, Panyu District, Guangzhou, 510006,
People's Republic of China
b
Guangdong Provincial Key Laboratory of Plant Resources Biorefinery, Guangdong University of Technology, Guangzhou, 510006, People's Republic of China
c
Guangzhou Key Laboratory of Clean Transportation Energy Chemistry, Guangdong University of Technology, Guangzhou, 510006, People's Republic of
China

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

Article history: This study investigated cellulosic ethanol production from sugar cane bagasse (SCB) pretreated by
Received 15 January 2021 triethylbenzyl ammonium chloride/lactic acid (TEBAC/LA) deep eutectic solvent (DES). The results
Received in revised form showed that the pretreatment of SCB with TEBAC/LA DES at 120
C for 4 h with 1:15 (solid to liquid ratio)
4 May 2021
resulted in the best cellulose digestibility (88.23 ± 1.24%), which was approximately 228% higher than
Accepted 24 May 2021
Available online 1 June 2021
that of untreated SCB. Furthermore, the maximum cellulosic ethanol concentration of 16.84 g/L was
achieved using glucose (36.06 g/L) and xylose (7.36 g/L). Moreover, the ethanol productivity and yield
were 0.70 g/(L$h) and 0.42 g/g fermentable sugar, respectively. The efficient bioconversion was ascribed
Keywords:
Cellulosic ethanol
to the remarkable delignification (88.72 ± 1.63%), xylan removal (73.93 ± 0.17%), along with optimum
Deep eutectic solvent cellulose recovery (95.89 ± 1.54%). Importantly, the enzymatic hydrolysis digestibility remained un-
Pretreatment changed after 5 times DES recycling process. Overall, it also provided an insight into the efficient SCB
Sugar cane bagasse biorefinery of DES systems.
© 2021 Elsevier Ltd. All rights reserved.

1. Introduction Sugar cane is an important crop cultivated in southern China,

The depletion of fossil fuels and several serious environmental
burdens caused by their utilization has gained increasing concern
worldwide [1e3]. Seeking an environment-friendly and pollution-
free alternative to fossil fuels has become an important research
topic in the field of biorefinery [4]. The conversion of sustainable
agro-industrial and forest waste into high value-added platform
chemicals or biofuels is an ideal way to reduce the environmental
pollution and the destruction of microorganisms in the soil caused
by direct combustion of agricultural residues [5,6]. Among the
suggested biofuels, cellulosic ethanol is considered as the potential
biofuel to power automobiles because it could avoid the conflict
between raw material and food production caused by the usage of
starchy grains to produce first-generation ethanol [7,8].
* Corresponding author. School of Chemical Engineering and Light Industry,
Guangdong University of Technology, No. 100 Waihuan Xi Road, Panyu District,
Guangzhou, 510006, People's Republic of China.
E-mail address: linxiaoqing@gdut.edu.cn (X. Lin).
which is the main raw source for sugar industry in China [1,9]. The
annual production of sugar cane in southern China reaches 70
million tons, resulting in approximately 20 million tons of sugar
cane bagasse (SCB) [10]. Currently, about 50% of SCB is directly
burnt as energy within the sugar factory; however, the utilization
rate is very low due to the high moisture content of SCB. In addition,
simple open-air centralized stacking of SCB will release toxic and
harmful substances in the humid and hot environments, which
directly cause air and soil pollution, and even enters the surface and
groundwater through infiltration and leaching, causing water
hypoxia and eutrophication. Therefore, the bioconversion of the
remaining accumulated SCB into cellulosic ethanol may have sus-
tainable economic and important strategic benefits [11,12].
Compared with straw agricultural waste, SCB possesses more
abundant cellulose and hemicellulose, containing 35e50% cellu-
lose, 20e30% hemicellulose, and 20e25% lignin [13]. Nevertheless,
the existence of lignin in the SCB could form a physical barrier that
limits the accessibility of enzymes to cellulose [14e16]. Thus, it is an
urgent need to develop a suitable pretreatment method to remove

https://doi.org/10.1016/j.renene.2021.05.131
0960-1481/© 2021 Elsevier Ltd. All rights reserved.
Y. Liu, X. Zheng, S. Tao et al. Renewable Energy 177 (2021) 259e267

lignin and disrupt the recalcitrance of SCB cell walls and improve
the accessibility of enzymes [17,18]. In this regard, various pre-
treatment methods, such as ball milling [19], laccase [20], steam
explosion [21], supercritical fluids [22], liquid hot water [23], dilute
acid [24], alkaline [25], organic solvents [26] and ionic liquids [27]
have been tested to overcome the recalcitrance of SCB and to
improve cellulose and hemicellulose digestibility. Although the
yields of glucose and xylose from pretreated SCB using liquid hot
water (LHW) combined with disk milling (DM) were
0.432 ± 0.009 g/g and 0.125 g/g dry SCB, respectively [28], this
pretreatment method usually required high pretreatment tem-
perature (160e200
C). Furthermore, acid and liquid hot water
pretreatments resulted in the formation of several inhibitors along
with fermentable sugars, including furans derivatives (furfural, 2-
furoic acid, and 5-hydroxymethylfurfural), aliphatic carboxylic
acids (acetic acid, formic acid, and levulinic acid), and phenolic
compounds (benzoic acid, cinnamic acid, coniferyl aldehyde, ferulic
acid, and 4-hydroxy-benzoic acid) [29,30]. Recently, ionic liquids
(ILs) were used to significantly improve the yields of fermentable
sugars and bioethanol [31,32]. Nevertheless, one issue for ILs pre-
treatment used in the industrial scale application for biomass
pretreatment is the high cost; another significant issue is that the
degraded products in the pretreatment liquor is hard to be recov-
ered or separated [33,34]. More recently, deep eutectic solvents
(DESs) [35] have been used as a promising alternative to ILs for
biomass pretreatment due to their excellent performance of low
cost, green, biocompatible, environmental friendliness, easy to
synthesize and recycle [36e38]. To date, there has been no current
report on the biorefinery of SCB using a DES system.
Previous study by our group demonstrated the use of TEBAC/LA
(1:9) DES to remove 79.73 ± 0.93% lignin from wheat straw and
then 0.550 g of sugar per gram of wheat straw was obtained [18].
Lin et al. used TEBAC/LA (1:7) DES to pretreat corn stover at 120
C
for 90 min, the removal of xylan and lignin reached 57.58% and
61.40%, respectively. Importantly, the cellulose recovery of DES
treated corn stover was 99.24% [39]. Xu et al. adopted TEBAC/LA
(1:2) DES to disturb the recalcitrance of corncobs at 100e140
C for
120 min; the highest glucose yield reached 89.2% [40]. Considering
that TEBAC/LA DES possesses an excellent removal rate of lignin
and hemicellulose and could retain a high recovery rate of cellulose,
herein, for the first time, we used TEBAC/LA based DES as an ideal
solvent to economically convert SCB into fermentable sugar and
produce cellulosic ethanol. Additionally, the effect of temperature
on the recovery and removal efficiency of cellulose, xylan, and
lignin was systematically investigated. The raw SCB and cellulose-
rich solids were characterized by scanning electron microscope
2. Materials and methods
2.1. Materials
SCB was generously provided by Guangxi Guitang Group Co.,
Ltd. (Guangxi, China). The main composition of SCB contains
37.72 ± 0.69% cellulose, 22.95 ± 0.42% xylan, and 22.34 ± 0.09%
lignin (dry weight basis) [41]. The cellulase 1.5 L (54 FPU/mL) and
AMG300L b-glucosidase (94 CBU/mL) were purchased from Novo-
zymes (China) Investment Co., Ltd. The alcohol-fermentation active
dry yeast was kindly supplied by Angel Yeast, Co. Ltd. (Hubei,
China), which was activated at a ratio of 1:50 (w/v) using 2 wt%
glucose solution. TEBAC (98% purity), and LA (purity85%) were
obtained from Macklin Biochemical Co., Ltd. (Shanghai, China).
2.2. DES synthesis and pretreatment of SCB
The detailed preparation of TEBAC/LA based DES and pretreat-
ment process was carried out according to our previous work [18].
In a typical run, the SCB pretreatment was carried out in a three-
neck bottle with 2.0 g of SCB and 30.0 g of TEBAC/LA based DES
at three various pretreatment temperatures (100, 120, and 140
C)
for 4 h at a stirring speed of 200 rpm. The purified DES was
repeatedly used in the pretreatment of raw SCB at 120
C for 4 h,
and the recycle number of TEBAC/LA DES was 5 times.
2.3. Enzymatic hydrolysis
Following the above pretreatment, the enzymatic hydrolysis
tests were conducted in a 150 mL Erlenmeyer flask containing 4.0 g
of SCB (untreated or pretreated) and 50 mL of citrate buffer (50 mM,
pH ¼ 4.8). The dosage of cellulase used was 15, 25, 35 FPU/g dry
biomass, respectively. The dosage of b-glucosidase was kept at 82
CBU/g dry biomass. The enzymatic hydrolysis process was placed at
50
C in a ZQZY-80BS constant temperature incubator shaker
(Shanghai Zhichu Instrument Co., Ltd., Shanghai, China) for 108 h
with a shaking speed of 200 rpm. The samples were withdrawn
after 12, 24, 36, 48, 60, 72, 84, 96, and 108 h and terminated after
inactivation in boiling water for 5 min. The supernatant was
centrifuged at the speed of 10000 rpm for 5 min and diluted twice
by mobile phase, and then filtered by 0.45 mm membrane to analyze
the fermentable sugar content by high performance liquid chro-
matography (HPLC). The polysaccharide digestibility and sugar
yields were calculated according to our previous study [18]:

ðglucose amount  0:9 þ Cellobiose  0:95Þ

Cellulose digestibility ¼ (1)
cellulose amount in pretreated SCB

(SEM), Fourier transform infrared (FT-IR) spectrometry, and X-ray

diffraction (XRD). Moreover, the effect of enzyme loading on
polysaccharide digestibility and sugar yields during the enzymatic ðhemicellulose amount  0:88Þ
hydrolysis process was also studied. At last, the production of
Xylan digestibility ¼
hemicellulose amount in pretreated SCB
cellulosic ethanol from untreated and treated SCB using TEBAC/LA (2)
based DES and ball-milling pretreatment were evaluated.

ðglucose amount  0:9Þ

Glucose yield ¼ (3)
cellulose amount in untreated SCB

260
Y. Liu, X. Zheng, S. Tao et al. Renewable Energy 177 (2021) 259e267

Table 2
ðxylose amount  0:88Þ Effect of different pretreatment temperatures on enzymatic hydrolysis of SCB.
Xylose yield ¼ (4) (Enzymatic hydrolysis conditions: pretreated SCB: 0.4 g; citrate buffer (50 mM,
xylan amount in untreated SCB
pH ¼ 4.8): 20 mL; Cellulase: 35 FPU/g substrate; b-glucosidase: 82 CBU/g substrate;
enzymatic hydrolysis temperature: 50
C; enzymatic hydrolysis time: 72 h; rotating
Fermented sugar content ¼ glucose content þ xylose content speed: 150 rpm).

(5) Temperature(oC) Enzymatic hydrolysis (%) Sugar yield (%)

Cellulose Xylan Glucose Xylose

where 0.90 and 0.88 are the conversion factors used to calculate the
digestibility of cellulose and xylan into glucose and xylose, Untreated 38.76 ± 2.10 12.23 ± 1.32 38.76 ± 2.10 12.23 ± 1.32
100 74.98 ± 1.17 65.92 ± 0.94 70.79 ± 1.19 24.97 ± 0.78
respectively. 120 88.23 ± 1.24 78.96 ± 1.41 71.42 ± 1.43 20.59 ± 1.09
140 85.40 ± 1.07 99.38 ± 1.03 64.27 ± 0.79 10.46 ± 1.47
2.4. Cellulosic ethanol fermentation

Fermentation of the cellulose-rich SCB solid after TEBAC/LA DES

pretreatment was carried out using separate hydrolysis and
fermentation (SHF). For the inoculum process, an active culture
(50 mL) containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L
glucose, 1.5 g/L KH2PO4, 4 g/L (NH4)2SO4, and 0.5 g/L MgSO4 was
prepared. Inoculum was grown on a rotary shaker for 24 h at 33
C
and 150 rpm. The SHF experiment was carried out in 100 mL
Erlenmeyer flasks with 50 mL of medium composed of untreated
and treated SCB enzymatic hydrolysate. The medium was supple-
mented with 3 g/L yeast extract, 4 g/L peptone, 3 g/L KH2PO4, 4 g/L
(NH4)2SO4, 0.5 g/L MgSO4, 0.05 g/L FeSO4$7H2O, and 0.05 g/L
ZnSO4$7H2O. After sterilization at 121
C for 15 min, the 10% V/V
Angel yeast inoculum was transferred aseptically into the fermen-
tation medium. Fermentation was performed at 33
C and 150 rpm
for 36 h using a ZQZY-80BS constant temperature incubator shaker
(Shanghai Zhichu Instrument Co., Ltd., Shanghai, China). The
Samples were taken at 0, 3, 6, 9, 12, 24, 28, and 36 h for sugar
consumption and cellulosic ethanol analysis.

2.5. Analytical methods

The contents of SCB including cellulose, xylan, and lignin before
and after pretreatment were determined by NREL method [41]. The
concentrations of glucose, xylose, cellobiose, and ethanol were
monitored by HPLC (Agilent Technologies 1200 Series, USA)
equipped with a refractive index detector (RID, Agilent Technolo-
gies 1260 Infinity II). An Aminex HPX-87H anion exchange column
(300 mm  7.8 mm, Bio-Rad Laboratories, USA) was used and the
column temperature was maintained at 55
C. Diluted sulfuric acid
(5 mM) was used as the mobile phase and the flow rate was 0.5 mL/
min. The volume of sample injection was 10 mL.
3. Results and discussion
3.1. Effect of pretreatment temperature on SCB composition
Table 1 shows the results of the SCB pretreated by DES at
different pretreatment temperatures ranging from 100 to 140
C for
4 h at the ratio of 1: 15 (solid to liquid). As can be seen from Table 1,
the recovery of solids was gradually decreased from 63.98 ± 0.04%
to 48.49 ± 0.66% with the reaction temperature increased from 100
Fig. 1. XRD patterns of untreated and treated SCB by DES at different pretreatment
temperatures.
to 140
C. Moreover, compared with the untreated SCB, the lignin
content in the treated SCB decreased from 22.34 ± 0.09% to
2.66 ± 0.72% with the temperature rise to 140
C. While the xylanse
content in the treated SCB was decreased from 22.95 ± 0.42% to
4.89 ± 1.03%. These above results suggested that high temperature
may enhance the bond fracture among lignin, cellulose, and xylan
[42], thereby facilitating the selective extraction of lignin, which
will enhance the enzymatic hydrolysis efficiency [43]. Similar re-
sults were also reported by a prior study using choline chloride/
glycerol (1:2), choline chloride/urea (1:2), choline chloride/imid-
azole (3:7), choline chloride/lactic acid (1:10) and choline chloride/
lactic acid (1:2, 1:4, 1:8) to pretreat sweet corncob, poplar, and
bamboo residues [44e46]. However, it should be noted that the
cellulose content in the treated SCB was increased from
37.72 ± 0.69% (untreated SCB) to 70.67 ± 2.12% (treated SCB at
140
C). Although the higher temperature (140
C) led to higher
xylan removal (89.48 ± 1.24%) and lignin removal (94.13 ± 1.14%),

Table 1
Solid recovery, cellulose, xylan and total lignin content as well as cellulose recovery and xylan and lignin removal after DES pretreatment. (Experiment condition: SCB: 2.0 g;
DES: 30.0 g; time: 4 h).

Pretreatment Residue recovery (%) Cellulose (%) Xylan (%) Total lignina (%) Cellulose recovery(%) Xylan removal(%) Lignin removal (%)

Untreated SCB 100 37.72 ± 0.69 22.95 ± 0.42 22.34 ± 0.09 e e e

TEBAC/LA 100
C 63.98 ± 0.04 55.66 ± 2.01 13.59 ± 3.75 11.46 ± 0.97 94.42 ± 2.11 62.12 ± 2.47 67.19 ± 1.33
TEBAC/LA 120
C 53.23 ± 0.44 68.74 ± 0.62 11.37 ± 0.04 4.79 ± 1.78 95.89 ± 1.54 73.93 ± 0.17 88.72 ± 1.63
TEBAC/LA 140
C 48.49 ± 0.66 70.67 ± 2.12 4.89 ± 1.03 2.66 ± 0.72 92.59 ± 1.48 89.48 ± 1.24 94.13 ± 1.14
a
Total lignin is sum of acid soluble and insoluble lignin.

261
Y. Liu, X. Zheng, S. Tao et al.
Fig. 2. FT-IR spectrum of untreated and treated SCB by DES at different pretreatment
temperatures.
the lower temperatures (120
C) resulted in higher cellulose re-
covery (95.89 ± 1.54%).
To further evaluate the pretreatment performance, the enzy-
matic digestibility of the untreated and treated SCB at various
temperatures was compared and the results are listed in Table 2.
Clearly, untreated SCB displays a low cellulose enzymatic hydrolysis
conversion rate of 38.76 ± 2.10%, mainly due to the presence of
lignin and xylan in SCB warped cellulose, which severely hinders
enzymatic hydrolysis [47]. By comparison, the cellulose conversion
rates of SCB pretreated by DES were dramatically increased to
88.23 ± 1.24% at 120
C, which was approximately 228% higher than
that of the untreated SCB. The enzymatic conversion rate of xylan
was also increased from 12.23 ± 1.32% (untreated SBC) to
78.96 ± 1.41% (120
C). Generally, the existence of lignin has a
negative impact on the yield of enzymatic hydrolysis [48,49]. Lignin
could adsorb enzyme via hydrophobic, electrostatic, and hydrogen
bonding interactions and the release of soluble lignin-derived
compounds may act as toxic inhibitors of enzyme [50]. Although
the digestibility of glucan and xylan in treated SCB by DES at 140
C
reach 85.40 ± 1.07% and 99.38 ± 1.03%, respectively, the cellulose
Fig. 3. SEM pictures of SCB before and after TEBAC/LA based DES pretreatment at various temperatures.  2000: A, C, E and G;  500: B, D, F and H.
262
Renewable Energy 177 (2021) 259e267
recovery was only 92.59 ± 1.48%. In contrast, when the pretreat-
ment temperature was 120
C, the cellulose conversion rate of
treated SCB could reach 88.237 ± 1.24%, meanwhile, the cellulose
recovery reaches 95.89 ± 1.54%. Furthermore, the less poly-
saccharide loss will enhance the yield of ethanol during the sub-
sequent fermentation process. Taken solid recovery, cellulose
recovery, xylan, and lignin removal together, DES treated SCB per-
formed best at 120
C, which was selected as the optimal reaction
temperature for the pretreatment process.
3.2. Characterization of solid fraction
Crystallinity of cellulosic materials is an important indicator for
evaluating the efficiency of enzymatic hydrolysis. Therefore, the
comparison of the CrI of untreated and DES treated SCB at different
temperatures were used to evaluate the enzymatic performance.
Fig. 1 shows the XRD curves of untreated and treated SCB at
different pretreatment temperatures using TEBAC/LA (1:9). Clearly,
the typical diffraction peaks of 16.2
, 21.8
and 34.8
were observed
in both untreated and treated SCB, revealing the cellulose I crystal
type of SCB [51,52]. In addition, the position of the characteristic
peaks of type I cellulose structure did not change after DES pre-
treatment. Moreover, the value of CrI of untreated raw SCB was
50.96% (see Fig. 1), while the CrI of the DES treated SCB was
increased to 69.47% after pretreatment at 140
C, which is mainly
attributed to the fact that the amorphous hemicellulose and lignin
was removed [44]. These above results are consistent with the
composition analysis of the untreated and treated SCB (see Table 1).
Furthermore, the values of CrI in the treated SCB were increased
from 65.62% (100
C) to 69.47% (140
C) with the increasing of
pretreatment temperature, suggesting that higher pretreatment
temperature possesses a strong ability to partially disrupt and
decrease the amorphous regions of cellulose, and resulted in the
exposure of crystalline cellulose and higher crystallinity [53e55].
FT-IR spectroscopy was used to identify the chemical structural
changes of SCB before and after DES pretreatment at different
temperatures. As displayed in Fig. 2, the stretching vibration peak at
890 cm1 (b-(1,4)-glycosidic linkage) still exists, suggesting that the
cellulose structure in the treated SCB was not significantly affected.
This is mainly because DES could stabilize the cellulose system by
hydrogen bond interaction and avoid cellulose loss [56]. However,
as the pretreatment temperature increased to 140
C, the peak at
890 cm1 slightly decreased. The main explanation was that some
Y. Liu, X. Zheng, S. Tao et al.
Fig. 4. Enzymatic saccharification of DES pretreated SCB under different enzyme loadings at a solids content of 8% (w/v) in citric buffer solution for 108 h (4.0 g solid, 50 mL Tris,
50
C, pH ¼ 4.8).
cellulose was damaged at high reaction temperature and reduced
the cellulose content, which is consistent with the results listed in
Table 1. It is worth noting that obvious changes were observed for
the peaks associated with hemicellulose and lignin. In comparison
with untreated SCB, the notable reduction of the peaks at
1033 cm1 (CeO bond stretching vibration in cellulose, hemicel-
lulose, and lignin) was found [57], verifying again that the DES
could significantly remove the hemicellulose and lignin from the
SCB (see Table 1). Moreover, compared with the absorption peaks at
1594 cm1 (C]C bond), 1468 cm1 (eCH2e bond) and 1240 cm1
(CeOeC bond between hemicellulose and lignin) in the untreated
SCB, the lignin aromatic skeletal vibrations were significantly
weakened, especially after DES pretreatment at 140
C. This result
could be explained by the depolymerization of lignin and hemi-
cellulose after DES pretreatment at different temperatures [58].
Therefore, based on the above results from the FT-IR spectra, the
pretreatment mechanism of SCB using DES was concluded that DES
could form hydrogen bonds with the components of hemicellulose
and lignin. As a result of effective delignification, the enzyme could
efficiently penetrate the cellulose and remaining hemicellulose,
resulting in higher hydrolysis digestibility of cellulose and hemi-
cellulose (see Table 2).
Fig. 3 demonstrates the morphology changes of untreated and
DES pretreated SCB. Clearly, the surface of untreated SCB exhibited
a rather smooth and dense outer surface layer with a high degree of
virgin fibrous structure, which hindered the penetration of
263
Renewable Energy 177 (2021) 259e267
enzymes. However, the shape of the fibrils in the treated SCB by
DES at different temperatures became disordered and rough,
revealing that DES efficiently removed the hemicellulose and lignin
from the outer layer of lignocellulose, and harshly stripped the
lignin layer. Specially, the structure of DES treated SCB at 140
C
suffered the most drastic rupture, implying that high pretreatment
temperatures facilitated the removal of lignin and hemicellulose.
Besides, most of the hemicellulose and lignin were removed, more
cellulose is exposed outside the surface and allowed enzymes to
penetrate, which can significantly improve the enzyme digestibility
efficiency.
3.3. Optimization of enzyme loading
The amount of enzyme occupies a major cost in ethanol
fermentation, so it is necessary to optimize the loading of enzymes.
Samples pretreated with DES at 120
C for 4 h were selected for
enzymatic hydrolysis. The substrate content was 8%, and cellulase
(Cellulase 1.5 L, Novozymes) loading varied from 15 to 35 FPU/g
solid, and the b-glucosidase (AMG300L, Novozymes) loading was
set at 82 CBU/g solid. The effect of enzyme loading on enzyme di-
gestibility is shown in Fig. 4. As can be seen, when the cellulase
loading increased from 15 to 35 FPU/g, the cellulose conversion
increased from 72.27 ± 1.78% to 82.79 ± 1.74% (See Fig. 4 A). The
conversion rate of xylan remained unchanged at about
74.47 ± 2.23%. This is mainly because the added loadings of b-
Y. Liu, X. Zheng, S. Tao et al.
Fig. 5. A) Enzymatic saccharification of pretreated SCB under various conditions at 8%
solid loading; B)SHF ethanol growth and sugar consumption.
glucosidase (AMG300L, Novozymes) are consistent. The final con-
centrations of fermentable sugar were 40.57, 44.68, and 47.04 g/L
for 15, 25 and 35 FPU/g enzyme cellulase loading, respectively.
Although increasing the amount of cellulase could effectively pro-
mote the cellulose conversion and the yield of total fermented
sugar, it will also increase the cost accordingly. As cellulase
increased to 25 FPU/g (substrate), the cellulose and xylan conver-
sion reached 77.93 ± 1.18% and 75.18 ± 3.12%, respectively, pro-
ducing 37.45 g/L of glucose and 7.77 g/L of xylose, which is only
2.49 g/L lower than the fermentable sugar content of 35 FPU/g
(substrate) cellulase loading. Therefore, 25 FPU/g cellulase and 82
CBU/g glucosidase were selected as the optimal enzyme loading to
improve the initial sugar accumulation.
3.4. Separate hydrolysis and fermentation (SHF) on DES treated SCB
As presented in Fig. 5 (A), DES pretreatment can significantly
increase the total fermentable sugar concentration during the
enzymatic hydrolysis process for 108 h, which gradually increased
to 44.09 g/L. Compared with untreated (18.64 g/L) and ball milling
264
Renewable Energy 177 (2021) 259e267
(20.87 g/L), the total fermentable sugar content of SCB after DES
pretreatment increased by approximately 237% and 211%, respec-
tively. This was attributed to the removal of most of the lignin after
DES pretreatment, which promoted the accessibility of enzymes
and released more sugar. SCB pretreated by ball milling could also
increase the enzymatic hydrolysis efficiency and release more
fermentable sugars. This may be because after ball milling, the SCB
has become more refined and increased the specific surface area.
However, ball milling pretreatment cannot significantly increase
the content of fermentable sugars. It should be ascribed to the
presence of lignin inhibits the effective contact between the
enzyme and cellulose. According to the findings mentioned above
and comparison with the other existing pretreatment methods
listed in Table 3, DES pretreatment of SCB used in the current work
was an efficient and cost-effective pretreatment method.
The fermentation of enzymatic hydrolysates of raw and pre-
treated SCB using ball milling and DES were conducted for 36 h
with alcohol-fermentation active dry yeast. The SHF ethanol
growth and sugar consumption were shown in Fig. 5 (B). After the
fermentation, the maximum ethanol concentrations attained from
untreated, ball milling, and DES pretreated SCB were 7.22 g/L,
8.57 g/L, and 16.84 g/L, respectively. The cellulosic ethanol pro-
ductivity reached 0.30, 0.36, and 0.70 g L1 h1, respectively.
Furthermore, the ethanol yields of untreated, ball-milling and DES
pretreated SCB were 0.39, 0.40, and 0.42 g/g. At the late stage of
fermentation, the sugar concentration remains almost constant;
this is because some cellobiose was not used in the enzymatic
hydrolysate. In summary, although both DES pretreatment and ball
milling pretreatment can improve the ethanol production from
SCB, DES can significantly increase ethanol production, which is a
cost-effective method of biomass processing and utilization.
3.5. Recovery and reuse of the DES
The TEBAC/LA DES was recovered and reused to evaluate its
repeatability during the SCB pretreatment process. The specific
regeneration flow chart is shown in Fig. 6. The water was rotary-
evaporated from the DES-water mixture at the end of each test,
and the DES was dried as before, then reused in a further five
successive pretreatment reactions. The enzymatic digestibility
analysis of the cellulose-rich substrate after five recycling cycles
were investigated and the results were displayed in Fig. 7. Clearly,
the cellulose digestibility of treated SCB was gradually decreased
with the increasing recycle of DES, from 88.23 ± 1.24% to
68.02 ± 2.41% after five recycling cycles. The reason for the decrease
in cellulose digestibility was because the carboxylic acids derived
by hemicellulose and cellulose decomposition and the phenolic
compounds produced by lignin decomposition during the DES
pretreatment [68]. Importantly, the cellulose digestibility of the
fifth recovered DES still reached 68.02 ± 2.41%, which is about 175%
higher than that of untreated SCB, revealing that DES recycling after
pretreatment not only improved the cellulose digestibility
(compared with untreated SCB), but also reduce chemicals con-
sumptions. Therefore, DES pretreated SCB provides a great poten-
tial in the cost reduction and recyclability of SCB biorefinery.
4. Conclusions
In this work, TEBAC/LA DES was used to pretreat SCB and pro-
duce cellulose-rich solid residues. As expected, the cellulose di-
gestibility of the treated SCB by DES at 120
C for 4 h reached
88.23 ± 1.24%, which was approximately 228% more than that of
untreated SCB. Additionally, the highest concentration of ethanol
was 16.84 g/L, and the ethanol yield and productivity reached
0.42 g/g fermentable sugar and 0.70 g/(L$h), respectively.
Y. Liu, X. Zheng, S. Tao et al. Renewable Energy 177 (2021) 259e267

Table 3
Comparative analysis of different pretreatment methods.

Method Pretreatment conditions Solid (%) Glucan (%) Xylan (%) Total lignin Enzyme dosage Enzymatic hydrolysis Reference
(%)
Conversion (%) Sugar yield (%)

BM 400 rpm, 60 min e 38.8 ± 0.66 23.5 ± 0.44 e 15 FPU FPase, e G: 78.7 ± 0.4 [59]
79.7 BGU b- X: 72.1 ± 0.3
glucosidase, 262.4 IU
CMCase,
576.3 IU xylanase,
1.39 IU b-xylosidase,
22.5 IU a-
Larabinofuranosidase.
UP UPþ2% NaOH, 50
C, 20 min e C: 44.61 H: 26.32 5.26 e e e [60]
SCP scCO2 187
C, 15.6 MPa, e e e e 15 FPU/g cellulose e 97.8 [61]
40 min þ1%, pH ¼ 11.5 H2O2,
333 K, 9 h
LHW LHW 200
C, 10 min þ DM 64.62 ± 0.31 41.68 ± 0.23 3.76 ± 0.19 11.37 ± 0.12 15 FPU/g substrate e 0.432 g/g dry [28]
0.054 mL/g substrate bagasse
Cellic® Htec2
SEP 8 min, 17 bar 77.6 ± 0.6 C: 52.8 ± 0.5 3.4 ± 0.1 29.4 ± 5.1 20 mg enzyme/g WS: 96 e [62]
glucan UW: 56


DA 1% H2SO4, 121 C, 150 min 60.30 ± 0.34 C: H: 31.98 ± 0.04 15 FPU/g cellulase and G: 60.80 G: 53.16; [63]
57.29 ± 0.01 6.57 ± 0.01 25 CBU/g b-
glucosidase
DA 1% H2SO4þ1% CH3COOH, 63.00 C: H: 32.96 ± 0.25 0.232 g cellulose/g C: 76 e [64]
190
C, 10 min 61.65 ± 0.50 2.79 ± 0.03 substrate
0.052 g b-glucosidase/
g substrate
AP 2% NaOH, 80
C, 120 min 73.2 63.5 ± 0.46 29.0 ± 0.81 9.7 ± 0.10 20 FPU/g cellulose Glucan: 74 [65]
ORG Ethanol þ H2SO4 (1.25%w/w), 87.18 ± 2.28 62.08 ± 1.03 8.71 ± 0.13 28.32 ± 0.57 15 FPU/g substrate e 20.87 ± 0.18 g [66]
175
C, 60 min 15 IU/g substrate glucose/100 g raw
material
IL [Ch][Lys], 90
C, 6 h 56.6 61.4 14.7 14.45 17.5 U mL1 cellulase e G: 80.7; [67]
X: 56.1


DES TEBAC/LA, 120 C, 4 h 53.23 ± 0.44 68.74 ± 0.62 11.37 ± 0.04 4.79 ± 1.78 25 FPU/g biomass C: 77.93 ± 1.18% G: 71.42 ± 1.43 This work
82 CBU/g biomass H: 75.18 ± 3.12% X: 20.59 ± 1.09

Abbreviations: BM: ball milling; UP: ultrasound pretreatment; SCP: supercritical carbon pretreatment; LHW: liquid hot water; DM: disk milling; SEP: steam explosion
pretreatment; DA: dilute Acid; AP: alkaline pretreatment; ORG: organosolv; IL: ionic Liquids; WS: washed; UW: unwashed; UP: ultrasound pretreatment; C: cellulose; H:
hemicellulose; G: glucose; X: xylose.

Fig. 6. The specific regeneration flow chart of TEBAC/LA DES.

265
Y. Liu, X. Zheng, S. Tao et al.
Fig. 7. Cellulose digestibility from recycled DES pretreated SCB. (Enzymatic hydrolysis
conditions: pretreated SCB: 0.4 g; citrate buffer (50 mM, pH ¼ 4.8): 20 mL; Cellulase:
35 FPU/g substrate; b-glucosidase: 82 CBU/g substrate; enzymatic hydrolysis temper-
ature: 50
C; enzymatic hydrolysis time: 72 h; rotating speed: 150 rpm).
Importantly, the enzymatic hydrolysis digestibility remained un-
changed after 5 times DES recycling process. Based on the
comprehensive analysis, DES can achieve efficient separation of
lignocellulosic biomass and conversion of ethanol.
CRediT authorship contribution statement
Yao Liu: Conceptualization, Methodology, Writing e original
draft. Xiaojie Zheng: Data curation, Formal analysis. Shunhui Tao:
Visualization, Investigation. Lei Hu: Visualization, Investigation.
Xiaodong Zhang: Visualization, Investigation. Xiaoqing Lin: Su-
pervision, Project administration, Methodology, Writing e review
& editing.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
X. Lin is very grateful to the financial support from the National
Natural Science Foundation of China (21978053, 51508547), the Key
Area R&D Program of Guangdong Province (2020B0101070001),
and the “One-Hundred Young Talents” Program of Guangdong
University of Technology (220413185). None of the authors have
any conflict of interest.
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