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Optimization of Liquid Hot Water Pretreatment and Fermentation for Ethanol

Production from Sugarcane Bagasse Using Saccharomyces cerevisiae

Article in Catalysts · April 2022

DOI: 10.3390/catal12050463

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 catalysts
Article
Optimization of Liquid Hot Water Pretreatment and
Fermentation for Ethanol Production from Sugarcane Bagasse
Using Saccharomyces cerevisiae
Punjarat Khongchamnan 1 , Nopparat Suriyachai 2,3 , Torpong Kreetachat 2 , Navadol Laosiripojana 4 ,
Khatiya Weerasai 4 , Verawat Champreda 3 , Kowit Suwannahong 5 , Chainarong Sakulthaew 6 ,
Chanat Chokejaroenrat 7 and Saksit Imman 2, *

1 School of Energy and Environment, University of Phayao, Tambon Maeka, Amphur Muang,
Phayao 56000, Thailand; punjarat-khongchamnan@hotmail.com
2 Integrated Biorefinery Excellence Center (IBC), School of Energy and Environment, University of Phayao,
Tambon Maeka, Amphur Muang, Phayao 56000, Thailand; nopparat.su@up.ac.th (N.S.);
torpong.kr@up.ac.th (T.K.)
3 BIOTEC-JGSEE Integrative Biorefinery Laboratory, National Center for Genetic Engineering and
Biotechnology, Innovation Cluster 2 Building, Thailand Science Park, Khlong Luang,
Pathumthani 12120, Thailand; verawat@biotec.or.th
4 The Joint Graduate School for Energy and Environment (JGSEE), King Mongkut’s University of Technology
Thonburi, Prachauthit Road, Bangmod, Bangkok 10140, Thailand; navadol_l@jgsee.kmutt.ac.th (N.L.);
k.weerasai@gmail.com (K.W.)
Citation: Khongchamnan, P.; 5 Department of Environmental Health, Faculty of Public Health, Burapha University,
Suriyachai, N.; Kreetachat, T.; Chonburi 20131, Thailand; kowit007@gmail.com
6 Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University,
Laosiripojana, N.; Weerasai, K.;
Champreda, V.; Suwannahong, K.; Bangkok 10900, Thailand; cvtcns@ku.ac.th
7 Department of Environmental Technology and Management, Faculty of Environment, Kasetsart University,
Sakulthaew, C.; Chokejaroenrat, C.;
Bangkok 10900, Thailand; chanat.c@ku.ac.th
Imman, S. Optimization of Liquid
* Correspondence: saksit.im@up.ac.th; Tel.: +66-8615-84845
Hot Water Pretreatment and
Fermentation for Ethanol Production
Abstract: Sugarcane bagasse can be considered a potential raw material in terms of quantity and
from Sugarcane Bagasse Using
Saccharomyces cerevisiae. Catalysts
quality for the production of alternative biofuels. In this research, liquid hot water (LHW) was
2022, 12, 463. https://doi.org/ studied as a pretreatment process to enhance the digestibility of pretreated material for further
10.3390/catal12050463 conversion into bioethanol. Different variables (temperature, residual time, and acid concentration)
were determined to predict the optimized condition. LHW pretreatment showed an impact on
Academic Editors: Maria Ricciardi,
the hemicellulose structure. The optimized condition at 160 ◦ C for 60 min with 0.050 M acid
Raffaele Cucciniello and Roberto
concentration reached the highest glucose yield of 96.86%. Scanning electron microscopy (SEM)
Esposito
showed conspicuous modification of the sugarcane bagasse structure. The effect of LHW pretreatment
Received: 22 March 2022 was also demonstrated by the changes in crystallinity and surface area analysis. FTIR techniques
Accepted: 14 April 2022
revealed the chemical structure changes of pretreated sugarcane bagasse. The prepared material was
Published: 21 April 2022
further converted into ethanol production with the maximized ethanol concentration of 19.9 g/L.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in Keywords: sugarcane bagasse; glucose yield; bioethanol-alcohol production; optimization; liquid hot
published maps and institutional affil- water pretreatment
iations.

1. Introduction
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
Lignocellulose biomass (LB) is predicted to be a substantial carbon-free energy source,
This article is an open access article
which can reduce carbon dioxide (CO2 ) emissions and atmospheric pollution. As a result,
distributed under the terms and it represents a viable option for limiting crude oil production, and can be used to produce
conditions of the Creative Commons bioenergy, chemicals, and biomaterials in industry for biorefinery products [1]. Bioethanol
Attribution (CC BY) license (https:// is a type of alcohol produced using the sugar alcohols from different biomass sources,
creativecommons.org/licenses/by/ mostly identified in starch crops such as sugar beet, corn biomass, rice grain, sugarcane
4.0/). bagasse, and wet sorghum [2]. Moreover, bioethanol sources include sucrose, starch, and

Catalysts 2022, 12, 463. https://doi.org/10.3390/catal12050463 https://www.mdpi.com/journal/catalysts

Catalysts 2022, 12, 463 2 of 11

lignocellulose material [3]. Today, a variety of bioproducts are produced according to

typical biorefinery processes (physical–chemical reaction, hydrolysis and fermentation) in
order to break down the biomass substrate into biofuels and biochemicals. An alternative
bioethanol is subsequently refined to blend with fuels for utilization in automobiles and
other vehicles. The fuel is typically 90 percent gasoline and 10 percent ethanol. Concerning
lignocellulosic resources, corn and sugarcane bagasse are reported to be the most widely
used to produce bioethanol [3].
Sugarcane bagasse is an agro-industrial biomass with significant energy resources,
which consists of agricultural lignocellulosic biomass. It is a low-cost, renewable, and
unique natural resource for renewable energy production or bioethanol production, which
can significantly reduce emissions [4]. Sugarcane bagasse is a low-cost agricultural waste
product. These biomass materials have a great deal of potential for the development of
useful products. Therefore, sugarcane bagasse characteristic data can contribute to the
development of sugarcane bagasse valorization in value-added applications. This presents
a huge challenge for industry and the environment. Typically, the major components of
lignocellulose biomass materials derived from agricultural waste residuals or economic
crops contain 40–50% cellulose content, 25–40% hemicellulose content, and 15–30% lignin
content depending on the biomass material [5]. In terms of structure, cellulose and hemi-
cellulose comprise polysaccharide structures [6]. In general, the cellulose structure is a
polysaccharide composed of linear glucan chains consisting of β-(1-4)-glycosidically linked
units, featuring numerous crystal structures of small molecules [7]. Bioethanol synthesis
requires the hydrolysis of lignocellulose biomass material for glucose release after the
pretreatment process to obtain optimal glucose yields.
A previous optimization study of enzymatic hydrolysis from sugarcane bagasse based
on organosolv pretreatment for glucose yield used a 1.25% H2 SO4 solution for 60 min,
achieving a high glucose concentration (~28.8 g/L) and yield (~25.1 g/100 g dry matter).
In addition, in studies on ethanol production after obtaining glucose yields under optimal
conditions, it was found that subsequent hydrolysis occurred using 10% (w/v) substrate
in a reaction medium supplemented with xylanase at 300 UI/g and Tween-20 (2.5% w/w)
after fermentation for around 24 h with Saccharomyces cerevisiae. Ethanol production was
achieved with up to 92.8% of the anticipated yield [1]. The optimization of alkaline pretreat-
ment conditions for rice straw was investigated using response surface methodology (RSM)
with NaOH concentration (1.0–4.0%), reaction time (30–90 min), and reaction temperature
(60–100 ◦ C) as the variables for enhancing glucose yield [8]. The results showed that the
maximum glucose yield of 254.5 ± 1.2 g/kg was obtained under conditions of 2.96% NaOH,
81.79 ◦ C, and 56.66 min. Later, Zhang et al. [9] interestingly studied the production of
bioethanol and xylooligosaccharides (XOS) from sugarcane bagasse (SCB) by autohydrol-
ysis pretreatment under optimal conditions, and found the maximized optimization for
the production of XOS yield of 50.53% at 200 ◦ C for 10 min and conversion into bioethanol
yield (66.67%). Milessi et al. [10] studied the hydrothermal and organosolv pretreatments
of hemicellulose mainly in the form of oligomers. The results showed that the maximized
xylooligosaccharide yield were determined at 185 ◦ C, 10 min at a 1:10 solid: liquid ratio.
For both hydrothermal and organosolv liquors, enzymatic conversion to XOs employing
an endoxylanase reached approximately 90%. Furthermore, Silva et al. [11] reported that
during enzymatic hydrolysis at 30 FPU cellulase g/L treated SCB within 72 h can result
in hemicellulose removal of 89.7%, glucan recovery of 97.8% and maximum ethanol con-
centration of 29.2 g/L. These results are promising particularly when compared with the
theoretical ethanol yield, which is equivalent to 58.9%. For an efficient fermentation process
to produce ethanol, the main concern is the optimization of the pretreatment process,
including various parameters. Therefore, pretreatment of biomass is required to obtain
optimal glucose yield conditions before being used for bioethanol production via enzymatic
hydrolysis. The aim of this study was to optimize the pretreatment of sugarcane bagasse
for the application of fermentation technologies to industrial biorefinery applications. Since
sugarcane bagasse is one of the crop residues according to the agricultural sector and sugar
Catalysts 2022, 12, 463 3 of 11

industry in Thailand, the use of by-products and excess materials are of much interest in
terms of the economy and consistent with the current situation of biorefineries.

2. Results and Discussion

2.1. Chemical Composition of Sugarcane Bagasse Substrates
The native sugarcane bagasse was mainly composed of cellulose (33.1 ± 0.51) hemi-
cellulose (19.2 ± 0.44), lignin (29.2 ± 0.20), ash (6.1 ± 0.55), and extractives (12.1 ± 0.25%).
However, more research into the structural and functional features of extractives at high
concentrations is needed.

2.2. Optimization of Reaction Temperature, Residual Time, and Acid Concentration for
Glucose Yield
In this study, all parameters were assessed according to the quality and quantity of
the solid fraction following liquid hot water pretreatment using the Box–Behnken design
matrix shown in Table 1. Along with the results of glucose yield, the experimental values
of stable conditions for cellulose, hemicellulose, and lignin content in the remaining solid
residue were examined utilizing response surface methodology (RSM).

Table 1. The effect of all response surface method in remaining solid residual were analyzed using
the Box−Behnken Design.

Factors Responses (%)

Acid Concentration Time Cellulose Hemicellulose Remaining Solid Glucose Yield
T (◦ C) (M) (min) (%) a (%) a Lignin (%) a Residual (%) a (%)
140 0.025 60 26.32 2.11 29.30 72.47 56.07
180 0.025 60 26.34 0.63 22.60 69.87 67.54
140 0.075 60 25.54 0.87 25.76 76.89 68.43
180 0.075 60 23.62 0.27 22.50 56.44 73.69
140 0.050 30 28.89 2.52 28.12 65.46 61.68
180 0.050 30 26.32 1.21 27.65 66.21 77.78
140 0.050 90 26.70 1.90 28.78 79.86 70.32
180 0.050 90 27.61 0.23 24.56 50.45 72.35
160 0.025 30 28.43 2.04 24.00 79.44 64.67
160 0.075 30 25.88 1.50 23.18 69.51 89.58
160 0.025 90 27.83 0.89 25.13 69.35 81.97
160 0.075 90 26.39 0.09 23.35 70.57 76.09
160 0.050 60 29.40 0.59 23.50 67.37 96.86
160 0.050 60 29.43 0.61 23.49 67.43 96.12
160 0.050 60 29.38 0.60 23.54 67.33 97.13
160 0.050 60 29.42 0.64 23.51 67.29 96.72
160 0.050 60 29.46 0.66 23.73 67.44 98.85
a Based on relative content of cellulose, hemicellulose and lignin content in remaining solid residual.

The experimental results obtained were analyzed using the Box−Behnken design
to assess the relationship among the reaction temperature (◦ C; X1 ), acid concentration
(M; X2 ), and time (min; X3 ). A glucose yield of 56.07–98.85% was obtained from the
Box−Behnken design. The resulting optimization of sugarcane bagasse following liquid
hot water pretreatment was analyzed using the analysis of variance. It was observed that
the optimization of reaction temperature (140–180 ◦ C), residence time (30–90 min), and
acid concentration (0.025–0.075 M) for maximum glucose yield (%) could be fitted to a
second-order polynomial multiple regression equation. The experimental value calculated
(Equation (1)) revealed the suitability of the quadratic model.
Glucose yield (%) = (−1360.96) + (16.02402 X1 ) + (3153.129 X2 ) + (2.47819 X3 ) − (3.10515 X12 ) − (0.00586)
(1)
− (10.2633 X23 ) − (0.04781 X2 1 ) − (−18,528.1 X2 2 ) − (−0.00830 X2 3 )
In addition, the predicted values of glucose yield after liquid hot water pretreatment
showed that all quadratic models of glucose yield were statistically significant at the 95%
confidence interval under optimal conditions. The R-squared value of the glucose yield
was greater than 0.90 for all results of this experiment, as shown in Table 2, indicating the
high accuracy of the model based on a comparison between predicted and experimental
values. It can be seen that the model predicted glucose yield with an accuracy of 99.63%
(Figure 1).
 values. It can be seen that the model predicted glucose yield with an accuracy of 99.63%
(Figure 1).

Table 2. The significance of all parameters in the regression models of glucose yield on ANOVA
analysis.
Catalysts 2022, 12, 463 4 of 11
Sum of Mean p-Value
Source DF F Value Comments
Squares Square Prob > F
ModelTable 2. The significance of all parameters in the regression models of glucose yield on ANOVA
A-Temperature
analysis. 151.99 1 151.99 233.95 <0.0001 Significance
B-Concentration 176.15 1 176.15 271.14 <0.0001 Significance
Sum of Mean p-Value
C-Time Source 6.15 Squares
1 6.15 DF 9.47Square 0.0179 a
F Value Prob > F
Comments
AB Model 9.64 1 9.64 14.84 0.0063 a

AC A-Temperature 49.56 151.991 49.56 1 76.29151.99 <0.0001233.95 Significance

<0.0001 Significance
B-Concentration 176.15 1 176.15 271.14 <0.0001 Significance
BC C-Time 237.01 1
6.15 237.011 364.816.15 <0.0001 9.47 Significance 0.0179 a
AB 9.64 1 9.64 14.84 0.0063 a
A*2 AC 1539.83 49.56 1 1539.831 2370.18 49.56 <0.0001 76.29 Significance
<0.0001 Significance
B*2 BC 564.62 237.01 1 564.621 869.09 237.01 <0.0001364.81 Significance
<0.0001 Significance
A*2 1539.83 1 1539.83 2370.18 <0.0001 Significance
C*2 B*2 235.48 564.62 1 235.481 362.47 564.62 <0.0001869.09 Significance
<0.0001 Significance
C*2 235.48 1 235.48 362.47 <0.0001 Significance
Note: a The non-significant
a
p-values.
Note: The non-significant p-values.

s 2022, 12, x FOR PEER REVIEW 5 of 12

(a)

(b)

(c)
Figure 1. Response surface
Figure plot of the
1. Response LHW
surface pretreatments
plot process: Effect
of the LHW pretreatments of reaction
process: Effecttime (30–90time (30–90 min),
of reaction
min), acid concentration (0.025 M–0.075 M) and temperature (140–180 °C) on glucose yield (a) Pre-
acid concentration (0.025 M–0.075 M) and temperature (140–180 ◦ C) on glucose yield (a) Predicted vs.
dicted vs. actual data plot (b) 2D contour plot (c) 3D surface plot on glucose yield under optimiza-
actual data plot (b) 2D contour plot (c) 3D surface plot on glucose yield under optimization.
tion.

2.3. Optimization of Glucose Yield after Liquid Hot Water Pretreatment of Sugarcane Bagasse
The second-order polynomial model obtained in Equation (1) for the target response
in this study to optimize the response following LHW pretreatment was determined using
 Catalysts 2022, 12, 463 5 of 11

2.3. Optimization of Glucose Yield after Liquid Hot Water Pretreatment of Sugarcane Bagasse
The second-order polynomial model obtained in Equation (1) for the target response
in this study to optimize the response following LHW pretreatment was determined using
Design Expert software 10.0. The results indicated that the optimal glucose yield (96.81%)
was predicted under conditions of 0.055 M acid, 162.24 ◦ C, and 57.506 min residence.
Thus, the optimal conditions produced >90% glucose yield. The regression analysis
identified the optimal conditions for glucose yield (96.86%) as 0.050 M acid, 160 ◦ C, and
60 min residence, following LHW pretreatment of sugarcane bagasse.
Furthermore, the HMF and furfural in the liquid fraction were discussed. Typically,
the hydrolysis of sugarcane bagasse in LHW pretreatment generates by-products in the
form of HMF and furfural, especially, under harsh conditions; a temperature above 180 ◦ C
marks the initial point to produce by-products. In addition, acid concentrations above
0.1 M also influence the process. Thus, this study determined the level of parameters as
lower than the criteria. This resulted in concentrations of HMF and furfural in the ranges
of 0.56–0.92 mg/L and 1.1–1.5 mg/L, respectively. It was observed that the concentration
of by-products fell within the same ranges. These concentration levels are not inhibitory to
Saccharomyces cerevisiae or Candida guilliermondii, which tolerate HMF and furfural up to
2 mg/mL [12].

2.4. SEM, XRD, BET Surface, and FTIR Characterization of Native Sugarcane Bagasse and Solid
Residue after LHW Pretreatment
Catalysts 2022, 12, x FOR PEER REVIEW 6 of 12
The physical structure of native sugarcane bagasse was modified compared with the
solid residue following LHW pretreatment under suitable conditions, as shown in Figure 2.
The surface characterization using SEM revealed an intact, smooth surface and a highly
ordered crystalline
cellulose structure. Inwere
and hemicellulose addition, it cancovered
densely be seen that the exterior
by lignin, thussurfaces of cellulose
concealing true cellulose
and hemicellulose were densely covered by lignin, thus concealing true cellulose before
before LHW pretreatment. Following pretreatment under suitable conditions, it can be
LHW pretreatment. Following pretreatment under suitable conditions, it can be noted
noted that the lignin and hemicellulose were removed, revealing the structure of cellulose
that the lignin and hemicellulose were removed, revealing the structure of cellulose and
and a rough
a rough surface
surface [13]. However,
[13]. However, these changes
these changes in the microstructure
in the microstructure of sugarcaneofbagasse
sugarcane ba-
gasse could increase
could increase thearea
the surface surface area of thereby
of cellulose, cellulose, thereby enzymatic
facilitating facilitating enzymatic
hydrolysis andhydroly-
sis and cellulose
cellulose degradation
degradation [14]. [14].

2.Scanning
Figure 2.
Figure Scanningelectron micrographs
electron of (A)
micrographs of native sugarcane
(A) native bagasse
sugarcane and (B)and
bagasse solid(B)
residue
solid after
residue after
pretreatment process.
pretreatment process.

The crystallinity and surface area of the solid residue have a significant impact on
The crystallinity and surface area of the solid residue have a significant impact on
enzymatic digestibility [15]. These parameters were directly affected by the efficiency of
enzymatic digestibility [15]. These parameters were directly affected by the efficiency of
LHW pretreatment, as shown in Table 3. One can see that a high crystallinity (66.8%)
LHW
was obtained for the as
pretreatment, shown
solid in Table
residue 3. Onepretreatment
after LHW can see thatunder
a highsuitable
crystallinity (66.8%) was
conditions
obtained for the
(0.050 M, 160 solid
◦ C, 60 residue
min) after LHW
in comparison pretreatment
to the underbagasse
native sugarcane suitable conditions
(49.6%). Before(0.050 M,
160 °C, 60 min) in comparison to the native sugarcane bagasse 2
pretreatment, the sugarcane bagasse showed a low surface area of 2.1 m /g. In contrast,pretreat-
(49.6%). Before
ment, the sugarcane
after pretreatment bagasse
under optimalshowed a low
conditions, surfacearea
the surface areawas
of 10.5
2.1 mm22/g.
/g.In contrast,the
Therefore, after pre-
treatment under optimal conditions, the surface area was 10.5 m2/g. Therefore, the acces-
sibility of the cellulose surface for enzymatic digestibility was significantly increased
when compared with native sugarcane bagasse.
Catalysts 2022, 12, 463 6 of 11

accessibility of the cellulose surface for enzymatic digestibility was significantly increased
when compared with native sugarcane bagasse.

Table 3. Surface area and XRD analysis of native sugarcane bagasse and solid residue after LHW
pretreatment under optimal conditions.

Order Surface Area (m2 /g) Degree of Crystallinity (%)

Native sugarcane bagasse 2.1 49.6
Solid residuals after LHW pretreatment 10.5 66.8

The FTIR spectra of the native sugarcane bagasse and solid residue after pretreatment
are shown in Table 4, revealing the chemical structure changes of lignocellulosic materials.
The results showed the O–H stretching of hydroxyl groups (3449–3431 cm−1 ) in lignin and
C–H stretching vibrations of methyl groups (2915–2895 cm−1 ) [16]. The C–O stretching at
1609–1602 cm−1 was attributed to the aromatic ring [17]. The absorption at 1375–1370 cm−1
was related to C–H deformation in cellulose and hemicellulose [18]. The intense peak
in the range 1429–1428 cm−1 was assigned to CH2 stretching vibrations in cellulose [19].
The vibrations at 1221–1220 cm−1 were related to the C–O stretching of syringyl rings in
lignin. The band at 1164–1162 cm−1 was attributed to the C–O–C vibration of cellulose and
hemicellulose [20]. The absorption in the range 1130–1128 cm−1 was related to the aromatic
structures in lignin [21]. The 1059–1043 cm−1 band was related to C–O stretching vibrations
in cellulose. Similarly, the peak at ~895 cm−1 indicated the C–H–O stretching vibrations of
β-(1-4)-glycosidic linkages in cellulose. ln addition, the intensity of the rocking vibration of
CH2 bands in cellulose Iα (~751 cm−1 ) was apparently increased after LHW pretreatment
under optimal conditions [22].

Table 4. FTIR spectra of the native sugarcane bagasse and solid residual after pretreatment process
under suitable conditions.

Order Frequency, (cm− 1 ) Functional Group

1 3449–3431 O–H stretching in phenolic compound
2 2915–2895 C–H stretching vibrations in methyl group
3 1609–1602 C–O stretching in lignin structure
4 1375–1370 C–H deformation in cellulose and hemicellulose
5 1429–1428 CH-2 stretching vibrations in cellulose
6 1221–1220 C–O stretch of syringyl rings in lignin structure
7 1164–1162 C–O–C vibration of cellulose and hemicellulose
8 1130–1128 automatic structure in lignin structure
9 1059–1043 C–O stretch vibrations in cellulose
10 ~895 C–H–O stretching vibrations of β-(1-4)-glycosidic linkage
11 ~751 CH-2 bands

2.5. Simultaneous Saccharification and Fermentation (SSF)

In the present study, ethanol production was obtained via the SSF process in S. cerevisiae
(TISTR 5339) yeast using the solid residue after LHW pretreatment as a starting material
(cellulose substrate). S. cerevisiae was grown in YPD medium supplemented with carbon
sources as the cellulose substrate (solid residual). It was previously reported that cellulose
is the best source of carbon for ethanol production [23]. After fermentation, the glucose
and ethanol production using the solid residue is shown in Figure 3. Using S. cerevisiae,
the final ethanol production ranged from 4.2–19.9 g/L on a 20–30% dry mass basis at pH
4.5 following 0–96 h of fermentation. The maximum amount of ethanol produced was
19.9 g/L using S. cerevisiae yeast for 72 h of fermentation. According to previous research,
Catalysts 2022, 12, 463 7 of 11

Fan et al. [24]. reported the use of various pretreatment strategies (liquid hot water, ethano-
solv, dilute acid and alkaline) in the presence of different surfactants for enhancement
of glucose production from sugarcane bagasse during enzymatic hydrolysis. The results
showed that dilute acid pretreatment could increase the glucose yield from 75.04 to 86.14%
in the presence of 1.5% PEG 6000. According to appropriate conditions, the maximum
ethanol concentration of 20.17 g/L was obtained via simultaneous saccharification fer-
mentation. Wang et al. [25] studied the effect of LHW pretreated for ethanol production
from sugarcane bagasse. The results showed that the pretreated sugarcane bagasse under
LHW pretreatment could enhance the ethanol production in both of batch and fed-batch
Catalysts 2022, 12, x FOR PEER REVIEW
hydrolysis in simultaneous saccharification and fermentation. The ethanol production 8 ofand
12
theoretical yield obtained from the process of SSF after fed-batch hydrolysis were 55.4 g/L
and 88.3% for 72 h, respectively.

30

25 Glucose Ethanol
Concentration (g/L)

20

15

10

0
0 6 12 24 48 72
Time (h)
Figure 3. Effect of glucose content on ethanol production of pretreated sugarcane bagasse.
Figure 3. Effect of glucose content on ethanol production of pretreated sugarcane bagasse.
3. Materials and Methods
3.3.1.
Materials
Materialsand Methods
3.1. Materials
Sugarcane bagasse was obtained from Ban Du, Chiang Rai, Thailand. The sugarcane
Sugarcane
bagasse bagasse
was dried ◦ C obtained
at 50was for 24 h infrom
a hotBanairDu, Chiang
oven. Rai, Thailand.
Afterward, Themilled,
it was cut, sugarcaneand
sieved to
bagasse a particle
was dried atsize
50 in
°Cthe
forrange 1–2.0
24 h in mm
a hot air(Retsch ZM200, Haan,
oven. Afterward, Germany),
it was followed
cut, milled, and
by grinding
sieved using 0.061–0.25
to a particle size in the mm sieves.
range 1–2.0Themmfinal moisture
(Retsch content
ZM200, Haan,ofGermany),
the milled sugarcane
followed
bybagasse wasusing
grinding 5%, as assessed mm
0.061–0.25 by weight
sieves.loss
Theafter
finaldrying in an
moisture oven at
content the ◦milled
of105 C for 5sugar-
h with
the goal
cane of maintaining
bagasse constantby
was 5%, as assessed weight.
weightThe lossprepared sugarcane
after drying bagasse
in an oven at 105was kept
°C for 5hin
sealed plastic bags at room temperature for the experiments. The composition
with the goal of maintaining constant weight. The prepared sugarcane bagasse was kept of sugarcane
inbagasse
sealed was determined
plastic according
bags at room to the National
temperature Renewable Energy
for the experiments. Laboratory [26].
The composition All
of sug-
chemicals and reagents, including sugar standards, were acquired from
arcane bagasse was determined according to the National Renewable Energy Laboratory major chemical
suppliers,
[26]. such as Sigma-Aldrich
All chemicals (St. Louis,sugar
and reagents, including MO, USA), which
standards, provided
were acquiredglucose,
from xylose,
major
arabinose,
chemical 5-hydroxymethyl-2-furaldehyde
suppliers, such as Sigma-Aldrich (St.(HMF), Louis, and
MO,furfural (S.M. provided
USA), which Chemical glucose,
Supplies
Co., Ltd.,
xylose, Bangkok,
arabinose, Thailand). Sulfuric acid was(HMF),
5-hydroxymethyl-2-furaldehyde purchased
and from
furfuralMerck
(S.M.(Merck
ChemicalLtd.
Bangkok, Thailand).
Supplies Co., Ltd., Bangkok, Thailand). Sulfuric acid was purchased from Merck (Merck
Ltd. Bangkok, Thailand).
3.2. Liquid Hot Water Pretreatment of Sugarcane Bagasse
The pretreatment
3.2. Liquid of raw sugarcane
Hot Water Pretreatment bagasse
of Sugarcane Bagassewas implemented in a stainless-steel
reactor with a capacity of 600 mL, heated using an electric jacket with a thermocouple to
The pretreatment of raw sugarcane bagasse was implemented in a stainless-steel re-
measure the temperature (Parr Reactor 4560, Parr Instrument Co., Moline, IL, USA). Initially,
actor with a capacity of 600 mL, heated using an electric jacket with a thermocouple to
the pretreatment was carried out using a 1 g/15 mL ratio of native sugarcane bagasse to
measure the temperature (Parr Reactor 4560, Parr Instrument Co., Moline, IL, USA). Ini-
water with various concentrations of H2 SO4 acid catalyst (0.025–0.075 M), temperatures
tially, the pretreatment was carried out using a 1 g/15 mL ratio of native sugarcane bagasse
(140–180 ◦ C), and residence times (30–90 min). The response time was initiated once the
to water with various concentrations of H2SO4 acid catalyst (0.025–0.075 M), temperatures
(140–180 °C), and residence times (30–90 min). The response time was initiated once the
target temperature was reached. Nitrogen gas (N2) was flowed into the reactor for purging
and adjusting the initial pressure to 20 bar, and the reaction was stirred at 100 rpm to
maintain a homogeneous system. At the end of the reaction time, the reaction was quickly
Catalysts 2022, 12, 463 8 of 11

target temperature was reached. Nitrogen gas (N2 ) was flowed into the reactor for purging
and adjusting the initial pressure to 20 bar, and the reaction was stirred at 100 rpm to
maintain a homogeneous system. At the end of the reaction time, the reaction was quickly
quenched in a water bath. The solid cellulose-enriched fraction was separated using
Whatman No. 4 filter paper and then washed with ~150 mL of DI water. The pretreated
substrate was dried overnight at 70 ◦ C and stored at room temperature for further research.
Monomeric sugars and inhibitory products were analyzed by high-performance liquid
chromatography (HPLC analysis).

3.3. Experimental Design and Optimization of Glucose Yield Using Box–Behnken Response
Surface Design
Box–Behnken response surface methodology (RSM) and statistical analysis were
performed using Design Expert software (Trial version 10.0, Stat-Ease, Inc., Minneapolis,
MN, USA) to study glucose yield optimization in the sugarcane bagasse residue. Three
variables were identified as the critical process parameters for glucose yield from after LHW
pretreatment of sugarcane bagasse: reaction temperature (X1 , 140–180 ◦ C), residence time
(X2 , 30–90 min), and acid concentration (X3 , 0.025–0.075 M), with three levels of each factor
(−1, 0, 1). Therefore, to estimate the model coefficients, 17 experiments were conducted
with three replications at the center point, processed in a random order. Box–Behnken
design with experimental design is shown in Table 5. The target response was glucose
yield, with the response surface regression equation fitted to a second-order polynomial to
investigate the interaction effect and optimal circumstances (Equation (2)):

Y = β0 + β1 X1 + β2 X2 + β3 X3 + β12 X1 X2 + β13 X1 X3 + β23 X2 X3 + β11 X2 1 + β22 X2 2 + β33 X2 3 (2)

where Y is the predicted response; X1 , X2 , and X3 are the independent variables; β0 is a

constant; β1 , β2 , and β3 are the linear coefficient terms; β12 , β13 , and β23 are the interaction
coefficient terms; and β11 , β22 , and β33 are the quadratic coefficient terms.

Table 5. Box–Behnken design with experimental design of glucose yield.

Run T (◦ C) Acid Concentration (M) Time (min)

1 −1 −1 0
2 1 −1 0
3 −1 1 0
4 1 1 0
5 −1 0 −1
6 1 0 −1
7 −1 0 1
8 1 0 1
9 0 −1 −1
10 0 1 −1
11 0 −1 1
12 0 1 1
13 0 0 0
14 0 0 0
15 0 0 0
16 0 0 0
17 0 0 0

3.4. Enzymatic Hydrolysis

Enzymatic hydrolysis was performed on the solid residue obtained after pretreatment
using commercial cellulase concentrate enzymes (Cellic Ctec2, Novozymes A/S, Bagvaerd,
Denmark). The sugar yields from the enzymatic hydrolysis were measured. The enzymatic
hydrolysis reaction experiment was performed in 25 mL-Erlenmeyer flasks consisting of 5%
w/v pretreated substrate with 25 FPU/g of enzyme in 50 mM sodium citrate buffer pH 4.8
and 1% w/v sodium azide. The reaction was carried out under incubation in a rotatory
Catalysts 2022, 12, 463 9 of 11

shaker at 150 rpm, 50 ◦ C, for 72 h. The hydrolysis experiments were performed in triplicate.
Profiles of released sugars were analyzed on a high-performance liquid chromatograph
(LDC Model 4100, Shimadzu, Kyoto, Japan) equipped with a refractive index detector and
an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) operating at 65 ◦ C with 5 mM
H2 SO4 as the mobile phase at a flow rate of 0.5 mL/min. Glucose yield was calculated
as the percentage of glucose release from enzymatic hydrolysis as a function of cellulose
content in the pretreated biomass according to Equation (3).

Amount of glucose after hydrolysis (g)

Cellulose hydrolysis (%) = × 100 (3)
Amount of glucose in pretreated material (g)

3.5. Analysis of Aqueous Phase

The soluble product in the liquid phase was interpreted using a liquid chromatograph
(LDC Model 4100, Shimadzu, Kyoto, Japan) equipped with a refractive index, UV/Vis
detector, and Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). Fermentable sugar
profiles and inhibitory byproducts were obtained at a specific temperature of 65 ◦ C, with a
constant flow rate of 0.5 mL/min (5 mM H2 SO4 ).

3.6. Characterization of Native Sugarcane Bagasse and Remaining Solid Residue

3.6.1. Scanning Electron Microscopy Analysis
Scanning electron microscopy (SEM; JSM-6301F, JEOL, Japan) was used to examine
the microstructure of native sugarcane bagasse and the remaining solid residue after the
pretreatment process. The samples were dried and coated with gold for analysis. An
electron beam energy of 20 kV was used for analysis.

3.6.2. X-ray Diffraction Analysis

The crystallinity of the native sugarcane bagasse and remaining solid residue was
determined by X-ray diffraction (XRD), using an X’Pert PRO diffractometer (PANalytical,
Almelo, The Netherlands). The samples were scanned at room temperature in a range of
2θ = 10◦ –30◦ with a step size of 0.004◦ at 500 kV, 30 mA. The crystallinity index (CrI) was
determined using the following Equation (4):

I002 − Iamorphous
CrI = × 100 (4)
I002

where I002 is the scattered intensity of the main peak of cellulose, which typically lies in the
002 plane, and Iamorphous is the scattered intensity of the amorphous portion evaluated in
the 101 plane.

3.6.3. BET Surface Area Measurement

The method of Brunauer, Emmett, and Teller (BET) was used to determine the total
surface area of materials. Native sugarcane bagasse and the remaining solid residue were
analyzed for their BET surface area, using a Belsorp-max TPDpro (BEL Japan, Tokyo, Japan)
with a thermal conductivity detector (semi-diffusion type, 4-element W–Re filament) at the
National Nanotechnology Center, Thailand.

3.6.4. Fourier-Transform Infrared Spectroscopy Analysis

The chemical composition of native sugarcane bagasse and the remaining solid residue
was analyzed using FT-IR measurements on a PerkinElmer instrument (Waltham, MA,
USA) with the KBr pellet technique. The measurement resolution was set at 4 cm−1 with
a mirror velocity of 0.6329 cm/s. Infrared spectra were collected within the range of
400–4000 cm−1 from ~32 scans. Commercial cellulose from Sigma-Aldrich was used as
a reference.
Catalysts 2022, 12, 463 10 of 11

3.7. Simultaneous Saccharification and Fermentation Process for Ethanol Production

The sample was evaluated in a 1.6 L reactor with a total operating volume of 1.0 L
(Biostat® b 2, B. Braun, Bangkok, Thailand). The fermentation medium for the fermentation
process was composed of yeast extract (1 g), (NH4 )2 SO4 (5 g), and MgSO4 ·7H2 O (0.025 g/L),
pH 4.8, with 6.25% of the remaining solid residue under optimal conditions following the
liquid hot water pretreatment process. Then, the yeast medium was sterilized using
an autoclave at 121 ◦ C for 15 min. The remaining solid residue was predigested using
25 FPU/g Cellic Ctec2 at 50 ◦ C by 6 h of hydrolysis at 300 rpm.
Saccharomyces cerevisiae No. TISTR 5339 was the yeast strain used in this work [27].
The leavening agent cells of this microorganism from inoculated agar plates (30 ◦ C for 24 h)
were transferred to 150 mL Erlenmeyer flasks containing YPD medium, consisting of H3 PO4
and NH4 OH, pH 4.8, incubated in a rotatory shaker at 35 ◦ C, 150 rpm, and fermented for
120 h. The concentrations of sugars (glucose and xylose) and ethanol were analyzed using
a high-performance liquid chromatograph equipped with an Aminex HPX-87H column
(Bio-Rad, Hercules, CA, USA). The ethanol yield was calculated as a percentage of the
potential ethanol yield of consumed glucose, which was 0.511 g/g sugar [26].

4. Conclusions
Liquid hot water pretreatment was identified as an effective method for the pretreat-
ment of sugarcane bagasse. The results indicated that 96.86% glucose yield was predicted
under conditions of 160 ◦ C, 60 min residence, and 0.050 M acid after LHW pretreatment
using the Box–Behnken response surface design. A high ethanol concentration (19.9 g/L)
was obtained via 72 h of the simultaneous saccharification fermentation process. Based
on the obtained results, this study provides a methodological framework to optimize the
alternative use of sugarcane bagasse for bioethanol production in biorefinery industries.
The point of view is considered in term of biomass supply chain, bioenergy manufacturing
chain and biofuel trade. In addition, bioethanol is suggested emphasizing sustainability,
localness and recycling principles.

Author Contributions: Methodology writing—original draft preparation, P.K.; Conceptualization,

N.S.; Visualization, T.K.; Writing—review and editing, N.L.; Supervision, K.W.; Writing—review and
Methodology, V.C.; Project administration, K.S.; Software, C.S.; Writing—review and editing C.C.;
Methodology, funding acquisition, S.I. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Thailand Research Fund (RTA 6280003), PMUB (B05F640093),
and Thailand Science Research and Innovation Fund and the University of Phayao (Grant No.
FF65-UoE008 and FF65-RIM001).
Data Availability Statement: Data sharing is not applicable to this article.
Acknowledgments: This research project was supported by the Thailand Science Research and
Innovation Fund and the University of Phayao (Grant No. FF65-UoE008 and FF65-RIM001). Navadol
Laosiripojana was supported from PMUB with grant number of B05F640093 and a research grant
(RTA 6280003) from the Thailand Research Fund.
Conflicts of Interest: The authors declare no conflict of interest.

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