fermentation

Article
Valorization of Bombax ceiba Waste into Bioethanol Production
through Separate Hydrolysis and Fermentation and
Simultaneous Saccharification and Fermentation
Misbah Ghazanfar 1 , Muhammad Nadeem 2 , Hafiz Abdullah Shakir 3 , Muhammad Khan 3 , Irfan Ahmad 4 ,
Marcelo Franco 5 , Lijing Chen 6, * and Muhammad Irfan 1, *

1 Department of Biotechnology, University of Sargodha, Sargodha 40100, Pakistan

2 Food & Biotechnology Research Center, PCSIR Laboratories Complex Ferozepur Road, Lahore 54600, Pakistan
3 Institute of Zoology, University of the Punjab New Campus, Lahore 54590, Pakistan
4 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University,
Abha 62529, Saudi Arabia
5 Department of Exact Science and Technology, State University of Santa Cruz Ilheus Brazil,
Ilheus 45662-900, Brazil
6 Key Laboratory of Agriculture Biotechnology, College of Biosciences and Biotechnology,
Shenyang Agricultural University, Shenyang 110866, China
* Correspondence: chenlijing1997@126.com (L.C.); irfan.ashraf@uos.edu.pk (M.I.)

Abstract: In this study, Seed pods of B. ceiba were used as a novel, cheap, and sustainable feedstock
for second-generation bioethanol production. B. ceiba waste was pretreated with NaOH under
different conditions using a Box–Behnken design (BBD) with three factors and three levels. Fourier
transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA),
and scanning electron microscopy (SEM) were used to investigate the chemical, structural, and
Citation: Ghazanfar, M.; Nadeem, M.;
morphological modifications made by pretreatment. NaOH pretreatment followed by steam was
Shakir, H.A.; Khan, M.; Ahmad, I.;
Franco, M.; Chen, L.; Irfan, M.
more effective as it offered 60% cellulose and 9% lignin at 10% substrate loading, 5% NaOH conc.,
Valorization of Bombax ceiba Waste and 4 h residence time. Samples with maximum cellulose were employed for ethanol production
into Bioethanol Production through by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation
Separate Hydrolysis and Fermentation (SSF) using indigenously produced cellulase as well as commercial cellulase. HPLC analysis revealed
and Simultaneous Saccharification and the best saccharification (50.9%) at 24 h and the best ethanol yield (54.51 g/L) at 96 h of fermentation
Fermentation. Fermentation 2022, 8, in SSF using commercial cellulose by Saccharomyces cerevisiae. SSF offered a better production of
386. https://doi.org/10.3390/ bioethanol from seed pods than SHF. The implications of the work support the notion that B. ceiba
fermentation8080386 waste could be utilized for large-scale bioethanol production.
Academic Editor: Andrzej Klasa
Keywords: B. ceiba; pretreatment; NaOH; bioethanol; saccharification
Received: 8 July 2022
Accepted: 9 August 2022
Published: 12 August 2022

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in The reserves of non-renewable energy, such as fossil fuels, have been declining day
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by day because of the growing population and vast industrialization. Serious ecological
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and environmental issues have appeared due to the immense exploitation of fossil fuels.
There is a need to search for an alternative renewable energy source to maintain the growth
of society. Ethanol from cellulosic biomass could be a promising substitute for petrol and
Copyright: © 2022 by the authors.
has good octane, which leads to lessened emissions of air pollutants [1–4]. Previously,
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sugar-rich biomasses from food crops, e.g., corn and sugarcane, have been employed for
This article is an open access article bioethanol production. However, the growing demand for bioethanol induced competition
distributed under the terms and for these food crops, resulting in increased prices of food products, and it also disturbed the
conditions of the Creative Commons food chain for animals and humans [5,6]. Lignocellulosic biomass could be an encouraging
Attribution (CC BY) license (https:// and non-food feedstock for bioethanol production. Such biomass is fairly low-priced as
creativecommons.org/licenses/by/ well as abundantly and locally accessible [7–9].
4.0/).

Fermentation 2022, 8, 386. https://doi.org/10.3390/fermentation8080386 https://www.mdpi.com/journal/fermentation

Fermentation 2022, 8, 386 2 of 22

Cellulose is the major component of lignocellulosic biomass and is considered to be a

perpetual source of raw material for green energy. It is the most ubiquitous waste matter
and inexhaustible biopolymer from agriculture and is abundantly found in nature [10,11].
It can be obtained from solid waste, remnants of forest and agriculture, and woody and
herbaceous crops. Bioethanol production from lignocellulosic material involves three major
steps, including pretreatment, saccharification, and fermentation [12–14]. Various physical,
chemical, and thermochemical pretreatment techniques have been employed to remove
lignin from lignocellulosic biomass and to enhance the accessibility of cellulase enzyme to
the cellulosic fibers [2]. Cellulases are hydrolytic enzymes with an important role in the
bioconversion of lignocellulosic biomass into fermentable sugars. Numerous microbes, e.g.,
bacteria and fungi, produce hydrolytic enzymes [15,16]. Bacteria are potential producers of
cellulases as they have a higher growth rate compared to fungi. Several bacterial genera,
including Bacillus, Cellulomonas, Micrococcus, Cellvibrio, and Pseudomonas sp., have cellulase-
degrading abilities [17].
Glucose obtained from saccharified material is converted into ethanol by the fer-
mentation process [18]. The yeast Saccharomyces can convert glucose into ethanol with
approximately 90% of the theoretical yield [19]. Separated hydrolysis and fermentation
(SHF) and simultaneous saccharification and fermentation (SSF) are the two most common
processes used in the fermentation of lignocellulosic hydrolysate. The optimization of
the pretreatment processes is required for greater ethanol yield and low production costs.
The Box–Behnken design (BBD) of response surface methodology (RSM) was used in this
study to optimize pretreatment conditions. RSM is a mathematical and statistical modeling
approach that is used to examine the effect of different parameters and their interactions on
productivity. Different biotechnological processes are optimized by using this technique.
The optimization of conditions by the commonly used one-factor-at-a-time approach is
time-consuming and strenuous and may cause inaccurate results in the end. However,
RSM delivers the interaction of multiple variables on the response in less time. Therefore,
scientists are now using this technique to optimize several process conditions for maximum
production [2].
Due to its large size and flaunting flowers, Bombax ceiba is known as King of the Forest
(Figure 1). It is a deciduous tree with a straight cylindrical stem and horizontally spreading
branches. Its large size, horizontally branching system, and buttress at the base are the
first perceived features to distinguish the species in the forest. The tree reaches up to 40 m
in height and 2 m in diameter with a vibrant trunk of 24–30 m [20]. It is locally found in
tropical regions of western Africa, Southeast Asia, Pakistan, Bangladesh, Sri Lanka, Bhutan,
India, Maldives, and Nepal (Aguoru et al., 2015). Its seeds are covered in seed pods that
fall on ripening. Seeds are dispersed via silky hairs, whereas seed pods remain as waste.
The enormous amount of fallen seed pods is usually found as waste around the B. ceiba tree.
We selected this tree as a feedstock because of its abundant, easy, and cheap availability.
The use of waste seed pods with good polysaccharide content (25% cellulose, 34% lignin)
would be a step toward waste management [21]. The aim of this study was to obtain
optimized conditions for the NaOH pretreatment of B. ceiba (seed pods), saccharification
with commercial and indigenous cellulase to obtain maximum sugars, and fermentation of
sugars for bioethanol production using SSF and SHF.
Fermentation 2022,8,8,386
Fermentation2022, x FOR PEER REVIEW 33 of
of 22
23

Figure1.1.Tree
Figure Treeof
ofB.
B.ceiba
ceiba[22].
[22].

2.
2. Materials
Materials and
and Methods
Methods
2.1. Biomass
2.1. Biomass
B.
B.ceiba
ceibaseed
seedpods
podswere
werecollected,
collected,washed,
washed,dried,
dried,and
andmilled
milledto
topowder
powderform
formand
andkept
kept
in plastic bags for further use [2].
in plastic bags for further use [2].
2.2. Pretreatment of Biomass
2.2. Pretreatment of Biomass
Powdered seed pods were pretreated by the method reported earlier [2]. Substrate (10 g)
Powdered seed pods were pretreated by the method reported earlier [2]. Substrate
was soaked in different concentrations of NaOH solution at the ratio of 1:10 (solid:liquid) for
differentwas
(10 g) soakedatin
durations different
room concentrations
temperature. Then, theofsamples
NaOH weresolution at thetoratio
subjected of 1:10
pressurized
heat treatment as per experimental design. After steaming, the samples were filtered, sub-
(solid:liquid) for different durations at room temperature. Then, the samples were and
jectedresidues
solid to pressurized heat treatment
were washed as per experimental
up to neutrality. design.are
Levels of variables After steaming,
mentioned in the sam-
Table 1.
ples were filtered, and solid residues were washed up to neutrality. Levels of variables
are mentioned
Table in Table
1. Box–Behnken 1. codes and levels of the variables.
design

Table 1. Box–Behnken design codes and levels of the variables.

Codes and Their Values
Independent Variables Symbols −1 0 +1
Codes and Their Values
Concentration of NaOH (%) X1 1 3 5
Independent Variables
Concentration of B. ceiba (%) X2
Symbols 5
−1 10
0 15
+1
Concentration
Time (h) of NaOH (%) X3 X1 4 1 63 85
Concentration of B. ceiba (%) X2 5 10 15
Time
2.3. Cellulose and (h) Estimation
Lignin X3 4 6 8
Cellulose was analyzed from solid residues (dried in oven) by method described by
2.3. Cellulose
Gopal and Lignin
and Ranjhan [23]. Estimation
Then, the samples containing maximum cellulose were estimated
Cellulose
for lignin [24]. was analyzed from solid residues (dried in oven) by method described by
Gopal and Ranjhan [23]. Then, the samples containing maximum cellulose were estimated
2.4. Analytical
for lignin [24].Methods
Total sugar (TS) content was analyzed as described by Dubois et al. [25]. Reducing
2.4. Analytical
sugar (RS) wasMethods
determined by DNS method [26]. Total phenolic (TP) contents found in
filtrate,
Total sugar during
liberated pretreatment,
(TS) content were estimated
was analyzed by the
as described by method of al.
Dubois et [27].
[25]. Reducing
sugar (RS) was determined by DNS method [26]. Total phenolic (TP) contents found in
2.5. Experimental
filtrate, liberatedDesign
during pretreatment, were estimated by the method of [27].
Optimization of NaOH pretreatment conditions was designed through BBD following
Ghazanfar et al. [2].
2.5. Experimental Design
Optimization of NaOH pretreatment conditions was designed through BBD follow-
2.6. Substrate Characterization
ing Ghazanfar et al. [2].
Treated and untreated substrates were characterized by X-ray diffraction (XRD) [28],
thermogravimetric analysis (TGA) [29], Fourier transform infrared spectroscopy (FTIR),
and scanning electron microscopy (SEM) [30].
Fermentation 2022, 8, 386 4 of 22

2.7. Saccharification and Fermentation Using SHF and SSF

Raw and pretreated substrates from each pretreatment with maximum cellulose
contents were employed for bioethanol production through SSF and SHF, as reported
earlier [21].

2.8. Statistical Analysis

The data collected from experiments were statistically analyzed through Minitab soft-
ware (State College, PA, USA), and ANOVA was also performed by Minitab software [31].

3. Results and Discussion

3.1. Pretreatment
In this study, B. ceiba seed pods were pretreated both chemically and thermochemically
with different concentrations of NaOH. An analysis of raw seed pods revealed 34% cellulose
and 25% lignin. The liberation of TP indicates the degradation of lignin, and the release of
RS and TS indicates the hydrolysis of hemicellulose and cellulose contents in the biomass
of B. ceiba. To optimize the pretreatment conditions for maximum lignin breakdown and
enhanced cellulose exposure, we applied BBD with three parameters and three levels.
Second-order polynomial regression equations (Equations (1)–(8)) were applied to calculate
the response (Tables 2 and 3).
Maximum TP (435.9400 mg/mL) liberated and cellulose (46%) exposed were obtained
at 3% (w/v) NaOH concentration and 15% (w/v) substrate loading while the residence
time was 8 h, whereas the maximum RS (28.0 mg/mL) and total sugars (1563.4 mg/mL)
released at a 5% (w/v) NaOH concentration and 15% (w/v) substrate loading while the
soaking time was 6 h. Autoclaving proceeded by NaOH pretreatment was found to be more
effectual, releasing maximum TP (246.8 mg/mL) after 4 h soaking time at a 15% substrate
concentration, 3% NaOH solution, and reducing sugars up to 87.44 mg/mL at a 1% NaOH
solution, 15% substrate concentration, and 6 h residence time. The highest cellulose content
exposed (60%) was obtained at a 10% substrate loading, 5% NaOH solution, and 4 h
residence time, and TS liberated were 259.57 mg/mL at a 6 h soaking time, 15% substrate
concentration, and 5% NaOH solution during thermochemical pretreatment.

Table 2. Observed values for RS, TS, cellulose, and TP after NaOH pretreatment optimized by
Box–Behnken design (BBD).

Run No. X1 X2 X3 Cellulose (%) TS (mg/mL) RS (mg/mL) TP (mg/mL)

1 5 15 6 32 1563.4 28 239.1
2 1 5 6 36 408.27 8.85 330.7
3 5 5 6 36 749.32 0.23 229.7
4 3 5 8 36 684.08 0.8 291.4
5 1 15 6 45 816.21 5.91 342.7
6 3 15 8 46 902.47 9.23 435.9
7 1 10 4 45 640.7 1.83 103.1
8 3 10 6 36 519.29 4.23 190.8
9 5 10 8 36 1066.4 7.26 106.2
10 3 10 6 39 521.6 3.31 191.6
11 5 10 4 34 1090.9 9.79 62.35
12 3 5 4 40 419.71 1.14 46.65
13 3 10 6 35 524.04 2.97 189
14 1 10 8 44 425.34 7.77 550.6
15 3 15 4 44 1412.1 14.3 26.54
Pretreatment condition = room temperature, X1 = base conc., X2 = substrate conc., X3 = retention time, TS = total
sugar, RS = reducing sugar, TP = total phenol.
Fermentation 2022, 8, 386 5 of 22

Table 3. Observed values for RS, TS, cellulose, and TP after NaOH + steam pretreatment optimized
by Box–Behnken design (BBD).

Run No. X1 X2 X3 Cellulose (%) TS (mg/mL) RS (mg/mL) TP (mg/mL)

1 5 15 6 54 259.5 53.5 119.3
2 1 5 6 58 52.55 13.6 66.6
3 5 5 6 56 131.9 8.74 47.7
4 3 5 8 60 84.43 10.2 67.7
5 1 15 6 36 169.1 87.4 204.6
6 3 15 8 32 156.7 63.1 186.1
7 1 10 4 52 117.3 29.4 122.7
8 3 10 6 45 162.4 20.5 116.1
9 5 10 8 54 180.1 17.4 108.5
10 3 10 6 46 163.3 23.6 117.7
11 5 10 4 60 233.7 17.8 128.1
12 3 5 4 54 55.8 6.58 41.59
13 3 10 6 47 164.1 19.6 115.3
14 1 10 8 45 128.5 43.8 121.9
15 3 15 4 54 223.6 59.9 246.8
Steam conditions: temperature = 121 ◦ C, pressure = 15 Psi, time = 15 min, X1 = base conc., X2 = substrate conc., X3
= retention time, TS = total sugar, RS = reducing sugar, TP = total phenol.

Data were analyzed statistically, and regression equations depicted that the results
were significant. The thermochemical pretreatment resulted in a greater breakdown of
hemicellulose contents and hence liberated more sugars. The Fisher’s F-test values of
31,568.92, 13.05, 28.84, and 38.21 were noticed for TS, cellulose, RS, and TP, respectively, for
chemical pretreatment (Tables 4 and 5). The F-test values were found to be 18.09, 101.29,
231.10, and 4398.87 for TP cellulose, RS, and TS, respectively, in the case of thermochemical
treatment (Tables 6 and 8). For the chemical treatment, the R2 values were 100.00%, 87.88%,
98.07%, and 96.65% for TS, cellulose, RS, and TP, respectively. Moreover, the integrity of
the model was assisted by adjusted R2 values (81.14%, 100.00%, 95.51%, and 93.30% for
cellulose, TS, RS, and TP, respectively). Thus, the validity of the model was revealed from
these values. Figures 2 and 3 illustrate contour plots for cellulose, RS, TP, and TS liberated
during different pretreatments. These plots depict different ranges of responses by keeping
one variable constant and varying the other two variables.

Table 4. ANOVA of cellulose (%) and TS (mg/mL) after NaOH treatment.

Source DF Adj SS Adj MS F-Value p-Value

Model 9 282.517 31.391 6.43 0.027
Linear 3 173.250 57.750 11.83 0.010
X1 1 128.000 128.000 26.21 0.004
X2 1 45.125 45.125 9.24 0.029
X3 1 0.125 0.125 0.03 0.879
Square 3 55.767 18.589 3.81 0.092
X21 1 1.256 1.256 0.26 0.634
Cellulose X22 1 5.026 5.026 1.03 0.357
(%) X23 1 49.641 49.641 10.17 0.024
2-Way Interaction 3 53.500 17.833 3.65 0.099
X1 *X2 1 42.250 42.250 8.65 0.032
X1 *X3 1 2.250 2.250 0.46 0.527
X2 *X3 1 9.000 9.000 1.84 0.233
Error 5 24.417 4.883
Lack-of-Fit 3 15.750 5.250 1.21 0.482
Pure Error 2 8.667 4.333
Total 14 306.933
Fermentation 2022, 8, 386 6 of 22

Table 4. Cont.

Source DF Adj SS Adj MS F-Value p-Value

Model 9 1,831,767 203,530 31,568.92 0.000
Linear 3 1,363,114 454,371 70,476.32 0.000
X1 1 593,903 593,903 92,118.65 0.000
X2 1 739,802 739,802 114,748.72 0.000
X3 1 29,409 29,409 4561.58 0.000
Square 3 268,561 89,520 13,885.27 0.000
X21 1 91,000 91,000 14,114.81 0.000
TS X22 1 156,204 156,204 24,228.44 0.000
(mg/mL) X23 1 59,779 59,779 9272.11 0.000
2-Way Interaction 3 200,091 66,697 10,345.18 0.000
X1 *X2 1 41,252 41,252 6398.43 0.000
X1 *X3 1 9105 9105 1412.25 0.000
X2 *X3 1 149,734 149,734 23,224.86 0.000
Error 5 32 6
Lack-of-Fit 3 21 7 1.24 0.476
Pure Error 2 11 6
Total 14 1,831,799
X1 = base conc., X2 = substrate conc., X3 = retention time, TS = total sugars.

Table 5. ANOVA of TP (mg/mL) and RS after NaOH treatment.

Source DF Adj SS Asj MS F-Value p-Value

Model 9 295,775 32,864 11.35 0.008
Linear 3 226,136 75,379 26.03 0.002
X1 1 59,469 59,469 20.54 0.006
X2 1 2656 2657 0.92 0.382
X3 1 164,010 164,010 56.64 0.001
Square 3 22,130 7377 2.55 0.169
X21 1 9328 9328 3.22 0.133
TP X22 1 7418 7418 2.56 0.170
(mg/mL) X23 1 4566 4566 1.58 0.265
2-Way Interaction 3 47,509 15,836 5.47 0.049
X1 *X2 1 2 2 0.00 0.982
X1 *X3 1 40,733 40,733 14.07 0.013
X2 *X3 1 6774 6774 2.34 0.187
Error 5 14,479 2896
Lack-of-Fit 3 14,476 4825 2721.01 0.000
Pure Error 2 4 2
Total 14 310,255
Model 9 678.748 75.416 22.97 0.002
Linear 3 324.558 108.186 32.95 0.001
X1 1 54.706 54.706 16.66 0.010
X2 1 269.352 269.352 82.04 0.000
X3 1 0.500 0.500 0.15 0.712
Square 3 94.885 31.628 9.63 0.016
X21 1 52.467 52.467 15.98 0.010
RS X22 1 44.576 44.576 13.58 0.014
(mg/mL) X23 1 1.376 1.376 0.42 0.546
2-Way Interaction 3 259.304 86.435 26.33 0.002
X1 *X2 1 235.776 235.776 71.81 0.000
X1 *X3 1 17.935 17.935 5.46 0.067
X2 *X3 1 5.593 5.593 1.70 0.249
Error 5 16.416 3.283
Lack-of-Fit 3 15.566 5.189 12.21 0.077
Pure Error 2 0.850 0.425
Total 14 695.164
X1 = Base conc., X2 = Substrate conc., X3 = Retention time, TP = Total phenol, RS = Reducing sugar.
Fermentation 2022, 8, 386 7 of 22

Table 6. ANOVA of cellulose (%) and TS (mg/mL) after NaOH + steam pretreatment.

Source DF Adj SS Adj MS F-Value p-Value

Model 9 957.15 106.350 101.29 0.000
Linear 3 579.250 193.083 183.89 0.000
X1 1 136.125 136.125 129.64 0.000
X2 1 338.000 338.000 321.90 0.000
X3 1 105.125 105.125 100.72 0.000
Square 3 81.650 27.217 25.92 0.002
X21 1 55.442 55.442 52.80 0.001
Cellulose X22 1 4.673 4.673 4.45 0.089
(%) X23 1 30.519 30.519 29.07 0.003
2-Way Interaction 3 296.250 98.750 94.05 0.000
X1 *X2 1 100.000 100.000 95.24 0.000
X1 *X3 1 0.250 0.250 0.24 0.646
X2 *X3 1 196.000 196.000 186.67 0.000
Error 5 5.250 1.050
Lack-of-Fit 3 3.250 1.08 1.08 0.513
Pure Error 2 2.000 1.000
Total 14 962.400
Model 9 50,737.2 5637.5 4398.87 0.000
Linear 3 44,404.2 14,801.4 11,549.41 0.000
X1 1 14,257.0 14,257.0 11,124.64 0.000
X2 1 29,330.2 29,330.2 22,886.10 0.000
X3 1 817.0 817.0 637.51 0.000
Square 3 2971.1 990.4 772.77 0.000
X21 1 568.6 568.6 443.66 0.000
TS X22 1 1853.0 1853.0 1445.89 0.000
(mg/mL) X23 1 426.9 426.9 333.08 0.000
2-Way Interaction 3 3361.9 1120.6 874.43 0.000
X1 *X2 1 30.4 30.4 23.75 0.000
X1 *X3 1 1048.7 1048.7 818.26 0.000
X2 *X3 1 2282.8 2282.8 1781.28 0.000
Error 5 6.4 1.3
Lack-of-Fit 3 4.8 1.6 2.06 0.343
Pure Error 2 1.6 0.8
Total 14 50,743.6
X1 = base conc., X2 = substrate conc., X3 = retention time, TS = total sugars.

Table 7. ANOVA of TS (mg/mL) and RS after NaOH + steam pretreatment.

Source DF Adj SS Adj MS F-Value p-Value

Model 9 42,001.4 4666.8 8.30 0.016
Linear 3 37,490.7 12,496.9 22.24 0.003
X1 1 1573.6 1573.6 2.80 0.155
X2 1 35,539.1 35,539.1 63.24 0.001
X3 1 378.0 378.0 0.67 0.449
Square 3 1436.1 478.7 0.85 0.523
X21 1 449.4 449.4 0.80 0.412
TP X22 1 65.6 65.6 0.12 0.746
(mg/mL) X23 1 826.9 826.9 1.47 0.279
2-Way Interaction 3 3074.6 1024.9 1.82 0.260
X1 *X2 1 1102.2 1102.2 1.96 0.220
X1 *X3 1 88.4 88.4 0.16 0.708
X2 *X3 1 1884.0 1884.0 3.35 0.127
Error 5 2809.7 561.9
Lack-of-Fit 3 2806.8 935.6 626.51 0.002
Pure Error 2 3.0 1.5
Total 14 44,811.1
Fermentation 2022, 8, 386 8 of 22

Table 7. Cont.

Table 8. ANOVA of TS (mg/mL) and RS after NaOH + steam pretreatment.

Source DF Adj SS Adj MS F-Value p-Value

Model 9 8159.46 906.61 131.94 0.000
Linear 3 7106.45 2368.82 344.73 0.000
X1 1 736.51 736.51 107.18 0.000
X2 1 6315.76 6315.76 919.13 0.000
X3 1 54.18 54.18 7.89 0.038
Square 3 787.37 262.46 38.20 0.001
X21 1 127.04 127.04 18.49 0.008
2 1 694.11 694.11 101.01 0.000
RS X 2
2 1 0.00 0.00 0.00 1.000
(mg/mL) X 3
2-Way Interaction 3 265.63 88.54 12.89 0.009
X1 *X2 1 210.83 210.83 30.68 0.003
X1 *X3 1 54.76 54.76 7.97 0.037
X2 *X3 1 0.04 0.04 0.01 0.939
Fermentation 2022, 8, x FOR PEER REVIEW Error 5 34.36 6.87 9 of 23
Lack-of-Fit 3 25.55 8.52 1.93 0.359
Pure Error 2 8.81 4.40
Total 14 8193.82
X1 =Xbase
1 = base conc., X2 = substrate conc., X3 = retention time, TP = total phenol, RS = reducing sugar.
conc., X2 = substrate conc., X3 = retention time, TP = total phenol, RS = reducing sugar.

Figure 2. Cont.
Fermentation 2022, 8, 386 9 of 22

Fermentation 2022, 8, x FOR PEER REVIEW 10 of 23

Figure Contourplots
2. Contour
Figure 2. plotsforfor
TS,TS, cellulose,
cellulose, TP, RS
TP, and andliberated
RS liberated after chemical
after chemical pretreatment
pretreatment of seed of
seed
pods.pods.

Figure 3. Cont.
Fermentation 2022, 8, 386 10 of 22

Figure 3.
Figure Contour plots
3. Contour plots for
for TS,
TS, cellulose,
cellulose, TP,
TP, and
and RS
RS liberated
liberated after
after thermochemical
thermochemical pretreatment
pretreatment of
of
seed pods.

Samples with
Samples with the
the maximum
maximum cellulose
cellulose content
content were
were analyzed
analyzed for lignin content.
for lignin content. It
It was
was
found that the sample from the chemical treatment had 15% lignin, while the sample
found that the sample from the chemical treatment had 15% lignin, while the sample from from
the thermochemical
thermochemical treatment
Fermentation 2022, 8, x FOR PEER REVIEW
the treatmenthad
had9%
9%lignin,
lignin,whereas
whereasthe
theuntreated
untreated sample
samplehad 25%
had 25%lignin
11 of 23
lig-
content
nin (Figure
content 4). 4).
(Figure

NaOH steam

NaOH

Raw

0 5 10 15 20 25 30
Lignin (%)

Figure4.4.Lignin
Figure Lignincontent
contentininraw,
raw,NaOH
NaOHpretreated,
pretreated,and
andNaOH
NaOH+ +steam
steampretreated
pretreatedB.B.ceiba
ceibabiomass.
biomass.

Pretreatmentofoflignocellulosic
Pretreatment lignocellulosicbiomass
biomassisisrequired
requiredtotomakemakesaccharifying
saccharifyingenzymeenzyme
accessibletotocellulose
accessible cellulosefor
forhydrolysis
hydrolysistotoyield
yieldhigh
highsugar,
sugar,asaslignin
ligninisisaabarrier
barrierprotecting
protecting
celluloseagainst
cellulose againstenzyme
enzymeattack
attack[30].
[30].Hence,
Hence,pretreatment
pretreatmentprior priortotosaccharification
saccharificationisisaa
mandatorystep
mandatory steptotominimize
minimizethe thelignin
lignincontent
contentand
andenhance
enhancethe thesurface
surfacearea
areaforforbetter
better
enzymeaction.
enzyme action.In Inthis
thisstudy,
study,wewefound
foundthatthatNaOH
NaOHpretreatment
pretreatmentfollowed
followedby bysteam
steamwas was
more effective in the delignification of seed pods of B. ceiba, revealing
more effective in the delignification of seed pods of B. ceiba, revealing 60% cellulose and60% cellulose and
9%9%lignin. Cellulose content
lignin. Cellulose contentincreased
increasedfrom from34%34%toto60%60%duedueto atoreduction
a reduction in lignin
in lignin con-
content
tent (from(from25% 25% to 9%).
to 9%). Similarly,
Similarly, totaltotal sugar,
sugar, phenol,
phenol, and and reducing
reducing sugars
sugars liberated
liberated were
were
also also greater
greater in thermochemical
in thermochemical pretreatment
pretreatment compared
compared to chemical
to chemical treatment
treatment alone.
alone. As-
Asghar
ghar etetal.al.[32]
[32]reported
reportedmaximum
maximumcellulose
celluloseexposure
exposure(60.6%)
(60.6%)atat2.5%
2.5%NaOH
NaOHafter after2424h hof
ofsoaking
soaking inin the
the cotton
cotton stalk,
stalk, whereas
whereas thethe maximum
maximum cellulose
cellulose (73.19%)
(73.19%) waswas obtained
obtained at
at 121
121 ◦ C after a 1 h soaking time with 2.5% NaOH, and we found 60% cellulose content
°C after a 1 h soaking time with 2.5% NaOH, and we found 60% cellulose content in seed
inpods
seedatpods at thermochemical
thermochemical conditions.
conditions. Ghazanfar
Ghazanfar and coworkers
and coworkers [2], using
[2], using another another
alkali,
alkali,
foundfound the maximum
the maximum releaserelease of RS mg/mL)
of RS (50.06 (50.06 mg/mL)
and TP and TP mg/mL)
(394.04 (394.04 mg/mL)
at a 15% at a
sub-
strate concentration, 8 h residence time, and 3% KOH concentration at 121 °C and reported
maximum TS (206.65 mg/mL) liberated and cellulose exposed (64%) at the same temper-
ature with a 10% substrate loading and 5% KOH concentration for a 8 h soaking time.
Their findings also collaborated with our results, as they obtained maximum cellulose
Fermentation 2022, 8, 386 11 of 22

15% substrate concentration, 8 h residence time, and 3% KOH concentration at 121 ◦ C

and reported maximum TS (206.65 mg/mL) liberated and cellulose exposed (64%) at the
same temperature with a 10% substrate loading and 5% KOH concentration for a 8 h
soaking time. Their findings also collaborated with our results, as they obtained maximum
cellulose (46%), RS (9.0075 mg/mL), TP (300.3901 mg/mL), and TS (146.1480 mg/mL)
at a 15% substrate concentration, using 3% KOH solution, and an 8 h soaking time at
room temperature.
Another study by Asghar and fellows [33] used BBD to optimize pretreatment condi-
tions and found that Saccharum spontaneum offered maximum cellulose (52.5%) after 24 h
of residence time with 2.5% NaOH. In the thermochemical pretreatment, S. spontaneum
was soaked in NaOH solution for 2 h followed by autoclaving for different durations
(15–60 min) at 121 ◦ C, suggesting 60 min duration as best for maximum cellulose exposure
up to 81.2%. Nadeem et al. [34] also found results similar to our findings in that physico-
chemical treatment resulted in a greater breakdown of hemicellulose and lignin and hence
liberated more sugars and phenolic contents. They soaked powdered bagasse for 1 h in a
NaOH solution of 2.5% and then autoclaved for 45 min at 126 ◦ C and achieved 9% lignin
and 74% cellulose content, whereas untreated substrate had 35% cellulose content and 25%
lignin. The results of Sarbishei and coworkers [35] were opposite to our results because
they noted a decline in cellulosic contents (from 44% to 27.6%) of tobacco product waste
after 10% NaOH pretreatment because of the hydrolysis of carbohydrates by alkali. A
recent study by Gunam et al. [36] found maximum cellulose of 65.46% from pretreatment
of corn straw with 4% NaOH.
Regression equations for NaOH pretreatment:

Cellulose (%) = 77.3 + 1.00X1 − 0.38X2 − 13.12X3 − 0.146 X21 + 0.0467X22

(1)
+ 0.917X23 − 0.325X1 ∗ X2 + 0.187X1 ∗ X3 + 0.150X2 ∗ X3

TS (mg/mL) = 1366.3 − 272.37X1 − 18.11X2 − 254.34 X3 + 39.248X21

(2)
+ 8.2273X22 + 31.810X23 + 10.155X1 ∗ X2 + 11.927X1 ∗ X3 − 19.348X2 ∗ X3
RS (mg/mL) = 12.0 − 8.85X1 − 3.213X2 + 4.48X3 + 0.942X21 + 0.1390X22
(3)
− 0.153X23 + 0.767 X1 ∗ X2 − 0.529 X1 ∗ X3 − 0.1183X2 ∗ X3
TP (mg/mL) = −379 + 33.5X1 − 56.7X2 + 211.6X3 + 12.57X21 + 1.79X22
(4)
− 8.79X23 − 0.06X1 ∗ X2 − 25.23X1 ∗ X3 + 4.12X2 ∗ X3
Regression equations for NaOH steam pretreatment:

Cellulose (%) = 76.91 − 9.13X1 + 0.500X2 − 3.63X3 + 0.969X21 + 0.0450X22

(5)
+ 0.719 X23 + 0.5000 X1 ∗ X2 + 0.062 X1 ∗ X3 − 0.7000 X2 ∗ X3

TS (mg/mL) = −357.19 + 24.02X1 + 43.538X2 + 63.24X3 + 3.102X21

(6)
− 0.8961X22 − 2.688X23 + 0.2758X1 ∗ X2 − 4.048X1 ∗ X3 − 2.3889X2 ∗ X3
RS (mg/mL) = 0.6 − 0.79 X1 − 3.11X2 + 4.18X3 + 1.466X21 + 0.5484X22
(7)
+ 0.000X23 − 0.726X1 ∗ X2 − 0.925 X1 ∗ X3 − 0.011X2 ∗ X3
TP (mg/mL) = −50 + 33.2X1 + 28.0X2 − 23.1X3 − 2.76X21 + 0.169X22
(8)
+ 3.74X23 − 1.66X1 ∗ X2 − 1.18X1 ∗ X3 − 2.17X2 ∗ X3

3.2. FTIR
FTIR analysis pointed out chemical changes in pretreated seed pods compared to
those untreated (Figure 5). The change in a peak from 3300.6 cm−1 to 3330.4 cm−1 showed
–OH band stretching. The intensity of –OH increased, which showed the influence of NaOH
on B. ceiba. The absorption band between 3200 and 3600 cm−1 is usually assigned to the
O–H stretching vibrations of alcohols, carboxylic acids, and hydroperoxides [5]. In this
 TP (mg/mL) = −50 + 33.2X1 + 28.0X2 − 23.1X3 − 2.76X + 0.169X + 3.74X − 1.66X1*X2 − 1.18X1*X3 − 2.17X2*X3

3.2. FTIR
FTIR analysis pointed out chemical changes in pretreated seed pods compared to
those untreated (Figure 5). The change in a peak from 3300.6 cm−1 to 3330.4 cm−1 showed
Fermentation 2022, 8, 386 12 of 22
–OH band stretching. The intensity of –OH increased, which showed the influence of
NaOH on B. ceiba. The absorption band between 3200 and 3600 cm−1 is usually assigned to
the O–H stretching vibrations of alcohols, carboxylic acids, and hydroperoxides [5]. In this
study,
study, the
thepeak
peakchanges
changes from
from1023.2 cmcm
1023.2 −1 to−1026.9
1 to 1026.9
cm−1 in −1 in samples
cmsamples are related
are to C–O and
related to C–O
C–H deformations that show cellulose breakdown. The peak in
and C–H deformations that show cellulose breakdown. The peak in untreateduntreated B. ceiba at 1593.4
B. ceiba
denoted
at 1593.4lignin ringlignin
denoted stretches,
ringbut these bands
stretches, were bands
but these stretched andstretched
were decreased in chemical
and decreased in
and thermochemical
chemical pretreatment,
and thermochemical respectively,
pretreatment, representing
respectively, the breakdown
representing of lignin of
the breakdown
due to pretreatment.
lignin The peakThe
due to pretreatment. at 896.4
peakcmat−1 896.4
in bothcm
treated
−1 in samples represented
both treated samples vibrations
represented
at β-glucosidic
vibrations bonds in C–O–C
at β-glucosidic bondsin in
hemicelluloses and celluloses.and celluloses.
C–O–C in hemicelluloses

Fermentation 2022, 8, x FOR PEER REVIEW 13 of 23

(a)

(b)

(c)
Figure
Figure5.5.FTIR
FTIRanalysis
analysisofof
(a)(a)
untreated, (b)(b)
untreated, NaOH-treated, (c) NaOH
NaOH-treated, + steam
(c) NaOH -treated seed pods.
+ steam - treated seed pods.

FTIR analysis suggested that NaOH pretreatment efficiently alters the linkages in bi-
omass. The peak 3334 cm−1 depicts the absorption of –OH of alcoholic hydroxyl [37]. An-
other study by Irfan et al. [38] reported a peak shift from 3336 cm−1 (raw) to 3315.26 cm−1
(pretreated). This change indicated –OH band extension in the pretreated substrate. The
peak at 1315 cm−1 depicted hemicellulose in the untreated sample. CH2 stretching in cel-
lulose is indicated by the peaks from 1370 to 1430 cm−1. The peaks at 1500 cm−1 are related
Fermentation 2022, 8, 386 13 of 22

FTIR analysis suggested that NaOH pretreatment efficiently alters the linkages in
biomass. The peak 3334 cm−1 depicts the absorption of –OH of alcoholic hydroxyl [37]. An-
other study by Irfan et al. [38] reported a peak shift from 3336 cm−1 (raw) to 3315.26 cm−1
(pretreated). This change indicated –OH band extension in the pretreated substrate. The
peak at 1315 cm−1 depicted hemicellulose in the untreated sample. CH2 stretching in cellu-
lose is indicated by the peaks from 1370 to 1430 cm−1 . The peaks at 1500 cm−1 are related to
the extension in the C=C bond from the lignin’s aromatic ring. The peak at 1030 cm−1 and
1034 cm−1 found in the untreated and pretreated substrate is associated with C–C–O, C=O,
and C–O of cellulose. The peak at 890 cm−1 showed C–O–C vibrations at β-glucosidic
bonds in cellulose. Zhang et al. [39] labeled the band at 1032 cm−1 with polysaccharides.
The reduction in the crystallinity of cellulose might be characterized by the breakdown of
structural hydrogen bonding of cellulose chains and the partial conversion of crystalline
parts to amorphous ones. The decreased band intensity around 3350 cm−1 could be linked
with the slackening of the intra- and inter-molecular O–H bond of cellulose, proposing that
the extremely ordered cellulosic system was converted to a more amorphous form.

3.3. XRD
Fermentation 2022, 8, x FOR PEER REVIEW 14 of 23
Figure 6 revealed the XRD spectra of raw and pretreated (both chemical and thermo-
chemical) samples. The crystallinity index (CI) demonstrated the crystalline structure of the
cellulose. The CI of the untreated substrate was 34.5%, which improved in NaOH- treated
treated (51.2%),
(51.2%), and NaOH and+NaOH + steam-treated
steam-treated seed(51.1%).
seed pods pods (51.1%). The increase
The increase in CI in CI showed
showed the
the removal
removal of amorphous
of amorphous components,
components, such such as hemicellulose
as hemicellulose and lignin,
and lignin, from from the bio-
the biomass.
mass. Earlier,
Earlier, it was described
it was described that thethat the concentrations
concentrations of the of thewere
peak peaklinked
were linked
with thewith the
crys-
tallinity, whichwhich
crystallinity, increases with pretreatment
increases and is associated
with pretreatment with thewith
and is associated decline
the in the surface
decline in the
area. Increasing
surface CI depicted
area. Increasing cellulosecellulose
CI depicted exposure.exposure .

Raw NaOH NaOH steam

400
350
Intensity (arb. units)

300
250
200
150
100
50
0
0 10 20 30 40 50
Angle (2θ)

Figure6.6.XRD
Figure XRDanalysis
analysisofofseed
seedpods
podsofofB.B.ceiba
ceibatreated
treatedwith
withNaOH
NaOHand
andNaOH
NaOH++steam.
steam.

XRDrevealed
XRD revealedthat
thatthetheCICIofofpretreated
pretreatedsamples
sampleswas wasimproved
improvedcompared
comparedto tothe
theraw
raw
samples,which
samples, whichindicated
indicatedthe theremoval
removalofofhemicellulose
hemicelluloseand andlignin
ligninfrom
fromthe
thecrystalline
crystallinepart
part
ofofthe
thebiomass,
biomass,cellulose
cellulose[40].
[40].Our
Ourresults
results were
were in in accordance
accordance withwith earlier
earlier studies
studies describ-
describing
ing increased CI after different types of pretreatments using various agricultural
increased CI after different types of pretreatments using various agricultural wastes [40–43]. wastes
A[40–43]. A recent
recent XRD study XRD study byetGunam
by Gunam et al. [36]changes
al. [36] revealed revealed inchanges in the crystallinity
the crystallinity degree of
degree of NaOH-pretreated
NaOH-pretreated corn straw. corn straw.
Research byResearch
Singh and bycoworkers
Singh and[44] coworkers
reported[44]
thatreported
the CI
(36.96%)
that the ofCIuntreated
(36.96%) of jute biomassjute
untreated declined to 23.61%
biomass declined andto 18.42%
23.61% after 2% NaOH
and 18.42% afterand
2%
2%
NaOH H2 SOand
4 treatment,
2% H 2SO 4 respectively.
treatment, This decrease
respectively. This in CI may
decrease be
in CIdue
mayto the
be degradation
due to the deg-
of intra- and
radation inter-hydrogen
of intra- bonding in
and inter-hydrogen the crystalline
bonding cellulose cellulose
in the crystalline resultingresulting
in an altered
in an
crystal structure. Awoyale and Lokhat [5] observed peaks of reduced
altered crystal structure. Awoyale and Lokhat [5] observed peaks of reduced intensities intensities in thein
pretreated
the pretreatedbiomass samples,
biomass a representation
samples, a representationof incomplete
of incomplete breakdown
breakdown of the
of cellulose
the cellu-
with
lose pretreatment.
with pretreatment.

3.4. TGA
To study the thermal degradation behavior of raw and treated (with maximum cel-
lulose from each pretreatment) B. ceiba, TGA was performed. Figure 7a revealed decom-
position in line with temperature and time; 9.194% decomposition was observed at 100–
Fermentation 2022, 8, 386 14 of 22

3.4. TGA
To study the thermal degradation behavior of raw and treated (with maximum cel-
lulose from each pretreatment) B. ceiba, TGA was performed. Figure 7a revealed de-
composition in line with temperature and time; 9.194% decomposition was observed at
100–200 ◦ C (first stage), 50.02% at 300–400 ◦ C (second stage), and 33.39% at 500–600 ◦ C
(third stage). During the first stage, the NaOH-treated substrate presented 7.94% con-
version, 44.50% during the second stage, and 28.81% during the third stage
Fermentation 2022, 8, x FOR PEER REVIEW
(Figure 7b),
15 of 23
whereas NaOH + steam-pretreated seed pods showed degradation of 7.291% during the
first stage, 65.62% during the second stage, and 26.38% at 500–600 ◦ C (Figure 7c).

(a)

(b)

Figure 7. Cont.
 Fermentation
Fermentation 2022,8,8,386
2022, x FOR PEER REVIEW 16 of 23 15 of 22

(c)
Figure
Figure7.7.TGA
TGAofof
B.B.
ceiba seed
ceiba pods.
seed (a) Raw,
pods. (b) NaOH-pretreated,
(a) Raw, (c) NaOH
(b) NaOH-pretreated, + steam-pretreated.
(c) NaOH + steam-pretreated.

TGA
TGAdepicted
depictedmaximum
maximum weight
weightloss at the
loss temperature
at the temperaturerange of the
range of second stagestage
the second in in all
all
substrates, whereas maximum degradation of 65.62% was observed in the NaOH ++ steam-
substrates, whereas maximum degradation of 65.62% was observed in the NaOH
steam-treated biomass. Another study found the highest (74.48%) degradation of Pinus
treated biomass. Another study found the highest (74.48%) degradation of Pinus ponderosa
ponderosa (sawdust), followed by Shorea robusta (sawdust) (70.03%) and Areca catechu (nut
(sawdust), followed by Shorea robusta (sawdust) (70.03%) and Areca catechu (nut husk)
husk) (69.09%) at a temperature range of 200–500 °C (second stage). Hemicellulose is de-
(69.09%) at a temperature range of 200–500 ◦ C (second stage).
graded at temperatures of ◦180–340 °C, cellulose at 230–450
Hemicellulose is degraded at
°C, and lignin decomposed at
temperatures of 180–340 C, cellulose at 230–450 ◦ C, and lignin decomposed at temperatures
temperatures greater than 500 °C [45]. A recent study by Tsegaye and others [29] reported
greater ◦
that the than
rate of500
lossCof[45]. A recent
weight study
was very highby(nearly
Tsegaye andatothers
80%) [29] reported
a temperature range that the rate of
of 200–
loss of weight was very high (nearly 80%) at a temperature range of 200–500 ◦ C for all the
500 °C for all the employed treatments.
employed treatments.
3.5. SEM
3.5. SEM
The structural modifications that appeared after pretreatment were observed by SEM
images.TheMicrographs
structural modifications that appeared
verified the surface after and
characteristics pretreatment
structural were observed
changes in seedby SEM
images.
pods of B.Micrographs
ceiba caused by verified the surface
pretreatment. characteristics
The NaOH and structural
reaction damaged changes
the surface of B. in seed
ceiba, of B. ceibasome
podsgenerating caused by pretreatment.
irregular cracks and pores.TheSEMNaOH reaction
images revealeddamaged the dif-
a significant surface of
ference
B. ceiba,between the surface
generating some structures
irregular of pretreated
cracks and untreated
and pores. B. ceibarevealed
SEM images (Figure 8).a The
significant
raw sample between
difference exhibited the
a complex
surfaceorder and dense
structures structure, while
of pretreated both the treated
and untreated speci-
B. ceiba (Figure 8).
mens
The rawrevealed
samplea greater degree
exhibited of porosity.
a complex The number
order and dense andstructure,
size of pores confirmed
while both thethe treated
effectiveness of NaOHa+greater
specimens revealed steam pretreatment in delignification.
degree of porosity. The number and size of pores confirmed
the effectiveness of NaOH + steam pretreatment in delignification.
Thus, pretreatment could efficiently lessen the crystallinity of the B. ceiba, as reported
earlier for corn leaf and cattail’s narrow leaf by Donghai et al. [46] and Ruangmee and
Sangwichien [47]. This shows that alkali pretreatment can remove a significant percentage
of lignin and hemicellulose. Jabasingh and Nachiyar [48] also detected such variations
in bagasse. Awoyale and Lokhat [5] reported that the micrographs of the alkali-treated
lignocellulosic substrate demonstrate more fragmentation and degradation, indicating
the efficacy of alkali pretreatment over the other pretreatment processes performed in
the research. Kusmiyati et al. [49] observed uneven surfaces with pores in treated palm
tree stem waste through SEM. Sindhu et al. [50] also observed differences in the surface
structure of native and pretreated bamboo substrates.
Fermentation 2022,
Fermentation 8, x8,FOR
2022, 386 PEER REVIEW 17 of 23
16 of 22

8. Scanning
Figure 8.
Figure Scanningelectron microscopy
electron images
microscopy of seedofpods.
images seed(a) NaOH-pretreated,
pods. (b) NaOH +(b)
(a) NaOH-pretreated, steam,
NaOH +
(c) raw.
steam, (c) raw.
3.6. Hydrolysis and Fermentation
Thus, pretreatment could efficiently lessen the crystallinity of the B. ceiba, as reported
Samples with maximum cellulose contents were used for saccharification and pro-
earlier for corn leaf and cattail’s narrow leaf by Donghai et al. [46] and Ruangmee and
duction of ethanol via SHF and SSF. In SHF, substrates with maximum cellulose content
Sangwichien [47]. This shows that alkali pretreatment can remove a significant percentage
from both treatments were saccharified by using indigenously produced cellulase as well
ofaslignin and hemicellulose.
commercial cellulase. SugarsJabasingh and Nachiyar
obtained from [48] also detected
these saccharifications such
were then variations in
fermented
bagasse.
by using Saccharomyces cerevisiae. During SSF, the pretreated biomass was added with lig-
Awoyale and Lokhat [5] reported that the micrographs of the alkali-treated
nocellulosic
indigenouslysubstrate
produceddemonstrate morethe
enzyme to form fragmentation
maximum sugarsand degradation,
needed for theindicating
produc- the
efficacy of alkaliand
tion of ethanol pretreatment over the
then incorporated with other
yeastpretreatment processes
culture. Similar SSF wasperformed in the re-
repeated with
commercial
search. cellulase.
Kusmiyati The[49]
et al. results of SHFuneven
observed with commercial cellulase
surfaces with showed
pores maximum
in treated palm tree
saccharification
stem waste throughafterSEM.
24 h Sindhu
in NaOH et+al.steam-pretreated substrate
[50] also observed (52.6%),
differences followed
in the surfacebystruc-
NaOH-treated (40.8%) and untreated substrates
ture of native and pretreated bamboo substrates. (16.4%). Maximum saccharification of
37% with indigenously produced cellulase was recorded in NaOH + steam-pretreated
seed pods. The fermentation of these hydrolysates resulted in the production of ethanol.
3.6. Hydrolysis and Fermentation
Hydrolysates obtained with indigenous cellulase offered maximum bioethanol titer (g/L)
Samples
of 28.7 in NaOHwith maximum cellulose
+ steam-treated, contents
16.01 in NaOH, andwere used
8.73 in for saccharification
untreated B. ceiba after 96 hand
of pro-
duction of ethanol
fermentation. Sugarsviaobtained
SHF and SSF.
with In SHF, substrates
commercial with maximum
cellulase presented a maximumcellulose content
ethanol
from
yieldboth treatments
of 48.8 were+saccharified
g/L in NaOH steam-treated,by39.63
usingg/L
indigenously produced
in NaOH-treated, and cellulase
15.6 g/L in as well
untreated biomass (Figure 9).
as commercial cellulase. Sugars obtained from these saccharifications were then fer-
mented by using Saccharomyces cerevisiae. During SSF, the pretreated biomass was added
with indigenously produced enzyme to form the maximum sugars needed for the pro-
duction of ethanol and then incorporated with yeast culture. Similar SSF was repeated
with commercial cellulase. The results of SHF with commercial cellulase showed maxi-
mum saccharification after 24 h in NaOH + steam-pretreated substrate (52.6%), followed
by NaOH-treated (40.8%) and untreated substrates (16.4%). Maximum saccharification of
37% with indigenously produced cellulase was recorded in NaOH + steam-pretreated
seed pods. The fermentation of these hydrolysates resulted in the production of ethanol.
Hydrolysates obtained with indigenous cellulase offered maximum bioethanol titer (g/L)
of 28.7 in NaOH + steam-treated, 16.01 in NaOH, and 8.73 in untreated B. ceiba after 96 h
Fermentation 2022, 8, x FOR PEER REVIEW 18 of 23

of fermentation. Sugars obtained with commercial cellulase presented a maximum etha-

Fermentation 2022, 8, 386 nol yield of 48.8 g/L in NaOH + steam-treated, 39.63 g/L in NaOH-treated, and
17 of15.6
22 g/L in
untreated biomass (Figure 9).

Figure 9.9.Separate
Figure Separatehydrolysis
hydrolysisandand
fermentation. (a) Saccharification
fermentation. after 24after
(a) Saccharification h. (b)24Ethanol producedproduced
h. (b) Ethanol
after 96
after 96h.h.

The results of SSF showed a decrease in sugar every 24 h; this could be due to
The results of SSF showed a decrease in sugar every 24 h; this could be due to the
the sugar consumption by yeast and subsequent ethanol production increase every 24 h
sugar consumption by yeast and subsequent ethanol production increase every 24 h due
due to fermentation. After adding indigenously produced cellulase and 1% S. cerevisiae,
to fermentation.
maximum After adding
saccharification indigenously
in untreated produced
lignocellulosic biomasscellulase
(17.3%)andwas 1% S. cerevisiae,
observed after max-
imum
48 h of inoculation, while chemically and thermochemically pretreated substrates showed after 48
saccharification in untreated lignocellulosic biomass (17.3%) was observed
h of inoculation,
maximum while chemically
saccharification (31.12% andand thermochemically
40.8%, respectively) after pretreated
24 h. Maximum substrates
ethanol showed
maximum
production wassaccharification
observed at 11.2(31.12% and
g/L, 18.7 40.8%,
g/L, respectively)
and 38.8 g/L in raw, after 24 h. treated,
chemically Maximum and ethanol
thermochemically pretreated substrates, respectively, after 96 h of fermentation
production was observed at 11.2 g/L, 18.7 g/L, and 38.8 g/L in raw, chemically treated, (Figure 10). and
Sugars obtained with commercial cellulase offered the highest ethanol
thermochemically pretreated substrates, respectively, after 96 h of fermentation production in (Figure
SSF. Maximum saccharification (50.9%) in SSF was seen with commercial cellulase in
10). Sugars obtained with commercial cellulase offered the highest ethanol production in
NaOH + steam-treated seed pods, which subsequently resulted in the highest ethanol
SSF. Maximum saccharification (50.9%) in SSF was seen with commercial cellulase in
(54.51 g/L) production after 96 h of fermentation. Maximum bioethanol yield in NaOH-
NaOH + steam-treated
treated (29.76 g/L) and rawseed pods,(19.87
seed pods which
g/L)subsequently resulted
was also obtained after 96inh the highest ethanol
fermentation
(54.51
(Figureg/L)
11). production after 96 h of fermentation. Maximum bioethanol yield in NaOH-
treated (29.76 g/L) and raw seed pods (19.87 g/L) was also obtained after 96 h fermentation
(Figure 11).
 Fermentation
Fermentation 8, 8,x 386
2022,
2022, FOR PEER REVIEW 18 of 22 19 of 23

24 h 48 h 72 h
50

40
Saccharification (%)
30

20

10

0
Raw NaOH NaOH steam (a)

24 h 48 h 72 h 96 h 120 h
50

40
Ethanol (g/l)

30

20

10

0
Raw NaOH NaOH steam
(b)

Figure 10. SSF

Figure 10. with indigenous
SSF with indigenouscellulase
cellulaseand
and
S. S. cerevisiae.
cerevisiae. (a)(a) Saccharification.
Saccharification. (%)(%)
(b) (b) Ethanol pro-
Ethanol
duced (g/L).
produced (g/L).

We used two different approaches, e.g., SHF and SSF, for saccharification and fermen-
tation of the pretreated substrate. In this study, the highest fermentable sugars in terms of
saccharification, 50.9% (after 24 h), and the highest bioethanol yield, 54.51 g/L (after 96 h),
were obtained from SSF with the commercial enzyme and S. cerevisiae. SSF with indigenous
cellulase gave a relatively low yield. In the case of SHF, results with commercial cellulase
were better than that of indigenous cellulase, but the overall yield was less than SSF. The
results of Sukhang et al. [51] and Vintila et al. [52] corroborated our findings that the SSF
contributed to higher ethanol production from lignocellulosic biomass than that of SHF.
A study by Triwahyuni [53] noticed a notable ethanol titer from SHF of oil palm empty
fruit bunch. Hydrolysis for 96 h produced 75.48% glucose, which subsequently yielded
78.95% ethanol. Ballesteros et al. [54] found the highest ethanol yield in SSF after 72 h of
fermentation. The reason for the good yield of ethanol in SSF could be the direct conversion
of produced sugars into ethanol, thus evading any feedback inhibition. Barron et al. [55]
employed other strains of yeast for ethanol production and found that Kluveromyces marxi-
anus produced 10 g/L ethanol after a 60 h fermentation period, and Pachysolen trannophylus
produced 11.8 g/L ethanol from the hydrolysate of wheat straw.
Fermentation 2022, 8, x FOR PEER REVIEW 20 of 23
Fermentation 2022, 8, 386 19 of 22

Figure 11.
Figure SSF with
11. SSF with commercial
commercial cellulase andS.S.cerevisiae.
cellulaseand cerevisiae.(a)(a)Saccharification.
Saccharification.(%)(%)
(b)(b)
Ethanol
Ethanol pro-
produced
duced (g/L).
(g/L).

Tan and Lee [56] noticed a better yield of bioethanol in SSF (90.9%) compared to SHF
We used two different approaches, e.g., SHF and SSF, for saccharification and fer-
(55.9%). SSF of seaweed biomass by means of S. cerevisiae had several advantages over SHF,
mentation
as the earlierofapproach
the pretreated substrate.
is a simple single-stepIn process
this study, thesave
that can highest fermentable
time, costs, and energysugars in
terms of saccharification,
while achieving 50.9%titer.
a high bioethanol (after 24 h),found
A study and athe highestethanol
maximum bioethanol
yield yield, 54.51 g/L
of 85.71%
(after ◦96 h), were obtained from SSF with the commercial enzyme
at 30 C with 2% wheat straw and 30 FPU of enzyme loading in SSF Ruiz et al. [57]. Findings and S. cerevisiae. SSF
with indigenous
of research cellulase
on bioethanol gave a relatively
production low yield.
from rice husk In the case
also supported ourof SHF,that
results results with com-
SSF was
better than
mercial SHF inwere
cellulase yielding a good
better thanethanol titer
that of [58]. Ahmad
indigenous et al. [59]but
cellulase, showed that sweet
the overall yield was
sorghum and sago biomasses yielded maximum ethanol at the end of 72
less than SSF. The results of Sukhang et al. [51] and Vintila et al. [52] corroborated ourh of fermentation.
Peace and
findings others
that the [60]
SSFused wood shavings
contributed for bioethanol
to higher production.from
ethanol production Theylignocellulosic
noticed that bio-
S. cerevisiae was able to convert 60.97% brix in wood extract into bioethanol at optimized
mass than that of SHF. A study by Triwahyuni [53] noticed a notable ethanol titer from
conditions of 15.66 g wood concentration, 4.47% NaOH concentration, 0.85 inoculum size,
SHF
72 h of oil palmtime,
incubation empty andfruit
a 40 bunch. Hydrolysis
◦ C incubation for 96 The
temperature. h produced 75.48%
ethanol titer glucose,
obtained was which
subsequently
1.68–2.25%. As various studies have used different plant biomasses in order to produce yield
yielded 78.95% ethanol. Ballesteros et al. [54] found the highest ethanol
inbioethanol,
SSF after this
72 hstudy
of fermentation.
suggested seed Thepods
reason
of B.for theasgood
ceiba yield of
a potential ethanol source
renewable in SSFof could be
the direct
green conversion
energy to be usedofon produced sugars into ethanol, thus evading any feedback inhibi-
a pilot scale.
tion. Barron et al. [55] employed other strains of yeast for ethanol production and found
that Kluveromyces marxianus produced 10 g/L ethanol after a 60 h fermentation period, and
Pachysolen trannophylus produced 11.8 g/L ethanol from the hydrolysate of wheat straw.
Tan and Lee [56] noticed a better yield of bioethanol in SSF (90.9%) compared to SHF
(55.9%). SSF of seaweed biomass by means of S. cerevisiae had several advantages over
SHF, as the earlier approach is a simple single-step process that can save time, costs, and
Fermentation 2022, 8, 386 20 of 22

4. Conclusion
The results of this study revealed that B. ceiba could be utilized as a potential lignocellu-
losic biomass source for bioethanol production. NaOH pretreatment significantly exposed
cellulose (60%), which was further hydrolyzed to fermentable sugars (50.9% saccharifi-
cation), which yielded better ethanol (54.51 g/L) production by Saccharomyces cervisae in
SSF. The findings of this research recommended that this biomass could be an encouraging
feedstock for the large-scale production of second-generation bioethanol.

Author Contributions: Conceptualization, M.I.; methodology, M.G.; software, M.N.; validation,

M.N.; formal analysis, H.A.S.; investigation, M.G; resources, M.K.; data curation, M.F.; writing—
original draft preparation, M.G.; writing—review and editing, M.I.; supervision, M.I.; funding
acquisition, I.A. and L.C. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Scientific Research Deanship at King Khalid University,
Abha, Saudi Arabia, via their financial support through the Small Research Group Project under grant
number (RGP.01-115-43), the National Key R & D Program of China (grant no. 2019YFD1001002), and
the discipline construction of a professional degree (grant no. 880220039) China.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Not Applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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