Bioresource Technology 297 (2020) 122484

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioethanol production from sesame (Sesamum indicum L.) plant residue by T

combined physical, microbial and chemical pretreatments
⁎
Pankaj Kumar, Vinod Kumar , Sachin Kumar, Jogendra Singh, Piyush Kumar
Agro-ecology and Pollution Research Laboratory, Department of Zoology and Environmental Science, Gurukula Kangri Vishwavidyalaya, Haridwar 249404, Uttarakhand,
India

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: This study explored the potential of sesame (Sesamum indicum L.) plant residue (SPR) for bioethanol production.
Bioethanol Three particle sizes, including 400, 850 and 1300 µm of SPR, were subjected to microbial degradation by
Combined pretreatment Phanerochaete chrysosporium followed by 1% H2SO4 pretreatments. FTIR and HPLC analyses showed that the
Kinetic modeling combined pretreatment which begins with microbial followed up by acid degraded SPR in the finest particle size
Renewable energy
(400 µm) resulted in the maximum contents of reducing sugars (370.23 mg·g−1). Kinetics studies of the pre-
Sesamum indicum
treatment process also confirmed the maximized rate of hemicellulose and lignin reduction with reducing sugars
production. The logistic model had better fitness as compared to the modified Gompertz model to predict
bioethanol production. SPR gave a maximum of 1.90 g·L−1 bioethanol yield after 60 h of fermentation using
Saccharomyces cerevisiae. This study is the first report on bioethanol production from SPR, which proposed its
suitability for sustainable energy production.

1. Introduction Plant-based lignocellulosic materials have been playing an im-

portant role in creating alternative fuel resources like bioethanol and
Global fuel demand has been increasing day by day due to rapid biogas (Carvalheiro et al., 2008; Paul and Dutta, 2018). Different types
population growth (Atabani et al., 2011; Wagner et al., 2016). It is of plant residue, such as leaves, seeds, stem, roots or fruits, and their
estimated that nearly 100.6 million barrels of crude oil are consumed peels have been successfully used to produce bioethanol (Hsu, 2018).
worldwide per day (USEIA, 2019). A rapid revolution of the motor- However, steps involved in the bioethanol production process may be
vehicle sector in the recent past has created an extra burden on the affected by a number of factors including available deductible sugars,
reserved stock of fossil fuels (Fischer-Kowalski et al., 2019). Over- the effect of the pretreatment process, chemicals used for pretreatment,
exploitation of naturally fossilized fuel stock is anticipated to lead to an temperature, pressure, etc. (Mood et al., 2013; Hassan et al., 2018).
energy crisis in the near future (Qureshi et al., 2016). Therefore, a Pretreatment of lignocellulosic biomass is the most important step
constant search of an alternative fuel technology has become a primary in the bioethanol production process (Sun and Cheng, 2002). In this
researchable area. step, available cross-linked cellulose and lignin are hydrolyzed into free

⁎
Corresponding author.
E-mail address: [email protected] (V. Kumar).

https://doi.org/10.1016/j.biortech.2019.122484
Received 29 September 2019; Received in revised form 18 November 2019; Accepted 21 November 2019
Available online 26 November 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
P. Kumar, et al. Bioresource Technology 297 (2020) 122484

sugars, which is further degraded using certain fermentative organisms the rural vicinity of Haridwar, Uttarakhand, India. Sesame plants were
to obtain bioethanol (Yao et al., 2018). Pretreatment of lignocellulosic harvested from the agricultural fields in Tanda Bhagmal village,
materials can be accomplished by a number of methods like diluted Haridwar, India (29°82′06″ N and 78°16′28″ E) and seeds were sepa-
acid and alkali pretreatment, microbial pretreatment, catalytic pre- rated. Whole plant residue was washed and dried in an electric oven
treatment, high-pressure pretreatment, ultrasonic pretreatment, etc. (KI-181, Khera Instruments, IN) at 60 °C until complete moisture was
(Hassan et al., 2018). However, the feasibility of the pretreatment removed. Sesame plant residue (SPR) was smashed using a mechanical
process depends on the composition of the material used. Therefore, grinder (Philips Amaze HL7576/00 600 W), and further sieved to ob-
there is a great need for research on the development of new and more tain the desired particle size (400 µm: No. 40; 850 µm: No. 20;
feasible biomass pretreatment methods to achieve sustainable fuel 1300 µm: No. 16; Elysian IN). This step was considered as the first
production goals (Kumar and Sharma, 2017). pretreatment (Step I) of SPR biomass.
Combined pretreatment of lignocellulosic materials has been found The SPR pretreatment was further done in two subsequent steps
to be more effective as compared to traditional methods (Hwang et al., including combined pretreatment with microbial (step II) followed up
2006). Crust fungi (Phanerochaete chrysosporium P. Karst) is one of the by acid (step III). For the microbial pretreatment, crust fungus
most commonly used microorganisms to breakdown the β-glucoside (Phanerochaete chrysosporium P. Karst) was obtained from Microbial
linkages present in lignin. It can sustain the biological release of re- Type Culture Collection Center (MTCC), Chandigarh, India (Accession
ducing sugars from the complex structure of plant lignin (Singh et al., No. MTCC787). Mother culture of the P. chrysosporium was prepared in
2011; Munir et al., 2015; Ansari et al., 2016), therefore minimizing the PDA (Potato dextrose agar) medium (pH: 6.5; temp. 28 °C; time: 84 h).
gross chemical requirement in pretreatment steps. This process re- Besides this, the fermentative yeast Saccharomyces cerevisiae (Accession
presents a cost-effective and sustainable method of pretreatment stra- No. MTCC170) was also obtained from MTCC, Chandigarh, India.
tegies. Combined pretreatment of plant biomass using P. chrysosporium Further, S. cerevisiae was cultured in a 500 mL broth solution having
and diluted H2SO4 has shown promising results (Zhang et al., 2018). 10 g anhydrous glucose (C6H12O6), 10 g tryptone and 10 g yeast ex-
Maximized pretreatment has been achieved by adjusting the biomass tract.
particle size in previous studies (Sarkar et al., 2012). The action of both
microbial cells and chemicals used for pretreatment of biomass is af- 2.2. Microbial and acid pretreatments of SPR
fected by the available interactive sites, which can be altered through
resizing the particle size (Liu et al., 2013). In the microbial assisted pretreatment (MA-PT), a total of 50 g
Sesame (Sesamum indicum) is one of the most common crops grown sieved SPR was taken in a 5 L beaker having a salt nutrient solution (E-
in South Asian countries (Pham et al., 2010). The essential oil con- supplementary data for this work can be found in e-version of this paper
taining seeds of sesame are useful for the production of a number of online). Salt nutrient solution and biomass ratio were adjusted to 1:20
medicinal, herbal, and their products (Saydut et al., 2008). After the (solid-liquid) during the MA-PT process. Contents were sterilized using
harvesting of sesame seeds from its plant, the whole plant is discarded an autoclave (KI- 174, Khera Instrument, IN) and P. chrysosporium was
or sometimes used as a soil conditioner or cattle feedstock (Xie et al., inoculated aseptically. Log phase cells (96 h) of P. chrysosporium were
2011). Occasionally, the biomass is burnt within the field, thereby used for the inoculation. Beakers were placed on a rotatory shaker and
causing inevitable environmental consequences (Zhou et al., 2017). MA-PT process was carried out for a period of 60 h at 30 °C at 150 rpm.
However, sesame comprises high contents of lignocellulosic material, After the microbial degradation, SPR was filtered from the beakers and
which can be utilized to produce low-cost biofuels like bioethanol after dried further at 60 °C. In the case of the dilute acid pretreatment (DA-
proper hydrolysis and making sugars available for fermentative yeasts. PT), contents were soaked in diluted sulfuric acid (1% H2SO4) for a
The reduced sesame plant residue (SPR) particle size through physical period of 60 min at 100 °C in a 250 mL conical flask.
pretreatment (grinding) may help to increase its surface area. It is well
reported that the smallest particle size of biomass has the maximum 2.3. Fermentation experiments for bioethanol production
surface area for chemical action (Jain et al., 2015). Moreover, the use of
P. chrysosporium growth action may give significant lignin breakdown The pH of pretreated SPR was adjusted to 7.0 using 1 N HCl and
before subjecting SPR to chemical pretreatment. Consequently, com- NaOH solutions. Neutralized SPR was then centrifuged (for 5 min at
bined physical, microbial and chemical pretreatments of SPR may result 5000 rpm) to release the remaining liquid contents (MC-12,
in a better release of reducing sugars, which could further be useful for Benchmark's Scientific, USA). The supernatant was separated and re-
bioethanol production. To the extent of our knowledge, no report is used for fermentation experiments. Fermentation was done in a ster-
available on the utilization of SPR to produce bioethanol. Only a few ilized bioreactor assisted with nitrogen aeration and agitation system,
reports are available on biogas production from its biomass using batch under 30 °C and 120 rpm agitation speed for a period of 60 h. The liquid
mode experiments and bioethanol production from its seeds (Saydut supernatant was inoculated with 6 g·L−1 of fermentative yeast (S. cer-
et al., 2008; Abada et al., 2018). evisiae), aseptically. During the fermentation process, contents from the
Therefore, the present study aimed to assess the potential of sesame reactor were carefully sampled at the period of every 5 h, centrifuged at
(Sesamum indicum) plant residue for bioethanol production by adjusting 5000 rpm (10 min), and afterward used for bioethanol estimation.
the biomass particle size and combined pretreatment with microbial
followed up by acid pretreatment. The pretreatment kinetics was de- 2.4. Analytical and instrumental methods
liberated using a first-order reaction-based model while the bioethanol
production was further simulated using two different growth based SPR was characterized by the presence of different bonds and
logistic and modified Gompertz models. The findings of this work are functional groups before and after pretreatments. Vacuum compressed
novel in terms of the production of bioethanol from sesame plant bio- SPR sample (1 g) mixed with diluent pellets (KBr, Laboratory-grade,
mass and for the sustainable agro-waste management of its crop re- Merck Inc., IN) was used to assemble the die-set and bond transmit-
sidue. tance was recorded using Fourier transform infrared (FTIR) spectro-
scopy (FTIR-8400S, Shimadzu Corp., USA).
2. Materials and methods During the MA-PT step, SPR samples were taken at intervals of
every 15 h, while samples were taken at intervals of every 15 min for
2.1. Description of plant biomass and microorganisms used in this study the DA-PT. Nine replicates of each treatment were performed to avoid
the changes in the solid-liquid ratio of experimental materials. Solid
Sesame (Sesamum indicum L.) is a common seasonal crop grown in samples were analyzed for cellulose, hemicellulose, and lignin, whereas

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P. Kumar, et al. Bioresource Technology 297 (2020) 122484

liquid samples were subjected to reducing sugars determination. 3. Results and discussion
Contents of cellulose, hemicellulose, and lignin were estimated by fol-
lowing standard methods suggested by Gessner (2005). Liquid SPR 3.1. Composition of SPR used in this study
samples were taken periodically and subjected to high-speed cen-
trifugation (12000 rpm and 5 min). Finally, the pinhole filtered su- The cellulose, hemicellulose, and lignin compositions of SPR bio-
pernatant was used for the estimation of reducing sugars. 3, 5-dinitro- mass (solid fraction) were presented in Table 3. The initial contents of
salicylic (DNS) was used to determine the total sugar content (Miller, SPR were characterized as 22.80% of cellulose, 37.76% of hemi-
1959). The other reducing sugars viz., arabinose, glucose, galactose, cellulose, and 7.35% of lignin, respectively. The content of total redu-
and xylose were determined using high-performance liquid chromato- cing sugars of SPR as determined by the DNS method in the liquid
graphy (Series 200, PerkinElmer, USA). The operating conditions were fraction was 60.02 ± 4.12 mg·g−1. Among the different reducing su-
maintained as described by Zhang et al. (2018). The content of bioe- gars, the contents of glucose, arabinose, galactose, and xylose were
thanol and sugars were determined using gas chromatography (Clasur 30.35 ± 0.92, 21.44 ± 0.41, 4.54 ± 0.07 and 6.44 ± 0.04 mg·g−1,
580, PerkinElmer, USA) equipped with mass spectroscopy (Campos and respectively (Table 3).
Sarkis, 2017). Certified reference methods and reagents were applied to Previously, Hahm et al. (2009) and Tenyang et al. (2017) studied
ensure the quality control step in all analyses. the proximate composition of sesame and found that whole-plant had
significant cellulosic contents which could be helpful for bioethanol
production.
2.5. Kinetics and prediction models

3.2. Effect of particle size on pretreatment efficiency of SPR

During the MA-PT and DA-PT pretreatments, the rate of SPR con-
stituent degradation/production process was determined using the
The effect of biomass particle size on SPR pretreatment efficiency
simplified first-order reaction model (Eq. 1). The fitness of the model
was investigated. Figs. 1 and 2 indicate time-course changes in SPR
was determined based on a log(C0/Ct) slope having a coefficient of
constituents (solid fraction) and reducing sugars (liquid fraction), re-
determination greater than 0.85. The dependency of initial constituent
spectively. For MA-PT, the process was stationary in the course of 45 to
concentration on pretreatment performance was evaluated using max-
60 h due to the aging of P. chrysosporium cells (Vandana et al., 2019).
imized rate constant (k). In both microbial and acid pretreatments, k
Besides this, the reactions of the DA-PT step were terminated after
was calculated as g·h−1, mg·g−1h−1, g·min−1, and mg·g−1min−1, re-
45 min as no more hemicellulose and lignin reduction along with cel-
spectively. For batch mode fermentation experiments, the rate of spe-
lulose, reducing sugars, glucose, arabinose, galactose, and xylose pro-
cific bioethanol production in relation to the growth of S. cerevisiae was
duction was observed (Table 3).
simulated using two different growth models.
According to the rate of cumulative bioethanol production, the lo-
3.2.1. Effect on cellulose, hemicellulose and lignin
gistic growth model and modified Gompertz model were implemented.
Hemicellulose is the heteropolymer found in plant cells to form a
Different kinetic variables were compared to choose the best fitting
cross-linked structure that can be hydrolyzed by using several microbial
model. The forms of non-linear logistic and modified Gompertz models
enzymes or chemical methods, while lignin act as a skeleton of plant
were given in Eqs. 2 and 3. Moreover, the models were validated using
cell supporting the tissue and holding cellulose. We found that after the
selected tools such as model efficiency (ME, Eq. 4), normalizing error
MA-PT step, the hemicellulose of SPR was reduced to 29.34%, 31.34%,
(NE, Eq. 5), and normalizing error bias (NEB, Eq. 6). Different model
and 34.35% from its initial value (37.76%), for the particle sizes 400,
equations and their terms were defined in Tables 1 and 2, respectively.
850, and 1300 µm, respectively. Again, after MA-PT, the subsequent
DA-PT reduced hemicellulose to 19.34%, 20.33%, and 26.44%. In this
2.6. Statistics study, MA-PT increased the cellulose contents (dry weight basis) of SPR
from initial (22.80%) to 29.53%, 27.45%, and 26.45% in 400, 850, and
The deviation of three standard measurements was presented as a 1300 µm particle treatments, respectively. After DA-PT, cellulose yield
sufficient parameter value. Nine datasets of each particle treatment was again increased to 41.23%, 35.75%, and 30.88% in respective
(n = 9 × 3 = 27) were used to validate the model obtained variables. treatment. Similarly, for lignin, the contents of residual lignin after MA-
Model quality was also validated using different model validation tools. PT were 6.04%, 6.45%, and 6.85%, while 3.66%, 4.53% and 5.03%
Statistical analyses and model simulations were done using SPSS 23.0 after DA-PT step. Compared to MA-PT, DA-PT gave the highest de-
(IBM Corp., USA) and Origin Pro 9.0 (Origin Inc., USA) software. gradation of cellulose, hemicellulose, and lignin for all particle sizes
Graphing work was done using Microsoft Office 2013 package (Fig. 1). However, among the three selected particle sizes, the order of
(Microsoft Corp., USA). hemicellulose and lignin degradation was 400 > 850 > 1300 µm
confirming the best capacity of both treatments in the finest particle
Table 1 size.
Models used in this study. It was observed that the particle size had the most determining ef-
Model Equation Reference Remark fect on the pretreatment of SPR. For the smallest particle size, the
maximum surface area was exposed to microbial cells and acid solution,
First-order Kumar et al. Eq. 1
k=
2.303
t2 − t1 ( )
C
∙log 0
Ct (2019)
which catalyzed the pretreatment reactions (Khullar et al., 2013; Li
et al., 2016; Gáplovská et al., 2018). Larger the particle size may have
Logistic y=
P Li et al. (2012) Eq. 2
(λ − t )
1 + exp ⎡4μm + 2⎤
lesser freely available reactive sites resulting in slow down of the pre-
⎣ P ⎦
treatment as revealed by two other treatments (850 and 1300 µm).
Modified Velmurugan and Eq. 3
Gompertz
y= { μm
Pexp −exp ⎡ (λ − t)
⎣ P
+ 1⎤
⎦ } Muthukumar Chundawat et al. (2007) and Liu et al. (2013) studied the effect of
(2012) particle size on enzymatic digestibility of milled corn stover, and they
Model efficiency ∑ (y −y ) 2 Eid et al. (2019) Eq. 4 noted maximum at smaller particle size.
1 − ⎡ model actual ⎤
(ME) 2
⎣ ∑ (ymodel − C) ⎦
Normalizing ∑ | ymodel − yactual | Eid et al. (2019) Eq. 5 3.2.2. Effect on reducing sugars formation
⎛ ⎞/n
error (NE) ⎝ yactual ⎠
The changes in reducing sugars content during pretreatment steps as
Normalized error ∑ (ymodel − yactual ) Eid et al. (2019) Eq. 6
bias (NEB) ∑ yactual affected by biomass particle size were shown in Table 3. Results ex-
hibited that the maximum release of reducing sugars (total reducing

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P. Kumar, et al. Bioresource Technology 297 (2020) 122484

Table 2
Definitions of the model expressions used in this study.
Expression Definition Unit

k Rate of constituent reduction/product formation g·h−1, g·min−1, mg·g−1h−1, mg·g−1min−1

t2-t1 Final and initial sampling times h, min
2.303 Reaction constant –
C0 Initial constituent concentration g·h−1, mg·g−1
Ct Constituent concentration at time t g·h−1, mg·g−1
y Predicted bioethanol production g·L−1
R2 Coefficient of determination –
P Maximum bioethanol production potential g·L−1
µm Maximum specific bioethanol production h−1, min−1
λ Lag phase h, min
t Experimental time of bioethanol production h, min
n Number of experimental replicates –
C Mean bioethanol production g·L−1

Table 3
Changes in constituents of SPR biomass and reducing sugars after selected pretreatments.
SPR Constituent Unit Before pretreatment After MA-PT After DA-PT

400 µm 850 µm 1300 µm 400 µm 850 µm 1300 µm

a
Dry weight g 50.00 ± 0.01 27.65 ± 0.59 34.25 ± 0.37 36.65 ± 0.40 11.70 ± 0.08 17.25 ± 0.13 23.85 ± 0.26
Cellulose a g 11.40 ± 0.15 8.17 ± 0.09 9.40 ± 0.03 9.69 ± 0.10 4.82 ± 0.02 6.17 ± 0.05 7.36 ± 0.02
% 22.80 29.53 27.45 26.45 41.23 35.75 30.88
a
Hemicellulose g 18.88 ± 0.23 8.11 ± 0.12 10.73 ± 0.06 12.59 ± 0.18 2.26 ± 0.07 3.51 ± 0.03 6.31 ± 0.09
% 37.76 29.34 31.34 34.35 19.34 20.33 26.44
a
Lignin g 3.36 ± 0.03 1.67 ± 0.05 2.21 ± 0.08 2.51 ± 0.02 0.43 ± 0.04 0.78 ± 0.01 1.20 ± 0.01
% 7.35 6.04 6.45 6.85 3.66 4.53 5.03
Reducing sugars b
mg·g−1 60.02 ± 4.12 120.13 ± 8.26 93.35 ± 8.54 80.55 ± 7.86 370.23 ± 10.20 310.95 ± 6.92 242.24 ± 11.58
Glucose b mg·g−1 30.35 ± 0.92 98.2 ± 5.01 70.32 ± 4.03 49.45 ± 2.04 190.34 ± 8.03 152.3 ± 9.76 120.3 ± 7.25
Arabinose b mg·g−1 21.44 ± 0.41 26.46 ± 0.89 23.45 ± 0.32 22.05 ± 0.65 68.46 ± 4.60 50.31 ± 4.49 40.33 ± 2.38
Galactose b mg·g−1 4.54 ± 0.07 8.45 ± 0.11 7.54 ± 0.04 7.01 ± 0.02 30.45 ± 0.42 26.43 ± 0.52 21.44 ± 0.27
Xylose b mg·g−1 6.44 ± 0.04 8.98 ± 0.09 8.01 ± 0.13 7.24 ± 0.03 14.35 ± 0.21 12.33 ± 0.82 9.24 ± 0.19

MA-PT: microbial assisted pretreatment for 60 h; DA-PT: dilute acid (1% H2SO4) pretreatment for 60 min; a: solid fraction of SPR; b: liquid fraction of SPR.

Fig. 1. Changes in percent cellulose, hemicellulose and lignin of SPR biomass (solid fraction) during selected pretreatments as affected by biomass particle size.

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P. Kumar, et al. Bioresource Technology 297 (2020) 122484

135 400 μm 105 400 μm 28 400 μm

Re du cing s uga rs ( mg· g-1 )

850 μm 850 μm 850 μm
1300 μm 1300 μm 1300 μm

Ara bi n ose (mg·g-1 )

G l u cose (mg ·g-1 )
115 85 26

95 65 24

75 45 22

55 25 20
0 15 30 45 60 0 15 30 45 60 0 15 30 45 60
Time (h) Time (h) Time (h)
420 400 μm 400 μm 80 400 μm
190
Re du cing s uga rs ( mg· g-1 )

850 μm 850 μm 850 μm

1300 μm 1300 μm 70 1300 μm

Ara bi n ose ( mg· g-1 )

350

G l u cose ( mg ·g-1 )
60
280 140
50
210
90 40
140 30
70 40 20
0 15 30 45 60 0 15 30 45 60 0 15 30 45 60
Time (min) Time (min) Time (min)

9 400 μm 10 400 μm
850 μm 850 μm
1300 μm 1300 μm
G a l a cto se ( m g·g -1 )

8 Microbial assisted
9
Xy l o s e ( mg ·g -1)

pretreatment (MA-
7 PT)
8
6
Dilute acid
7 pretreatment (DA-PT)
5

4 6
0 15 30 45 60 0 15 30 45 60
Time (h) Time (h)
35 400 μm 15 400 μm
850 μm 850 μm
30 1300 μm 1300 μm
G a l a cto se (m g·g -1 )

13
Xy l o s e (mg ·g -1)

25
20 11
15
9
10
5 7
0 15 30 45 60 0 15 30 45 60
Time (min) Time (min)
Fig. 2. Changes in reducing sugars of SPR biomass (liquid fraction) during selected pretreatments as affected by biomass particle size.

Table 4
Changes in the major FTIR peaks of SPR biomass before and after selected pretreatments.
Wavenumber group (cm−1) Marked peak Transmittance (%) Major chemical bonds Functional groups

Before treatment After treatment

400 µm 850 µm 1300 µm

3600–3400 3470.05 76.97 86.48 84.44 81.11 NeH, OeH Overlapping and stretching of amides
3000–2800 2970.47 93.54 87.42 92.62 90.82 CeH Alkyl chain
1680–1550 1627.97 66.27 74.37 71.39 69.58 NeH, CeC, C]O Primary amines, Unconjugated Alkenes, lignin
1470–1350 1350.21 86.59 74.64 78.01 81.69 CeH Bending cyclic alkenes
1400–1000 1028.09 75.48 52.05 60.38 67.39 COeOR Hemicelluloe and cellulose
850–550 781.19 85 53.12 70.89 77.27 CeH Saccharides
500–400 428.21 72.25 45.16 53.92 65.69 CeI Organoiodides

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P. Kumar, et al. Bioresource Technology 297 (2020) 122484

Table 5
Rate constant of changes in constituents of SPR biomass and reducing sugars during selected pretreatments.
SPR constituent kmax

MA-PT DA-PT

400 µm 850 µm 1300 µm 400 µm 850 µm 1300 µm

Cellulose formation a 0.1129 0.1127 0.1121 0.1069 0.1057 0.1026

Hemicellulose reduction a 0.0979 0.0978 0.0975 0.1055 0.1036 0.1007
Lignin reduction a 0.1778 0.1776 0.1775 0.1986 0.1917 0.1848
b
Reducing sugars formation 0.0855 0.0849 0.0846 0.0797 0.0769 0.0726
Glucose formation b 0.1017 0.1000 0.0976 0.0893 0.0813 0.0752
Arabinose formation b 0.1150 0.1143 0.1140 0.1100 0.1086 0.1054
Galactose formation b 0.2233 0.2165 0.2115 0.1717 0.1669 0.1533
Xylose formation b 0.1876 0.1854 0.1830 0.1722 0.1624 0.1560

kmax: first-order rate constant; MA-PT: microbial assisted pretreatment for 60 h; DA-PT: dilute acid (1% H2SO4) pretreatment for 60 min; a: solid fraction of SPR with
units g·h−1 or g·min−1; b: liquid fraction of SPR with units mg·g−1·h−1 or mg·g−1·min−1.

sugars, glucose, arabinose, galactose, and xylose) from SPR occurred in 69.58% (1300 µm), exhibiting successful deformation of lignin of se-
400 µm particle size. MA-PT achieved a maximum of same. Peak number 2970.47 cm−1 was marked to represent the
120.13 ± 8.26 mg·g−1 reducing sugars. Moreover, DA-PT attained changes in chain bearing alkyl compounds. This peak had an initial
370.23 ± 10.20 mg·g−1 reducing sugars. The finally available sugars transmittance of 93.54%, which later changed to 87.42% (400 µm),
after MA-PT and DA-PT in 400 µm treatment were 92.62% (850 µm), and 90.82% (1300 µm). The final marked peak
190.34 ± 8.03 mg·g−1 glucose, 68.46 ± 4.60 mg·g−1 arabinose, 3470.05 cm−1 (for amides) changed from 76.97% to 86.48 (400 µm),
30.45 ± 0.42 mg·g−1 galactose and 14.35 ± 0.21 mg·g−1 xylose 84.44 (850 µm), and 81.11 (1300 µm) revealing deformation of H-O
which is useful for fermentation experiments. It was observed that DA- bearing amides of SPR.
PT was more effective in terms of the production of cellulose and re- It was reported that the finest particle size promoted the maximum
ducing sugars during the pretreatment as compared to MA-PT (Fig. 2). deformation of lignin and hemicellulose and production of reducing
Therefore, particle size adjustment and two combined pretreatments sugars bearing compounds after the pretreatment process. Appearing of
were helpful to maximize net sugar yield from SPR. weaker peaks (high transmittance) after pretreatment indicated suc-
A lesser amount of released reducing sugars in larger particle sizes cessful deformation of associated compounds, whereas stronger peaks
after pretreatments might be due to decreased exposure of P. chrysos- (less transmittance) showed the formation of the respective compound
porium cells and low acid action. Most recognized and easy to consume within the sesame biomass after pretreatment. The maximum de-
substrate for S. cerevisiae is glucose (Childs et al., 2015). The optimum formation of the lignocellulosic content of SPR was due to the breaking
concentration of available reducing sugars gives improved bioethanol of β-glucoside bonds along with the formation of specific sugars during
yields. Sahoo et al. (2018) found that dilute acid pretreatment released the pretreatment process (Singh et al., 2011). Therefore, the efficiency
a maximum of 457 mg·g−1 reducing sugars from wild rice grass bio- of SPR hydrolysis was significantly affected by the particle size, as re-
mass. Similarly, P. chrysosporium assisted acid pretreatment of water vealed from the FTIR analysis. Previously, Sahoo et al. (2018) reported
hyacinth was helpful to release maximum reducing sugars as earlier FTIR analysis of wild rice grass biomass before and after chemical
reported by Zhang et al. (2018). pretreatment confirming significant changes in the major peaks asso-
ciated with compounds.

3.2.3. Changes in FTIR spectra of SPR

The effect of particle size on the FTIR spectra of SPR was studied
3.2.4. Effect on kinetic rate constant
before and after combined pretreatment (E-supplementary data for this
Kinetic rate constant (k) for changes in the SPR constituent during
work can be found in e-version of this paper online). Seven peaks
step II and step III pretreatment process was given in Table 5. The
(Table 4) were marked to represent net transmittance changes before
pretreatment process followed the first-order reaction for both MA-PT
and after pretreatment.
and DA-PT as suggested by the fitness plot (R2 > 0.85). It was ob-
The initial marked peak of 428.21 cm−1 was meant for certain C-X
served that k was correspondingly associated with the efficiency of the
(carbon-halogen) bond bearing compounds. The initial transmittance
pretreatment process. The finest particle size exhibited maximum va-
(72.25%) of this wavenumber was changed to 45.16%, 53.92%, and
lues of k for cellulose and lignin reduction with a maximum production
65.69% for particle sizes of 400, 850, and 1300 µm, respectively. A
of reducing sugars. However, the maximum rate constant was observed
decrease in the transmittance means the increase in the concentration
in DA-PT as compared to the MA-PT step. The maximum rate constant
of such compounds. This enables the pass of lesser light through it. The
in the finest particle size was due to maximum available reaction sites
second marked peak as 781.19 cm−1 stands for C–H bearing plant cell
on SPR particle surface which catalyzed the rapid deformation of the
skeleton forming saccharide compounds. The transmittance of this
lignin network and releasing free reducing sugars. Growth of P. chry-
group changed from the initial 85.00% to 53.12%, 70.89%, and 77.27%
sosporium cells improved the initial breaking of bonds associated with
for 400, 850, and 1300 µm, respectively. Similarly, peak 1028.09 cm−1
hemicellulose and lignin compounds, as revealed by FTIR analysis.
destined for hemicellulose and cellulosic (CO-OR bonds) had final
A recent report by Sarto et al. (2019) studied the kinetics of water
transmittance of 52.05% (400 µm), 60.38 (850 µm) and 67.39
hyacinth biomass pretreatment using power-law based on the first-
(1300 µm) as compared to initial i.e. 75.48%. Bending cyclic alkene
order model. They confirmed that the rate constant of most of the
group peak (1350.21 cm−1), changed from 86.59% to 74.64% (850 µ),
pretreatment processes can be successfully determined using the first-
78.01% (850 µm) and 81.69% (1300 µm) exhibiting the successful
order model, which further may be helpful to maximize the pretreat-
formation of C–H bearing sugars. Primary amines, unconjugated al-
ment efficiency. This report also verified the results proposed by us.
kenes, and lignin are characterized by wavenumber group of
1680–1550 cm−1, the marked peak (1627.97 cm−1) had an increase in
transmittance from 66.27% to 74.37% (400 µm), 71.39% (850 µm) and

6
P. Kumar, et al. Bioresource Technology 297 (2020) 122484

Table 6
Comparison of estimated model parameters.
Model Particle size (µm) Measured bioethanol yield (g·L−1) Model parameter

ymax P µm λ SE ME NE NEB R2

Logistic 400 1.90 ± 0.10 1.91 1.92 32.06 0.17 0.01 0.99 0.01 0.01 0.9997
850 1.31 ± 0.06 1.31 1.32 31.62 0.18 0.01 0.99 0.01 0.01 0.9996
1300 0.87 ± 0.08 0.88 0.89 34.52 0.19 0.02 0.99 0.01 0.01 0.9964
Modified Gompertz 400 1.90 ± 0.10 1.94 2.02 28.57 0.10 0.08 0.97 0.03 0.02 0.9963
850 1.31 ± 0.06 1.34 1.38 28.27 0.11 0.05 0.98 0.03 0.02 0.9956
1300 0.87 ± 0.08 0.88 0.90 31.71 0.14 0.05 0.99 0.01 0.01 0.9891

SE: standard error; ME: model efficiency; NE: normalizing error; NEB: normalizing error bias.

3.3. Bioethanol production from SPR using S. cerevisiae lag phase (λ), and experimental time of bioethanol production (t) also
had optimum values for logistic model. For the 400 µm particle size,
Bioethanol production from SPR by fermentation using S. cerevisiae maximum predicted yield of 1.91 g·L−1, less standard error (0.01),
was given in Table 6. S. cerevisiae showed efficient propagation in se- minimum normalizing error (0.01), reduced model normalized bias
same biomass for the initial 45 h which later become stationary to 60 h. (NEB: 0.01) and high model efficiency (0.99) were obtained. The ad-
We observed that the volume of bioethanol increased with a decrease in justed particle size should be taken considerably for maximized bioe-
particle size. This might be a result of the fed-back-inhibition of pro- thanol production and minimizing the error in prediction.
duced alcohol content within the reactor (Jönsson and Martín, 2016). Previously, growth function-based models have been successfully
However, among the different particle size, growth pattern was similar, implemented to simulate the bioethanol production process.
but because of the net availability of reducing sugars, the net bioe- Phukoetphim et al. (2017) simulated the bioethanol production po-
thanol production ranged as 1.90 ± 0.10, 1.31 ± 0.06 and tential of S. cerevisiae using sorghum and found that the logistic model
0.87 ± 0.08 g·L−1 in 400, 850, and 1300 µm size particle treatments, best fits to simulate the process.
respectively. It confirmed a significant effect of biomass particle size on
net bioethanol yield from SPR. 4. Conclusion
This is the first report on bioethanol production from sesame plant
residue; therefore, we were unable to find any previously published For the very first time, bioethanol production using sesame plant
research to compare our results. residue was investigated. Overall results described that 400 µm size of
sesame biomass particle gave the highest yield of reducing sugars after
3.4. Model fitting results of bioethanol production from SPR the microbial (P. chrysosporium) assisted diluted acid pretreatment and
promising yield of bioethanol contents 1.90 ± 0.10 g·L−1. The logistic
Simulation results of bioethanol production using logistic and model gave the best-fitting results to simulate the cumulative bioe-
modified Gompertz models as a function of S. cerevisiae growth were thanol production process as compared to the modified Gompertz
presented in Table 6. Both models showed strong fitness in the obtained model. Therefore, findings of this research are novel in terms of bioe-
data. Non-linear fitness curves of cumulative bioethanol production thanol production from sesame plant residue through combined phy-
were illustrated in Fig. 3. sical, microbial, and chemical pretreatments.
We assume that the logistic model had more accurate fitness as
compared to the modified Gompertz model. Various model variables CRediT authorship contribution statement
such as coefficient of determination (R2), maximum bioethanol pro-
duction potential (P), maximum specific bioethanol production (µm), Pankaj Kumar: Conceptualization, Methodology, Software, Writing

Fig. 3. Measured and model predicted bioethanol production using SPR biomass.

7
P. Kumar, et al. Bioresource Technology 297 (2020) 122484

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Declaration Competing of Interest sugar mill effluent by water hyacinth (Eichhornia crassipes) in a Continuous Stirred
Tank Reactor (CSTR). Environ. Technol. Innovation 14, 1–11. https://doi.org/10.
The authors declare that they have no known competing financial 1016/j.eti.2019.100327. 100327.
Li, P., Cai, D., Luo, Z., Qin, P., Chen, C., Wang, Y., Tan, T., 2016. Effect of acid pre-
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Vishwavidyalaya, Haridwar-249404 (Uttarakhand), India. The authors 1016/j.indcrop.2012.11.009.
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