Bioresource Technology Reports xxx (xxxx) xxx-xxx

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Bioresource Technology Reports

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Compressed hot water pretreatment enhanced bioethanol production from corn stalk
Abiodun E. Adekunle a ,b ,⁎ , Taposhi Rabeya a , Farjana Jehadin a , Mohammad A. Asad a ,
Olubunmi O. Ayodele b ,c , Md Saiful Islam a ,⁎⁎

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a
Institute of Fuel Research and Development, Bangladesh Council of Scientific & Industrial Research, Dhanmondi, Dhaka 1205, Bangladesh
b
Biotechnology Center, Forestry Research Institute of Nigeria, PMB 5054, Dugbe, Ibadan, Nigeria
c
Nanoscience Department, The Joint School of Nanoscience & Nanoengineering, University of North Carolina, NC 27401, USA

ARTICLE INFO ABSTRACT

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Keywords Finding alternative use for corn stalk (CS), an abundant agricultural waste, in energy production would solve
Bioethanol the problem of it becoming environmental encumbrance and increases energy availability. CS used in this study
Agricultural wastes was exposed to compressed hot water (CHW) pretreatment at 100, 120, 140, 160, 180 and 200 °C for reaction
Compressed hot water time 10 min and the slurry was used for production of ethanol in a simple bioreactor kept at 30 °C. Optimal
Bioenergy
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performance was recorded with the 180 °C pretreated corn stalk (PCS). Filtration of the slurry improved ethanol
production to 31.06 g/L. Under optimal conditions, 73.8% degradation of hemicellulose was recorded. Scanning
Electron Microscope images showed induced porosity and increased surface area in the PCS. Crystallinity index
of the PCS increased as revealed by X-ray diffraction analysis. CHW pretreatment shows potential as a promising
pretreatment method for lignocellulosic materials use for large-scale production of bioethanol that is useable as
fuel.
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1. Introduction overdependence on fossil fuel (Duangwang and Sangwichien, 2015).

Energy experts and researchers have predicted a promising future for the
Availability of energy plays vital role in the existence and develop- utilization and investment in production of bioenergy from renewable
ment of human life. According to the International Energy Agency, the sources (IEA, 2006; Limayem and Ricke, 2012). Lignocellulosic ma-
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world's energy outlook is at deficit comparing energy demand to its pro- terials are excellent renewable resources for bioenergy production. They
duction and availability (IEA, 2006). This crisis will continue due to the are abundant, cheap and their constituents are convertible to bioenergy
problems facing fossil fuels. Fossil fuels, which are the major contributor via biological and chemical processes.
to the global energy supply, are currently facing challenges of depletion Corn stalk is an agricultural waste that is generated around the
in its reserves and instability in the global oil prices (Wilcox, 2014). world in large quantity; with the United State and China being the
Another problem which discourages the reliance on fossil fuels is the largest producers (Ranum et al., 2014). These are abundant wastes
threat it poses to the environment. This is due to emissions from these whose utilization in the production of second generation of bioethanol
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fuel's resources which contribute to greenhouse gasses, global warming poses no threat to food chain (Balat, 2011; Balat et al., 2008).
and climate change (Saravanakumar and Kathiresan, 2014; Wilcox, Corn stalk is a lignocellulosic material whose chemical composition in-
2014). cludes hemicellulose, cellulose and lignin. These chemical components
The global effects of fossil fuel and its derivatives on our planet of cornstalk are polysaccharides whose monomers could be utilized as
make research and investment in clean and renewable energy necessary substrates to support growth and metabolic activities of organism for
(UNEP, 2019). One of such alternative energy resource is bioethanol, the production of desired metabolite (Wyman et al., 2005). How-
a bioenergy obtained from biological sources via fermentation. It is an ever, the greatest impediment to accessing these monomeric sugars is
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alternative fuel with many advantages over fossil fuel. It is less toxic, the complex structure and the recalcitrant nature of the lignocellu-
applied in blends to reduce emission, higher octane rating, biodegrad- losic material (Agrawal et al., 2017). Several pretreatment methods
able, useable in industrial processes, transportation fuel and medical have been developed and utilized for production of second generation
procedures (Mohd Azhar et al., 2017). In some parts of the world, bioethanol from lignocellulosic biomass (Rastogi and Shrivastava,
bioethanol is widely used in mixed proportions with gasoline to reduce 2017; Xu and Huang, 2014). Compressed hot water pretreatment uti

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Correspondence to: A.E. Adekunle, Biotechnology Center, Forestry Research Institute of Nigeria, PMB 5054, Dugbe, Ibadan, Nigeria.
⁎⁎ Corresponding author.
E-mail addresses: [email protected] (A.E. Adekunle); [email protected] (M.S. Islam)

https://doi.org/10.1016/j.biteb.2020.100595
Received 5 August 2020; Received in revised form 15 September 2020; Accepted 12 October 2020
Available online xxx
2589-014/© 2020.
A.E. Adekunle et al. Bioresource Technology Reports xxx (xxxx) xxx-xxx

lized for this study is environmentally friendly and a cost effective pre- ment, slurry of 180 °C PCS was filtered by using Buchner funnel coupled
treatment method for lignocellulosic material (Yan et al., 2016). It is to a vacuum pump (Rocker 400, 0.5 A). The filtrate was supplemented
also referred to as liquid hot water pretreatment. Although this method with the media-salts described above and inoculated with 1% (w/v) or-
has been utilized for enhanced bioethanol production from lignocellu- ganism. Effect of organism loading was carried out by utilizing various
losic materials, these studies adopted single severe temperature for the amount of organism used for inoculation. Media-supplemented filtrate
pretreatment of the lignocellulosic material used (Jiang et al., 2015; of 180 °C PCS was inoculated with 1, 2, 3, 4 and 5% (w/v).

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Li et al., 2013). In each case, The pH of the mixture was adjusted to 5.0 with 1 N
This study therefore sought to study the effect of temperature varia- NaOH/HCL using digital pH meter (HI 2211, HANNA Instruments) and
tion of CHW pretreated corn stalk on bioethanol production. The study contents of the flasks were autoclaved at 121 °C for 20 min prior to in-

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utilized the slurry of the CS that was pretreated at various tempera- oculation. Inoculation was carried out under aseptic conditions, fermen-
tures and further optimized some fermentation conditions for improved tation flasks were tightly plugged to maintain anaerobic condition and
bioethanol production in fermentation process. kept at 30 °C. All experiments were performed with triplicate sample
bottles for each group.
2. Materials and methods
2.4. Analytical methods
2.1. Organism and chemicals
The liquid broth was withdrawn from fermentation flask and cen-

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Saccharomyces cerevisiae used for this study was commercial beakers' trifuged at 5000 rpm for 5 min. Bioethanol concentration, total sugar,
yeast. Absolute ethanol, Anthrone reagent and Potassium dichromate Furfural and 5-HMF were quantified spectrophotometrically using UV
were purchased from Merck KGaA (Germany). Furfural and 5-Hydrox- spectroscopy GBC central 2020. Concentration of bioethanol in the
ymethylfurfural (5-HMF) were purchased from Sigma-Aldrich. All other supernatant was determined following the principle of oxidation of
chemicals used were of analytical grade and used without further purifi- ethanol in the presence of acidified potassium dichromate and the color
cation. change from orange to green was monitored using spectrophotome-
ter at 600 nm (Sumbhate et al., 2012). Anthrone-sulfuric acid assay

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2.2. Corn stalk: source, characterization and pretreatment was used for determination of sugar concentration in the supernatant
(Plummer, 1990). Furfural and 5-HMF were determined by reading ab-
CS was obtained from a farm in Dhamrai, near Dhaka, Bangladesh. sorbance of samples at 277 and 285 nm respectively. These values were
CS was sun-dried and thereafter stored in a cool and dry place. Prior used to determine the concentration of the inhibitors present in the sam-
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to usage, CS was pulverized to particle size of 2 mm and oven-dried at ples (Chi et al., 2009).
60 °C to constant weight.
Hemicellulose, cellulose and lignin content of the CS were deter- 2.5. Microstructural analysis of lignocellulosic biomass
mined as described by Goering and Goering and Van-Soest (1970).
Briefly described as follows. 1 g of CS was treated with neutral detergent CS was subjected to morphological examination by Scanning Elec-
to remove the extractives; leaving behind the holocellulose. The residue tron Microscope (SEM; EVO18, Carl Zeiss, UK). SEM images of the
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was treated with acid detergent (2 M HCl) for digestion of hemicellu- material were taken at magnification of 1.00k×, with EHT of 5.0 kV
lose. The remaining solid was placed in a 50 mL beaker. 5 mL sulfuric and working distance (WD) of 10.5 mm. Crystallinity index (CI) of the
acid (72%) was added to the solid fraction and left to stand for 3 h for treated and CHW pretreated CS was determined using X-ray diffrac-
digestion of the cellulose content. During each step, treated materials tion (XRD) patterns were obtained at 2θ, 40 V and 40 mA (XRD Instru-
were washed, filtered, dried and weighed to obtain the weight lost used ment: Model D8 Advance) (Bruker Germany). Structural effects of CHW
in calculating the hemicellulose, cellulose and lignin content. pretreatment on CS were investigated with Fourier-Transform Infrared
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CHW pretreatment of CS was carried out in a thermal catalytic re- (FTIR) spectroscopy. These effects were monitored by comparing spec-
actor-PARR 4843, Parr Instruments Company, USA. The reactor was tra of PCS and untreated CS. Samples were scanned within wavenum-
equipped with digital thermometer, mechanical stirrer with tachome- ber 4000 to 600 cm−1 using spectral resolution 4 cm−1 (Frontier Perkin
ter, 1 L stainless autoclave and purging gas cylinder filled with Nitrogen Elmer, USA).
(N2) gas. CS was placed in the chamber and sealed. The chamber was
degassed by passing N2 gas through it at 1.0 MPa. The temperature was 3. Results and discussion
adjusted to the desired set and monitored as it rises. When the desired
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temperature is attained, the chamber is allowed to operate for 10 min 3.1. Effect of CHW pretreatment temperature on bioethanol production
and cooled thereafter. The slurry was removed from the chamber dry
at ambient temperature (60 °C). Different sets of CS were treated as de- Variation of CHW pretreatment temperature of CS had effect on
scribed above at 100, 120, 140, 160, 180 and 200 °C. These conditions bioethanol production as observed in media culture. The product was
were chosen on the basis of low to mild to high temperatures for the detected in all fermentation broths after 24 h and attains individual
CHW pretreatment process (Brodeur et al., 2011; Yan et al., 2016). maximum concentration after 48 h (Fig. 1). Maximum bioethanol con-
centrations recorded were 7.04, 7.22, 8.15, 11.23, 17.41 and 14.63 g/L
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2.3. Fermentation and optimization at 100, 120, 140, 160, 180 and 200 °C respectively.
Highest concentrations of bioethanol recorded increased with in-
Fermentation media used for this study consist of KH2PO4 - 0.75, crease in pretreatment temperature while the optimal performance was
(NH4)2SO4 - 0.15, MgSO4 - 0.25, yeast extract - 9.0 all in g/L. Effect of recorded at 180 °C. Increase in severity of the CHW pretreatment tem-
various CHW pretreatment temperature on bioethanol production was perature above this threshold did not support higher product formation.
determined by using PCS that was pretreated at various temperatures The CHW pretreatment is a physicochemical pretreatment method
of 100–200 °C. For each treatment, 5 g of PCS was placed in 250 mL which utilizes high temperature and pressure to break down of chem-
Erlenmeyer flasks and supplemented with 100 mL fermentation media. ical (hemicellulose, cellulose and lignin) and physical structure of CS
The media was inoculated with 1% (w/v) organism. To determine the (Brodeur et al., 2011). Breakdown of these structures results in re-
effect of filtration of the optimal performing PCS from the first experi lease of fermentable sugars which are accessible to Saccharomyces cere

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Fig. 2. Effect of filtration of 180 °C pretreated CS on bioethanol production.

Fig. 1. Effect of compressed hot water pretreatment of corn stalk on bioethanol produc-
tion. Fig. 3. Organism loading at 1, 2, 3, 4 and 5% gave corresponding yield
of 20.81, 21.85, 25.39, 27.41 and 31.06 g/L respectively. Selection of
optimal inoculum size for bioethanol production could reduce cost of

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visiae for metabolic activity and production of secondary metabo-
production and fermentation period. It has also been associated to im-
lite-bioethanol (Goshima et al., 2013). Increase in severity of the pre-
prove organism's sugar consumption and enhance higher product forma-
treatment temperature to 200 °C in this study probably enhanced in-
tion (Mohd Azhar et al., 2017).
creased generation of inhibitory compounds. Few of such compounds in-
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The optimal performing inoculum size recorded in this work was
clude furfural, 5-HMF, acetic acid, formic acid and phenolic compounds
similar to those documented by Li et al. (2011) using hydrolysate of
(Tesfaw and Assefa, 2014). To properly understand this phenomenon,
steam-exploded corn stover for bioethanol production by S. cerevisiae. Li
the concentration of 5-HMF and furfural in the untreated and PCS (180
and co-workers further concluded that there was no significant improve-
and 200 °C) were determined. While these compounds were detectable
ment in product formation when the inoculums concentrations were in-
in trace amount in the raw CS, the 180 °C PCS contained 0.618 and
creased from 5 to 10%.
0.448 mg/L of 5-HMF and furfural respectively. These values increased
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Time-course for sugar consumption and bioethanol production of

by ≈2-fold in the 200 °C PCS (5-HMF-1.288 mg/L, furfural - 0.981 mg/
Saccharomyces cerevisiae under optimal fermentation condition are
L). Presence of these compounds at high concentration in fermentation shown in Fig. 4. Prior to commencement of fermentation experiments,
media have been implicated in the inhibition of organism's growth and initial sugar concentration in the fermentation media was
other metabolic activities (Bhadana and Chauhan, 2016). This proba- 78.16 ± 3.32 g/L. This was reduced to 8.98 ± 1.92 g/L at the end of
bly explains the lower concentration of bioethanol (14.63 g/L) recorded the experiment.
at the ultimate CHW pretreatment temperature when compare to those The highest bioethanol obtained in this work under the optimized
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recorded (17.41 g/L) at the penultimate pretreatment temperature. condition compared favourably with some previous studies on

3.2. Effect of filtration of PCS slurry on bioethanol production

Utilization of supplemented-filtrate obtained from the 180 °C PCS

showed improvement in the production of bioethanol in fermentation
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broth. In the filtered media, bioethanol concentration increased with

time and attained a maximum of 20.81 g/L after 48 h (Fig. 2). This
connotes 1.26-fold (26.27%) increase in bioethanol concentration ob-
tained when compared to the unfiltered slurry previously used for fer-
mentation under the same condition. One of the important factors af-
fecting production of biochemical in fermentation media is the rheol-
ogy of the system. The presence of solid material in the slurry used in
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the preceding fermentation must have impeded organism's movement,

access to nutrient and decreased in the amount of dissolved oxygen
(Chen, 2013). Improved rheological parameters in the filtrate media
thus enhanced higher production of bioethanol recorded in this experi-
ment (Núñez-Ramírez et al., 2012).

3.3. Effect of organism loading on bioethanol production

Result of the experiment to determine the effect of increase in organ-

ism inoculums' size used in production of bioethanol is shown in
Fig. 3. Effect of organism loading on bio ethanol production.

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perature raises from low to high. Hot water in the pretreatment cham-
ber becomes polar and form hydrogen bonds at low temperature. At ele-
vated temperatures, hot water dissociates and become acidic due to the
formation of hydronium ion (H3O+) (Yan et al., 2016). The ion forma-
tion triggers the cleavages of linkage bonds in polysaccharides and the
release of monomeric sugars. This phenomenon is responsible for hemi-

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cellulose degradation (Kumar et al., 2010).
Fig. 6a shows the effect of CHW pretreatment on micro-structural
changes of CS used for bioethanol production. At low pretreatment tem-

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perature, CHW induced pores, higher surface area and fiber extension
on the surface of the PCS when compared to the untreated (Fig. 6a i
& ii). Under sever heat pretreatment, the compact structure of the CS
was distorted and the three-dimensional structure of the PCS collapsed
(Fig. 6a iii). The structural collapse of the CS orchestrated the degrada-
tion recorded in the polysaccharide (hemicellulose) molecules and the
release of monosaccharide molecules necessary for fermentation.
Application of CHW at 180 °C for the pretreatment of CS increased

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cellulose crystallinity as shown by the X-ray diffraction pattern (Fig.
Fig. 4. Time-course for sugar consumption and bioethanol production. 6b). FTIR spectroscopy revealed changes in chemical bonds of the com-
ponents of the PCS when compared to the untreated and control CS (Fig.
bioethanol production using various lignocellulosic materials (Table 1). 6c). The little peak observed on the absorption spectra around 896 cm−1
in the untreated CS is due to C-O-C stretching of ?-glucosidic linkages
present in Cellulose and hemicellulose (Xiao et al., 2011). This peak
3.4. Effect of CHW pretreatment on physical, chemical composition and appeared weaker in the spectra of the control samples and vanished in

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micro-structure of CS the spectra of the 180 °C CHW pretreated CS. Absence of this peak in
the PCS suggests loss of these linkages in the polymer (CS) and the re-
The CHW method applied for pretreatment of CS had effect on the lease of its monomeric units. Distortion of peak orientations were also
physical appearance of the lignocellulosic materials as shown in Fig. 5. noticed around wave length 1328–1239 cm−1 which are regions due to
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This effect is observed to be commensurate with the intensification of fingerprints of phenolic OH, syringyl and guaiacyl ring condensation,
the pretreatment temperature. The appearances of the CS increasingly and C O stretching of lignin (Kucharska et al., 2019). This distor-
turn brown, dark brown to black with increase in pretreatment temper- tions also suggests the release of phenolics due to disintegration in lignin
ature applied. This color change might have been due to caramelization structure in the PCS. These losses and changes in chemical bonds in
effect of the constituent sugars present in the lignocellulosic material the PCS are further evidence of the effect of CHW pretreatment on the
and increase in the amount of fixed carbon content of the PCS (Quintas chemico-structural units of lignocellulosic material used for bioethanol
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et al., 2007; Zhao et al., 2017). production (Himmelsbach et al., 2002).

Effect of CHW pretreatment on the chemical composition of CS used
for optimal bioethanol production is shown in Table 2. Under optimal 4. Conclusion
condition of 180 °C, CHW pretreatment degraded 73.8% hemicellulose.
CHW pretreatment has its most severe effect on the hemicellulose con- CHW pretreatment of CS with various pretreatment temperature and
tent of the lignocellulosic material used for this study. Mosier et al. the effect on bioethanol production was investigated. Optimal perfor-
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(2005) earlier reported that the CHW pretreatment method resulted in mance of CHW pretreatment on PCS recorded with 180 °C PCS which
degradation of large proportion of hemicellulose. The increase in cellu- produced 16.48 g/L bioethanol after 48 h. Yield of bioethanol was fur-
lose and lignin content may be due to re-polymerization and condensa- ther improved using filtrate of CHW pretreated CS and 5% organism
tion products formed after hemicellulose removal (Lü and Saka, 2010). load. We ascribed yield of bioethanol to degradation of hemicellulose
present in CS and this proves that CHW is an efficient method to degrade
Generally, the mechanism via which CHW degrades lignocellulosic chemical constituents present in biomass. The pretreatment applied to
lignocellulosic material in this study proved effective and adaptable for
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biomass has been reported to be anomalous as the pretreatment tem

scale-up production of bioethanol.

Table 1
Comparison of several pretreated methods and lignocellulosic materials for used for bioethanol production.

Lignocellulosic Pretreatment Organism Ethanol (g/L) Ref

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Rice NaOH Mucor circinelloides 30.60 (Takano and Hoshino, 2018)

Palm wood H2SO4 + HNO3 + H3PO4 Kluveromyces marxianus 22.90 (Raja Sathendra et al., 2019)
Sugarcane bagasse NaOH S. cerevisiae 14.00 (Bhadana and Chauhan, 2016)
Oil palm bunch hydrolysate NaOH + H2SO4 S. cerevisiae 8.49 (Duangwang and Sangwichien, 2015)
Cassava pulp H2SO4 + Ca(OH)2 S. cerevisiae 11.90 (Akaracharanya et al., 2011)
Corn stover Steam explosion S. cerevisiae 44.55 (Li et al., 2011)
Corn stover Steam explosion S. cerevisiae 50.00 (Tian et al., 2013)
Corn stalk H2SO4 + 160 °C Trichoderma reesei 16.20 (Aboagye et al., 2017)
Corn stalk NaOH + 160 °C Trichoderma reesei 21.30 (Aboagye et al., 2017)
Corn stalk CHW S. cerevisiae 31.06 This study

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Declaration of competing interest

The authors declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.

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Acknowledgements

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We thank the Commission on Science and Technology for Sustain-
able Development in the South (COMSATS) and Bangladesh Council of
Scientific and Industrial Research (BCSIR) for the award of COMSAT-BC-
SIR Post-Doctoral fellowship to Dr. Adekunle E.A.

Fig. 5. Physical appearance of untreated and compressed hot water treated corn stalk at
different temperature.

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Table 2
Compositional analysis and bioethanol yield of the compressed hot water pretreated corn stalk at the optimal conditions.

Hemicellulose (%) Cellulose (%) Lignin (%) Ethanol

Before treatment After treatment Before treatment After treatment Before treatment After treatment Produced (g/L) Yield (g/g)

33.55 ± 0.76 8.78 ± 0.34 38.70 ± 3.44 42.14 ± 1.83 18.94 ± 2.98 25.56 ± 1.11 31.06 ± 1.52 0.449
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Fig. 6a. Scanning Electron Microscope (SEM) images: i - Untreated CS; ii - Control; iii - 180 °C PCS.

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Fig. 6b. Degree of crystallinity of the untreated CS, Control and 180 °C PCS.

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Fig. 6c. Fourier-transform infrared (FTIR) spectroscopy of the raw corn stalk, Control (water treated corn stalk) and 180 °C pretreated corn stalk.

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