Journal Pre-proof

Bioethanol Production from Characterized Pre-

treated Sugarcane Trash and Jatropha Agrowastes

Naglaa A. Elnagdy, Tamer I.M. Ragab, Mohamed

A. Fadel, Mohamed A. Abou-Zeid, Mona A.
Esawy

PII: S0168-1656(24)00062-2
DOI: https://doi.org/10.1016/j.jbiotec.2024.02.015
Reference: BIOTEC9339

To appear in: Journal of Biotechnology

Received date: 5 December 2023
Revised date: 25 February 2024
Accepted date: 25 February 2024
Please cite this article as: Naglaa A. Elnagdy, Tamer I.M. Ragab, Mohamed A.
Fadel, Mohamed A. Abou-Zeid and Mona A. Esawy, Bioethanol Production
from Characterized Pre-treated Sugarcane Trash and Jatropha Agrowastes,
Journal of Biotechnology, (2024)
doi:https://doi.org/10.1016/j.jbiotec.2024.02.015
This is a PDF file of an article that has undergone enhancements after acceptance,
such as the addition of a cover page and metadata, and formatting for readability,
but it is not yet the definitive version of record. This version will undergo
additional copyediting, typesetting and review before it is published in its final
form, but we are providing this version to give early visibility of the article.
Please note that, during the production process, errors may be discovered which
could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2024 Published by Elsevier.
Bioethanol Production from Characterized Pre-treated Sugarcane Trash and Jatropha
Agrowastes
Naglaa A. Elnagdya, Tamer I. M. Ragabb,*, Mohamed A. Fadelc,
Mohamed A. Abou-Zeid a,d, Mona A. Esawyb
a
Department of Microbiology, Faculty of Science, Ain Shams University.
b
Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries and
Drug Research Institute, National Research Centre, Dokki 12622, Cairo, Egypt.
c
Microbial Chemistry Department, Biotechnology Research Institute, National Research
Centre, Giza 12622, Egypt.

of
d
Faculty of Science, Galala University.
Corresponding author: [email protected]

ro
Abstract

-p
Low production costs and a potential feedstock supply make lignocellulosic ethanol
(bioethanol) an important source of advanced biofuels. The physical and chemical preparation
re
of this kind of lignocellulosic feedstock led to a high ethanol yield. In order to increase the
yield of fermentable sugars, pretreatment is an essential process step that alters the
lP

lignocellulosic structure and improves its accessibility for the expensive hydrolytic enzymes.
In this context, the chemical composition of sugarcane trash (dry leaves, green leaves, and tops)
a

and jatropha (shell and seed cake) was determined to be mainly cellulose, hemicellulose, and
lignin. Hydrogen peroxide and sodium hydroxide were applied in an attempt to facilitate the
rn

solubilization of lignin and hemicelluloses in five agrowastes. The extraction of hydrogen

u

peroxide was much better than that of sodium hydroxide. A comparative study was done using
SEM, EDXA, and FTIR to evaluate the difference between the two methods. The pretreated
Jo

wastes were subjected to saccharification by commercial cellulases (30 IU/g substrate). The
obtained glucose was fortified with nutrients and fermented statically by Saccharomyces
cerevisiae F-307 for bioethanol production. The results revealed the bioethanol yields were
325.4, 310.8, 282.9, 302.4 and 264.0 mg ethanol/g treated agrowastes from green leaves of
sugarcane, jatropha deolied seed cake, tops sugarcane, dry leaves of sugarcane, and jatropha
shell, respectively. This study emphasizes the value of lignocellulosic agricultural waste as a
resource for the production of biofuels as well as the significance of the extraction process.

Keywords: Agrowastes, Pretreatment, Cellulases, Sacchrification, Saccharomyces cerevisiae,

Bioethanol.
 1. Introduction

Biofuels have environmental friendliness, attracting interest from all over the world. As
a carbon-neutral energy source that is renewable, unlike fossil fuels, which would cause global
warming, biofuels do not upset the balance of air molecules in the atmosphere. Reducing
reliance on traditional fossil fuels by employing biofuels is among the most feasible
approaches. The world's most abundant biomass, lignocellulose, may be found in practically
all extant plants as leaves, peels, bodies, branches, etc. As fossil fuels are used up, there is a
growing need for renewable energy, particularly biofuels (Palmqvist et al., 2000). Therefore,
the manufacturing of lignocellulosic bioethanol is unquestionably a method of supplying

of
energy, particularly for nations.

ro
Agriculture and agro-industrial wastes, as well as inexpensive lignocellulosic biomass
supplies. These materials can range from sawdust to poplar trees, sugarcane bagasse to brewer's
-p
leftovers, grasses and straws to grain stems, leaves, husks, shells, and peels from corn,
sorghum, and barley. Despite using these materials to create valuable products, lignocellulose
re
wastes continue to accumulate annually in enormous amounts, posing environmental issues
(Sheehan, 2001). The polysaccharides in lignocellulose wastes are inherently shielded by their
lP

structure from enzyme and chemical hydrolysis, making the chemical and biological
conversion of lignocellulose to other products, including ethanol, more challenging. Lignins
a

are involved in the cross-linking of cellulose and hemicellulose in the matrix. These
rn

characteristics of lignins increase the strength and hardness of the lignocellulose structure.
Pretreatment, which removes lignins from lignocellulose and improves the penetration of
u

hydrolysis agents, is therefore an essential stage in the process of turning biomass into
Jo

bioethanol (Sheehan, 2001). Before lignocellulose is hydrolyzed and fermented, pretreatment

procedures are done. Before-hand care To make cellulose more easily hydrolyzed, more
amorphous areas should be present. To pretreat lignocelluloses, many physical, chemical, and
biological techniques are applied. use of chemicals It is well known that lignin can be dissolved
in aqueous, acidic and alkaline solutions. In order to potentially co-ferment hemicellulose with
a tailored yeast strain and produce the highest bioethanol output in history—up to 29% (%) dry
matter—the ideal K3PO4 pretreatments could leave nearly all of the xylose in the
hemicellulose (Fu, et al., 2022). For the most efficient and practical lignocellulosic bioethanol
production method, acidic and alkaline pretreatments of lignocellulose are used (Szczodrak et
al., 1986; Farid et al., 1983). Acidic pretreatments: sulfuric acid and hydraulic acid are
frequently used, although they are not particularly advised due to the production of furfural
compounds during the pretreatment process, which prevents the growth of microorganisms
during the fermentation process (Zhang et al., 2016; Zhang et al., 2023). The loss of
carbohydrates from hydrolysis is reduced when lignocellulose is first treated with an alkaline
solution. Additionally, it facilitates a later hydrolysis, inhibits the production of furfural, and
helps to eliminate acetyl groups. Because of its affordability and excellent efficacy, NaOH is
the most often used alkali for lignin removal in lignocelluloses (Rolz et al., 1986; Nazarpour,
2013). The single pretreatment technique doesn't seem to be able to produce the desired
outcome. It has long been practiced to combine several pretreatment techniques. The
polysaccharide-enriched material is hydrolyzed to hexoses by enzymes after lignocellulose has

of
undergone pretreatment (Chen et al., 2017; Fernandes et al., 2015). Cellulases, a general term
for a variety of enzymes that are isolated from microorganisms, are what are used in

ro
commercially available products to hydrolyze cellulose. These enzymes cleave glycosidic
bonds in carbohydrates, often via inverting or retaining processes, the latter of which advances.
-p
Microorganism development during fermentation is facilitated by enzymatic hydrolysis. The
single sugars that were released as a result of enzymatic hydrolysis are metabolized by
re
fermentation microorganisms to produce bioethanol. The enhanced cellulose nanofibrils
lP

demonstrate efficacy in stimulating the release of lignocellulose-degradation enzymes from

fungi, as seen by the 100% and 138% increase in the activity of the two cellulases
(exoglucanases and β-glucosidases), as well as the 44% rise in total protein content. Our
a

findings thus point to a unique environmentally friendly method of combining precise genetic
rn

alteration of lignocellulose substrates with effective biomass process technology to achieve

high-quality diversified bioproduction (Zhang et al., 2023).
u

The aim of the work is to produce bioethanol as renewable energy by using different
Jo

agricultural wastes (dry leaves, green leaves, tops of sugarcane, shell, and deoiled seed cake of
jatropha). The main biopolymers (cellulose, hemicellulose, and lignin) were extracted by using
different alkaline solutions (sodium hydroxide and hydrogen peroxide). SEM, EDXA and
FTIR were used to compare the effects of the two methods. The results showed that the
pretreatment with H2O2 was more efficient than NaOH. The application of extracted methods
was a new trend that improved the biomass of bioethanol production by decreasing the
inhibitors of fermentation processes.

2. Materials and Methods

2.1. Materials
 2.1.1. Collection of agrowastes

Sugarcane trash (dry leaves, green leaves, and tops) and jatropha seed cake will be collected
from the El Menia area of Upper Egypt. The collected agricultural wastes will be dried in open
air (in direct sunlight) and then grinding to a 0.5 mm mesh. After the sugarcane harvest, the
trash remaining consists of three main components, namely, dry leaves, green leaves, and tops
(Canilha et al., 2012), which dried in open air for almost seven days to remove water. Jatropha
seeds were collected after separating from the shell; these seeds squeezing in EL Hussein
presses of the kernel cold to remove oil to obtain jatropha seed cake.

2.1.2. Cellulase enzyme

of
Enzyme , cellulase, was gained from commercially available sources and were of

ro
industrial grade from Stern Enzym GmbH and Co.KG.Germany.

2.2. Methods
-p
2.2.1. Chemical composition of the untreated and treated agrowastes
re
According to AOAC (2012), we will determine the chemical composition (e.g., moisture,
lP

ash, wax, crude lipids, and low-molecular-weight carbohydrates (LMWC) of agricultural

wastes. According to Jayme and Knoll (1956), low-molecular-weight carbohydrates can be
determined by dissolving in 85% ethanol for 24 hours and examined by paper chromatography,
a

which is done after soxhlet extraction. Spots were detected by spraying the papers with aniline-
rn

phthalate reagent (Partridge, 1949) and aniline-xylose. Quantitative screening of glucose,

arabinose, and xylose was determined using the phenol-sulfuric acid method (Dubois et al.,
u

1956). Thereafter, the color density was measured at wavelengths of 480 nm and 490 nm for
Jo

pentoses and hexoses, respectively.

2.2.2. Total carbohydrate determination

Using strong acid hydrolysis, which extracted cellulose and hemicellulose as glucose
(Dubois et al., 1956), measured the total carbohydrates in agricultural wastes using the
following procedure: Carefully, 0.5 mg of sample was stirred with 0.5 ml of ice-cold 80%
H2SO4 at ambient temperature for 15 hours. It was then diluted with a mixture of cold distilled
water (up to 13 mL). The solution was hydrolyzed, then heated in a sealed tube for 6 hours in
a water bath that had previously been boiled. To neutralize the hydrolyzate solution, the
determined amount of BaCO3 was added. Washing with water was used to filter the neutralized
solution. A cation exchange resin called Amberlit IR-120 (H+) was used to treat the filtrate.
Using the phenol-sulfuric acid technique, the extract's total carbohydrate content was
calculated. This method's specifics were as follows: 1 ml of the resulting diluted solution was
added to 1 ml of a 5% phenol solution after the appropriate dilution. After blending, 5 ml of
concentrated H2SO4 was quickly added to the mixture, shook, and left to sit for 10 minutes at
room temperature before being heated to 20 to 30°C (in a water bath) for 20 minutes. Then,
using a 490 nm spectrophotometer, the color density was determined. UNICO 7200.

of
2.2.2.1.Qualitative examination

ro
Using a solvent system of n-butanol-acetone-water (4:5:1), the ethanol extract was
decolored by boiling it with charcoal, concentrating it under low pressure at 45°C, and

-p
examining the results using paper chromatography Whatman No.1Reference (Jayme and
Knoll, 1956). Chromatography was used to analyze real samples of xylose, arabinose,
re
glucuronic acid, and glucose. By spraying the sheets with the aniline-phthalate reagent, which
is made up of 1.66 g of o-phthalic acid and 0.91 ml of aniline diluted in a solution of 48 ml n-
lP

butanol, 48 ml diethyl ether, and 4 ml water, (Partridge, 1949) asserts that spots were observed.
The chromatogram was air dried following chromatographic separation, dipped in 50 ml of
a

aniline-phthalate reagent, and heated in an oven for 10 minutes at 105°C to produce the colored
rn

parts.

1.1.1.1. Quantitative Determination

u

According to the modified approach of (Wilson, 1959), the hydrolysis sugars were
Jo

quantitatively determined. The individual chromatography spots were separated into short
strips, dropped into a test tube containing 4 ml of eluting agents (0.7 N HCl in 80% ethanol),
and shaken for thorough elution. Using the spectrophotometer UNICO 7200, the absorbance
of the resulting colored solutions was measured at 390 nm for pentoses and 490 nm for hexoses
sugars. By comparing the sugar amounts to relevant standard curves created under the same
circumstances, the sugar amounts were identified.
 2.2.3. Alkali pretreatment

2.2.3.1. Sodium hydroxide

Five grams of sugarcane (dry leaves, green leaves, and tops) and jatropha (shell and deoiled
seed cake) were treated individually at 90°C for one hour (15 ml/g of liquid to solid ratio) three
times in a 75 ml solution of sodium hydroxide with a pH of 12. The reaction mixture was
filtered, the residue (crude cellulose) was washed with water several more times until it reached
neutrality, and the filtrate was neutralized pH 7–6 by hydrochloric acid while being cooled.
After filtering, hemicellulose is left behind and lignin is present in the filtrate; to extract lignin,
hydrochloric acid was added until pH 1.5. There was lignin left over after filtering. At 105 °C,

of
all of the separated residues (cellulose, hemicelluloses, and lignin) were dried.

ro
2.2.3.2. Hydrogen peroxide

An alkaline hydrogen peroxide (H2O2) solution was prepared by adding 5% H2O2 in

-p
distilled water and adjusting the pH to 11.5 with NaOH (liquid to agrowaste, 20 ml/g), then the
reaction mixture was placed on a shaker at 40°C and 150 rpm for 6 h. The reaction mixture
re
was filtrated. The crude cellulose was washed with distilled water and dried in a hot-air oven
lP

at 60°C (Niju et al., 2020).

2.2.3.3. Bleached cellulose

a

Sodium hypochlorite (2-1 w/w) in an appropriate quantity of distilled water at 80°C for two
rn

hours, followed by filtering, was used to create bleached cellulose from crude cellulose. In
accordance with (Ragab et al. 2014), the cellulose residue was completely dried at 70°C after
u

being extensively rinsed with distilled water until neutralization.

Jo

2.2.4. Chemical analysis

2.2.4.1. FTIR spectroscopy

The isolated products were characterized and identified using FT-IR. Using a Bruker
Vectra 22 FT-IR Spectrometer with a Dura Sample IR IITM detector, IR spectra were
immediately acquired from the powdered cellulose, hemicelluloses, and lignin onto a detector
prism. In the wave number range of 4000-400 cm-1, all spectra were recorded at a spectral
resolution of 4 cm-1.

2.2.4.2. SEM analysis

 Three-dimensional (3D) structural information is of central importance in biological
research on many long-scales. There are excellent methods to obtain atomic, molecular
structures, electron microscopic organelles and light-microscopic- resolution tissue. The
resolution is enough to trace even the thinnest axons and to distinguish synapses. Stacks of
several hundred parts were collected, 50–70 nm thick (Denk and Horstmann, 2004). The
surface morphology of samples was examined using a scanning electron microscopy (JEOL
5410) microscope with an accelerating voltage conducted of 10 kV. Samples were gold coated
using a Hitachi coating unit IB-2 coater under a high vacuum, 0.1 Torr, high voltage, 1.2 kV
and 50 mA.

of
2.2.4.3. Energy Dispersive X-Ray analysis (EDXA)

ro
EDXA is an x-ray spectroscopic method for determining elemental compositions
(qualitative and quantitative analysis)

-p
re
a lP
u rn
Jo
 2.2.5. Assay of cellulolytic enzymes

2.2.5.1. Exo-1, 4- glucannase (FP-ase) assay

The determination of FP-ase activity was measured according to Mandels et al. (1976) by
mixing 1 ml of supernatant with 1ml of 1% Whatman filter paper no. 1 (strips of 1x6 cm)
suspended in 0.05M sodium citrate buffer pH 4.8 and incubated at 50oC for 30 min. The color
of the reaction was developed by adding DNS reagent and the produced fermentable sugars
were measured at 540 nm against a reagent blank (Miller, 1959). One unit of enzyme was
defined as the amount of enzyme that released 1 µg of glucose.

of
2.2.5.2. Endo-1, 4- glucannase (CMC-ase) assay

ro
The determination of carboxymethylcellulase (CMC-ase) was measured according to
(Mandels et al., 1976) by mixing 1ml of supernatant, 1% CMC dissolved in 0.05M sodium
-p
citrate buffer pH 4.8) and incubating at 50oC for 30 min. The color of the reaction was
developed by adding DNS reagent and for the produced fermentable sugars the absorbance was
re
measured at 540 nm against a reagent blank (Miller, 1959). One unit of the enzyme was defined
as the amount of enzyme that released 1 µg of glucose per ml per minute.
lP

2.2.6. Enzymatic saccharification of treated lignocellulosic

a

The materials were shaken at 130 rpm and 50 °C to initiate saccharification processes. The
first thing that was done was. Using 10–50 IU/g of cellulosic material, the enzymatic reactions
rn

were carried out in 100 ml of 0.5 M sodium citrate buffer solutions at pH 4.8. Following the
u

hydrolysis reaction's incubation period, the substance was filtered through filter paper, and the
Jo

hydrolyzed sample was separated for further analysis to identify the reducing sugars. Three
duplicates of the experiment were carried out.

2.2.7. Quantification of reducing sugars

Miller's technique (Miller, 1959) was used to calculate the total content of reducing sugars
produced after hydrolysis. Based on the absorbance at 490 nm, this technique operates. Using
glucose as the standard, a calibration curve was compared to quantify the quantity of reducing
sugars contained in each sample in triplicate.
 2.2.8. Bioethanol Fermentation

After scarification using commercial cellulase, the glucose syrup was evaporated to
concentrate the sugar content to 10% (w/v). There were 100 mL of YPM medium (containing
(g/L) malt extract, yeast extract, peptone, and sucrose) in each of four conical flasks with a 250
mL capacity was inoculated with a loop of the yeast strain S. cerevisiaeF-307, sterilized with
steam at 121 °C for 15 min, cooled to room temperature, and then let stand at 34 °C for 24 h.
Inoculating the ready fermentation vessels at 1% v/v with the preceding yeast culture. After
sterilization, the aforementioned sugar syrup was fortified with (g/L) yeast extract, malt extract,
magnesium sulfate, and diammonium phosphate. It was then inoculated with the

of
aforementioned Saccharomyces cerevisiae inoculum at 1% v/v and allowed to stand at 34 °C
for 72 hours. By applying the following formula to determine how much sugar is consumed

ro
during fermentation and conversion to ethanol:

-p
1 g of glucose produces 0.51 g of ethanol, which equals 0.51 ×100 81 = 0.62 mL ethanol, as
described in (Fadel, 2013).
re
2.2.9. Fermentation Efficiently

The calculation of fermentation efficiency involved dividing the amount of generated

lP

ethanol by the theoretical amount of ethanol and multiplying the result by 100 (Fadel, 2014).
% Hydrolysis = glucose amount (w) divided by initial substrate weight x 0.9 x1000
a
u rn
Jo

3. Results and Discussion

 3.1. Physiochemical composition

3.1.1. Chemical composition of sugar cane

The results were obtained from dry leaves, as shown in (Table 1) such as moisture
content (5.52%), wax (2.46%), low molecular weight (7.66%), ash (3.50%), lipid content
(1.14%) and total carbohydrate (14.88%). The percentage of ash in this study is lower than that
published in Gómez et al. (2014). The moisture on dried leaves measured in this investigation
was in close agreement with the findings reported by Franco et al. (2012) and lower than
published by Gómez et al. (2014) and the percentage of wax determined was higher than the
published results by Attarda et al. (2015). The obtained results of low molecular weight were

of
higher than those published by Gómez et al. (2014). While the results of green leaves in (Table
1) were an average of 11.19% of moisture content, 1.13% wax, 5.24% low molecular weight,

ro
0.74% total lipid, ash (2.20%), and 11.15% total carbohydrate. The result recorded in the ash

-p
determination was approximately what was determined in Gómez et al. (2014). The
percentages of wax, low molecular weight, total lipid, ash, and total carbohydrate of the tops
re
in this work were (15.00%, 0.83%, 10.84%, 0.12%, 2.70%, and 16.08%). The ash result was
lower than the result mentioned by Franco et al. (2012) where, ash was on average 5.4% , 4.1%
lP

and 4.6% for green tops, dry leaves, and bulk straw, respectively. The moisture content of
tops, which between 15.00% and 5.52%, was lower than (Franco et al., 2012). Tops have seven
a

times more moisture than dry leaves. Similary, Menandro et al. (2017) reported that green tops
contained six times more moisture than dry leaves (68% and 11%, respectively). The levels of
rn

ashes (4.7% on average) were similar in both the tops and the dry leaves. However, the
u

extractive content is higher in tops (25.7%) than in dry leaves (13.7%) (Franco et al., 2012).
Jo

The section of ash was, on average, 5.4% and 4.1% for green tops and dry leaves, respectively,
and 4.6% for bulk straw. There were also significant differences for extractives: 67% for green
tops and 33% for dry leaves, allowing (Menandro et al., 2017). Green tops also store up to four
times as many nutrients as dry leaves. Green tops also had six times higher humidity and a
higher chlorine content, which reduced the efficiency of the milling operation. In addition, the
quality of dry leaves was higher in lignin, cellulose , and hemicellulose and tended to be a
better feedstock for ethanol production in the second generation. Overall, the results show that
dry leaves are preferable for the production of bioenergy, while green tops are left in the field
for nutrient recycling (Menandro et al., 2017).

3.1.2. Chemical composition of jatropha

 Results obtained for jatropha shell, moisture content, wax, low molecular weight, total
lipid, ash and total carbohydrate were (5.46%, 5.27%, 5.61%, 1.65%, 5.00% and 12.74%),
respectively. The moisture content of the jatropha shell was lower than published in Inekwe et
al. (2012), and the part was roughly similar to the published results of (Murata et al., 2011).
The measurement of low molecular weight (Table 1) was lower than that issued by Singh et al.
(2008). Direct combustion of jatropha shell was also characterized by flame front instabilities
and short combustion periods due to its higher ash content (Ewunie et al., 2021). The firm
results in seed cake with an average 4.63% of moisture content, 4.51% wax, 4.27% low
molecular weight, 1.98% total lipid, 2.00% of ash and 11.57% total carbohydrate. Moisture of

of
jatropha seed cake was higher than the result of (Ricardo et al., 2014) but almost equal to result
the of (Inekwe et al., 2012). Consistent with the results of (Ricardo et al., 2014) the extraction

ro
of lipids for the seed cake preparation was highly efficient, leaving only 1.15% residual lipids,
which was something like a fraction of this study. The percentage determined of total
-p
carbohydrate which was similar to the result (Inekwe et al., 2012). The published (Ricardo et
al., 2014) amount of ash was higher than determined in this study.
re
3.2. Alkali treatment
lP

3.2.1. Sodium hydroxide

The results were obtained from dry leaves, as shown in (Table 1), Plus, the percentages
a

determined for cellulose, hemicellulose and lignin were (44.00%, 9.00% and 14.00%),
rn

respectively. In this study, the result of cellulose was approximately to the authors published
(Pereira et al., 2015) and higher than (Franco et al., 2012). The obtained results for
u

hemicellulose were lower than the results published (Gómez et al., 2014). In this study, the
Jo

percentage of lignin was higher than published (Gómez et al., 2014) but was lower than the
result obtained by Menandro et al. (2017). In (Table 1) results of green leaves were determined
by the calculation of cellulose, hemicelluloses and lignin (41.09%, 11.05%, and 18.04%),
respectively. In addition, the percentage of low- molecular-weight-carbohydrates (Table 1) was
higher than published in (Gómez et al., 2014). According to Gómez et al. (2014), the results
were lower than the obtained results for cellulose and lignin. Because the straw has two or three
times the quantity of silica as the green leaves do, the plant's hardening and mechanical
resistance are increased in these areas. The amount of lignin in dry leaves was lower than that
in green leaves in this study. Like, the amount of lignin in dry leaves was approximately 60%
smaller than that found in the green leaves according to Gómez et al. (2014).
 In this study, the percentages of cellulose, hemicellulose and lignin in the tops were
shown in (Table 1). By comparison, the levels of ashes (4.7% on average) were similar in both
the tops and the dry leaves. However, the extractive content is higher in tops (25.7%) than in
dry leaves (13.7%) (Franco et al., 2012). The proportion of cellulose in this study was lower
than published in Franco et al. (2012) and approximately to the result of (Menandro et al.,
2017) but the published results of hemicellulose were higher than the result shown in (Table 1)
and the percentage of lignin was lower than (Menandro et al., 2017).

Table 1 Chemical composition of sugarcane trash (dry leaves, green leaves &tops) and jatropha
(shell and seed cake) after pretreated alkaline sodium hydroxide and hydrogen peroxide

of
Treatment with NaOH Treatment with H2O2
Cellulose Hemicellulose Lignin Cellulose Hemicellulose Lignin LMWT

ro
Dry 44.00 9.00 14.00 80.62 3.24 16.14 7.66
leaves

Green
±0.56

41.09
±0.50

11.05
±0.24

18.04
-p±0.49

75.41
±0.48

1.48
±0.42

23.11
±0.27

5.24
re
leaves
±0.74 ±0.49 ±0.78 ±0.55 ±0.09 ±0.33 ±0.65
lP

Tops 33.53 12.48 13.93 71.42 3.29 25.34 10.84

±0.50 ±0.27 ±0.36 ±0.52 ±0.11 ±0.34 ±0.29

a

Jatropha 41.53 09.84 13.00 67.42 18.61 13.97 5.61

Shell ±0.77 ±0.62 ±0.57 ±0.60 ±0.36 ±0.44 ±0.23
u rn

Jatropha 36.97 7.23 26.95 72.42 7.72 19.86 4.27

Seed ±0.88 ±0.25 ±0.69 ±0.35 ±0.20 ±0.19 ±0.13
Jo

cake

In (Table 1) results of shell, in keeping with Odetoye et al., (2018) the percentage of
cellulose was approximately as obtained in this study and higher than determined by Singh et
al.( 2008). And the amount of hemicellulose in this work, 9.84% was average to publish in
Singh et al. and lower than published in (Ewunie et al., 2021). The section of lignin in Table
13.00% was higher than circulated in Singh et al.(2008) and lower than the result mentioned
by (Ewunie et al., 2021). Seed cake in this work, the percentages determined for cellulose,
hemicellulose and lignin were (36.97%, 7.23% ,and 26.95%) respectively (Table 1). The
percentage of cellulose determined in this work was higher than obtained in (Ricardo et al.,
2014; Shuhairi et al., 2015). While the result of hemicellulose in this work was similar to
(Shuhairi et al., 2015) and the obtained result of lignin in (Table 1) was approximately
according to that of (Shuhairi et al., 2015) and lower than that result of (Ricardo et al., 2014).

3.2.2. Hydrogen peroxide

In Table 1 below, we saw that the results of cellulose, hemicelluose and lignin were
treated by alkaline hydrogen peroxide, which gave the highest percentage of holocellulose and
removed a high amount of lignin compared to the other traditional method, which removed a
small amount of lignin that would become an inhibitor to microorganisms in fermentation. The
percentage of cellulose, hemicellulose and lignin in the dry leaves of sugarcane was 80.62%,
3.24% and 16.14% respectively, while the green leaves contained 75.41% cellulose, 1.48% of

of
hemicellulose and 23.11% lignin. In connection with the result, sugarcane tops contain 71.42%
cellulose, 3.24% of hemicellulose and 25.34% lignin. According to the results published by

ro
(Niju et al., 2020), the percentage after alkaline hydrogen peroxide was 50.55%, which means

-p
the result of this work was higher than published. After pretreatment of jatropha shell and
deoiled seed cake with H2O2, the result obtained with that jatropha shell contained 67.42% of
re
cellulose and 18.61% of lignin, while deoiled seed cake contained 72.42%, 7.72% and 19.86%
were the percentage of cellulose, hemicellulose and lignin, respectively. H2O2 has been used
lP

to disrupt and destroy the lignocellulosic structure of biomass for decades. However, because
it creates potent hydroxyl radicals, its impact on biomass digestibility and delignification was
a

pH-dependant. This classic chemical pretreatment approach has been used to produce
fermentable sugar and bioethanol from lignocellulosic feedstocks such as sugarcane bagasse,
rn

rice husks or hulls, wheat straw, and other lignocellulosic feedstocks (Niju et al., 2020). The
u

authors suggested that high the alkalinity of sodium hydroxide deformed and soluble the
Jo

amorphous cellulose in the agrowastes so that the cellulose yield was low. The yield of
extracting cellulose by hydrogen peroxide (H2O2) was higher than by the sodium hydroxide
methodology. In contrat, lignin percentage was higher than in the case of the hydrogen peroxide
(H2O2) method, so the process of hydrogen peroxide (H2O2) is better than the sodium hydroxide
process because we decrease the lignin percentage, which plays an inhibitor in the fermentation
process.
3.3. Monosaccharide constituents
3.3.1. Sugar cane trash (dry leaves, green leaves and tops)
Dry leaves Monosaccharide constituents in this study were 15% D-xylose, 10% L-
arabinose, 20% D-fructose, 25% D-glucose, 20% D-galactose, 5% D-glucoronic acid and 5%
D-glactouronic acid, which are presented in (Table 2). While the monosaccharide constituents
of green leaves were (30%, 20%, 40%, and 10%) D-xylose, D-fructose, D-glucose, and D-
glactouronic acid, respectively. Chloroplast degradation and eventual depletion of complete
chlorophyll are among the earliest occurrences during the senescence of the leaves (Hendry
and Grime, 1993). In the dry and green leaves, the neutral monosaccharides fucose, rhamnose,

of
arabinose, galactose, glucose, and xylose were examined. Arabinose and xylose are
monosaccharides released from arabinoxylan, which is one of the most abundant

ro
hemicelluloses in the cell walls of sugarcane. Arabinoxylan (dry leaves) was not damaged in
the senescence process. Thus, it was very likely that the detected glucose in sugarcane leaves
-p
was a derivative of the mixed linkage β-glucan (β-glucan), another hemicellulose found in
sugarcane cell walls. However, measures have not been taken to remove starch from the cell-
re
wall preparation; thus, starch can also contribute to glucose. Furthermore, it is probable that a
lP

small portion of the glucose and xylose could not be discarded from xyloglucan, which also
occurred in sugarcane leaf cell walls. A small amount of galactose (~2.5 percent) and only
traces of fucose and rhamnose have been found. In these cases, it was presumed that these
a

monosaccharides are derived from pectin polymers (apart from some of the arabinose described
rn

above). Our findings show that no substantial changes in large hemicellulose or pectin cell-
wall polymers occur during sugarcane leaf senescence (yellowing of leaves) (Martins et al.,
u

2016). In the co-variance study, according to the authors, shown this in the 'in-leaf' gradient of
Jo

senescence, the monosaccharides rhamnose, arabinose, galactose and glucose are positively
correlated, while xylose is negatively correlated with all the above monosaccharides. The
percentage of tops Monosaccharide constituents were 5% D-xylose, 5% L-arabians, 30% D-
fructose, 30% D-glucose, 15% D-galactose, 10% D-glucoronic acid and 5% D-glactouronic
acid, which exposed in the (Table 2).

3.3.2. Jatropha (shell and seed cake)

The ratio shown in Table 2 monosaccharide constituents of jatropha shell was 5% D-

xylose, 5% D-fructose, 80% D-galactose and 10% D-glactouronic acid. The predominant
hemicellulosic sugar was xylose, followed by mannose, galactose, and arabinose. That's in
alignment with most of the previous studies. A significantly higher content of arabinose was
recorded only for Brazilian shell than that of mannose and galactose (García et al., 2014).
Galactose represented 31.6% of the sugars contained in the extract, followed by glucose
(21.7%), arabinose (16.3%) and rhamnose (11.9%) (García et al., 2014). In (Table 2)
represented jataropha seed cake monosaccharide constituents were 85% D- glucose, 10% D-
galactose and 5% D-galactouronic acid.

Table 2. Monosaccharide constituents of raw biomass after acid hydrolysis (% w/w)

Monosaccharide
constituents Sugarcane trash Jatropha
Dry leaves Green Tops Seed cake Shell

of
leaves

ro
D-xylose 15.00 30.00 5.00 ----- 5.00

L-arabinose 10.00 ------- 5.00 ---- ----

D-fructose 20.00 20.00 -p 30.00 ----- 5.00

re
D-glucose 25.00 40.00 30.00 85.00 ------

D-galactose 20.00 ----- 15.00 10.00 80.00

lP

D-glucoronic 5.00 ------ 10.00 ------ -----

acid
a

D-glactouronic 5.00 10.00 5.00 5.00 10.00

rn

Acid
u

3.4. Scanning electron microscopy (SEM)

Jo

3.4.1. Sugarcane trash

Scanning Electron Microscopy (SEM) was utilized to analyze the morphology of
untreated and treated sugarcane leaves and the surface of the treated sample showed broken
fibrils. SEM on dry leaves of sugarcane observed that the fibers were smooth like (Fig. 1A).
Ultrastructure observation in green leaves found that the chloroplasts were near the cell wall
and well organized in most instances, and the chloroplast thylakoids were organized in the
chloroplasts in order. As a result of the continuous stress of the drought, the leaves became dry,
plasmosis developed, the chloroplasts moved toward the center of the cell and eventually
changed from long and oval to nearly round, and the amount of starch increased as the water
content of the leaves decreased and the tips of the leaves began to curl. The native samples are
rigidly packed and compact (Sindhu et al., 2014).
 SEM dry leaves of sugarcane, which are represented in (Fig. 1B), appeared to have
cellulose fibers and very small fibers of hemicellulose and lignin, that indicating the removal
of them after the NaOH pretreatment process. The surface of dry leaves is ruptured after
pretreatment by the aggregate impact test apparatus setup on the leaves, and rough and oval-
shaped form fibers, such as material, have been elongated (Patil and Deshannavar, 2019). In
green leaves of sugarcane after pretreatment cellulose fibers aggregated with the removal of
hemicellulose, lignin, and chloroplast (Fig. 1E). While sugarcane tops (Fig. 1H) observed that
the structure slanted with fibers of cellulose and small fibers of hemicellulose and lignin after
pretreatment. The unpretreated samples have a compact, rigid structure, while the pretreated

of
samples show a distorted structure. The distorted structure and increase in surface area of the
pretreated sugarcane tops improve the hydrolysis efficiency (Sindhu et al., 2014).

ro
SEM analysis of dry leaves after pretreatment with H2O2, appears to show that of

-p
crystanilty of cellulose and hemicellulose removed a high percentage of lignin and the same
result obtained in green leaves and tops (Fig. 1C, F, I). The internal structures of the pretreated
re
sample showed distortions that indicate the breakdown of the lignin, hemicellulose, and
cellulose complex. The deformed shape can enhance the effectiveness of acid hydrolysis by
lP

increasing the accessible surface area (Sindhu et al., 2014).

a
u rn
Jo
 A B C

D E F

of
ro
-p
re
G H I
a lP
u rn
Jo

Fig. 1 SEM analysis ( Sugarcane – (A) raw materials of dry leaves, (B) dry leaves treated by
sodium hydroxide, (C) dry leaves by H2O2, (D) raw materials of green leaves, (E) green
leaves by sodium hydroxide, (F) green leaves by H2O2, (G) tops of raw materials, (H) tops
treated by sodium hydroxide and (I) tops by by H2O2
3.4.2. Jtropha (shell and seed cake)
When applied to the SEM analysis of the jatropha shell, it was presented that rod-like fiber
with groove slightly appearance in (Fig. 2A) which agrees with puplished of the untreated shell
(control) micrographs, which showed an intact rod-like fibril structure and a smooth exterior
surface. From the internal view of the shell, a surface layer covering the structure of the fibril
cell can be seen. In the untreated shell, no singular or loose fibers were found. SEM pictures
of shell that were handled showed a big difference between pretreated and untreated. According
to (Fig. 2D) which appeared in the SEM analysis of jatropha deoiled seed cake, fibers of
cellulose, hemicellulose and lignin were accepted as smooth fibers with the papper (Shuhairia
et al., 2015).
After NaOH pretreatment of the jatropha shell, which resulted in the removal of lignin
and hemicellulose to obtain pure cellulose fibers which appeared as rods with grove fibers (Fig.
2B). Pretreated shells had a loose, twisted fiber structure and pores that were possibly due to
biomass delignification. The study of the SEM supports the enzyme hydrolysis information,
which showed high yields of fermentable sugar. There is better interaction between the enzyme

of
and biomass because hydrolysis of the jatropha shell, destruction of the outer surface layers of
the jatropha shell, distortion and loosening of the fiber structure, and formation of pore

ro
structure help. Though the removal of not need fibers of hemicellulose and lignin to take
cellulose fibers, jatropha deoiled seed cake was considered in this study after pretreatment.
-p
The extracted sample displayed an irregular and disturbed surface structure substantially
different from the raw seed sample, primarily due to the breaking up of the lignin carbohydrate
re
matrix during the extraction phase. This results in the aggregation of such lignin complexes
lP

that are concentrated on the surface of the sample collected. In order to delignify the biomass
and increase the usable surface area of cellulose and hemicellulose to increase enzymatic
hydrolysis (Shuhairia et al., 2015). SEM analysis of the shell and deoiled seed cake of jatropha
a

after pretreatment by H2O2, appears to show the of crystanilty of cellulose and hemicellulose
rn

and the removal of a high amount of lignin.

u
Jo
 A B C

D E F

of
ro
-p
re
Fig. 2 jatropha shell- (A) raw materials, (B) treated by sodium hydroxide, (C) treated by
H2O2 and jatropha deoiled seed cake- (D) raw materials, (E) treated by sodium hydroxide, (F)
lP

treated by by H2O2
3.5. EDXA analysis
a

Qualitative and quantitative elements of sugarcane trash (dry leaves, green leaves and
rn

tops) are determined by EDXA and given as percentages. Firstly, dry leaves of sugarcane
(47.19% C, 0.76% N, 45.03% O, 0.35% Na, 0.20% Mg, 0.10% Al, 1.13% Si, 0.69% P, 1.15%
u

Cl and 3.40% K) and elements of carbon and oxygen had high percentages observed. In
Jo

addition to the dry leaves, this study determined sugarcane green leaves and a small ratio of
Mg, Na, Al, P, Cl, K which presented in (Table 3) and high percentages of C, N, O and Si.
EDXA analysis showed the tops of sugar cane contained the percentages of quantitative
elements such as C, which were was approximately equal to the ratio of O and minor traces of
Mg, Na, Al, P, Cl, K, which presented in (Fig. supplementary data). The tops had the maximum
N, K, P and Ca, while the dry leaves contained more of Mg. This work showed that the highest
N content in tops, whereas dry leaves had the lowest N content, which is equivalent to the
results of (Franco et al., 2012). The content of N, P and K in green leaves was much higher
than in other parts of sugarcane greatly, but the content of Ca, Mg and S in bone-dry leaves
was higher than in other parts. The highest content of Zn and Cu appeared in green leaves, and
Fe appeared in underground parts and the accumulation of N, P and K in stems accounted for
about 60%, and in green leaves and tip accounted for 20%-25%. Ca, Mg and S mainly
accumulated in the stem, bone-dry leaves, green leaves and tip (Xie et al., 2010). In sugarcane
dry leaves the nutrients Ca, S, Mg, B, Mn and Al were seen to have accrued. The leaves had a
comparatively higher abundance of Ca, S, Mn and Al. In the various areas of the leaf, Ca, and
Al had varying accumulation amounts between 50 and 75 percent from the tip to the base of
the leaf. Mg and B showed modest accumulation (25% on average). Interestingly, Mg showed
also high accumulation levels in the base of the leaves, about duplicate a compared to the other
portions of the leaf.
Qualitative and quantitative elements of sugarcane trash crude cellulose dry leaves after

of
NaOH pretretment (Fig. supplmentry data) (42.11% C, 2.13% N, 52.19% O, 0.72% Na, 0.69%
Mg, 0.16% Al, 1.02% Si, 0.19% P, 0.12% Cl and 0.11% K). Crude ellulose green leaves contain

ro
elements differentiated namely carbon (56.54%), which was the highest percentage, and the
small were N, Na, Mg, Si, P and Cl. EDXA analysis of crude cellulose tops showed that they
-p
contained mainly C, N, O, Na, Mg, Al, Si, P, Cl, K which presented in (Table 3).
In (Table 3) presents the quantitative elements after H2O2 pretreatment of dry leaves
re
(45.30% C, 1.04% N, 48.41% O, 0.16% Na, 0.13% Mg, 0.12% Al, 1.43% Si, 0.22% P, 0.10%
Cl- and 0.12% K). While the green leaves have variations, as determined in (Table 3) (53.06%
lP

C, 1.74% N, 32.80 % O, 0.06% Na, 0.04% Mg, 0.14% Al, 3.28% Si, 0.25% P, 0.05% Cl and
0.29% K).
a
u rn
Jo
Table 3 Qualitative and quantitative elements of sugarcane trash (Dry leaves, Green leaves
and Tops) (weight %).
Biomass Raw materials NaOH treatment H2O2treatment
Element Dry Green Tops Dry Green Tops Dry Green Tops
leaves leaves leaves leaves leaves leaves
C 47.19 49.58 42.70 42.11 56.54 42.38 45.30 53.06 50.43
N 00.76 01.59 2.20 2.13 0.99 1.94 1.04 1.74 1.43
O 45.03 38.50 41.07 52.19 32.56 53.62 48.41 32.80 42.10
Na 00.35 0.05 00.89 0.72 0.87 0.55 0.16 0.06 0.54

of
Mg 00.20 0.01 0.27 0.69 0.04 0.23 0.13 0.04 0.16
Al 00.10 0.12 0.29 0.16 2.97 0.06 0.12 0.14 0.16

ro
Si 01.13 8.96 10.96 1.02 0.26 0.12 1.43 3.28 0.29
P 0.69 0.72 0.59 0.19 0.22 0.21 0.22 0.25 0.21
Cl
K
1.15
3.40
0.09
0.37
0.47
0.56
0.12
0.11
-p
0.25
1.39
0.12
0.18
0.10
0.12
0.05
0.29
0.15
0.21
re
lP

The obtained results of jatropha shell NaOH pretretment were 40.30% C, 1.23% N, 51.58%
O, 2.81% Na, 0.31% Mg, 0.09% Al, 0.19% Si, 0.21% P, 0.20% Cl and 0.73% k, while deoiled
seed cake had the highest percentages of C and small traces of Na, 0.95% Mg, 0.26% Al, 0.17%
a

Si, 1.31% P, 0.15% Cl and 0.37% K. The quantitative element of nitrogen was equal in both
rn

jatropha shell and deoiled seed cake, which as observed in (Table 4) and shown in (Fig.
supplementary data). According to Kratzeisen and Müller, (2012) the ratio of the C jatropha
u

shell was 50.9% higher than determined in this work, but the O quantitative element was
Jo

approximately as published.
Analyzed crude cellulose samples of shell and deoiled seed cake H2O2 pretreatment by
using EDXA; however they were enclosed with different ratios as listed in (Table 4), these
elements were C, N, O, Na, Mg, Al, Si, P, Cl, and K. Qualitative and quantitative elements of
the jatropha shell were shown in (Table 4) (48.97% C, 1.81% N, 44.39% O, 0.05% Na, 0.02%
Mg, 0% Al, 0.03% Si, 0.25% P, 0.07% Cl, and 0.58% K). The percentage C element was the
highest compared to the other method of pretreatment. While the result of jatopha seed cake,
which was presented in different elements with different percentages, is shown in (Fig.
supplementary data).
Table 4. Qualitative and quantitative elements of jatropha (shell and deoiled seed cake)
(weight %)
Biomass Raw materials NaOH treatment Hydrogen peroxide
Element Shell Deoiled Shell Deoiled Shell Deoiled
seed cake seed cake seed cake
C 46.45 71.21 40.30 43.33 48.97 55.48
N 2.69 2.84 1.23 4.56 1.81 3.62
O 42.47 22.1 51.58 42.51 44.39 31.91
Na 1.94 0.90 2.81 4.27 0.05 0.58

of
Mg 0.53 0.21 0.31 0.95 0.02 0.38
Al 0.23 0.14 0.09 0.26 00 0.30

ro
Si 0.37 0.27 0.19 0.17 0.03 0.43
P 0.75 1.07 0.21 1.31 0.25 0.30
Cl
K
1.00
3.58
0.46
0.79
0.20
0.73
-p 0.15
0.37
0.07
0.58
0.13
0.30
re
lP

3.6. FT-IR analysis

FT-IR spectroscopy yields “fingerprint” spectra usable as structural evidence. Common
“nonpolymer” signals observed by means of FTIR spectroscopy are adsorbed water at
a

about1630- 1640 cm−1 and CO2 at about 2340- 2350 cm−1. The spectra of the agro-wastes
rn

before and after NaOH and H2O2 pretreatment were affected by the FTIR spectra. In sugarcane
and jatropha NaOH pretreatment (Fig. 3A and B), the most representative band is summarized
u

as follows. The band widening at 1318 cm-1can be ascribed to CH2 wagging vibrations in
Jo

cellulose. A similar observation was reported earlier by Spiridon et al. (2011). An absorption
band at 897 cm-1 is assigned to C-O-C stretching at the β- (1, 4)-glycosidic linkage in cellulose.
The asymmetric bending of CH3 was said to be responsible for the band at 1467 cm-1.
The guaiacyl ring of lignin, which is present in the spectra of native biomass, has a
characteristic band at 1516 cm-1 that was attributed to it and is associated with lignin removal.
This band is absent in the pretreated sample, showing that the lignin was eliminated during
pretreatment. The depolymerization of lignin during pretreatment may be the cause of the
bands at 1516 cm-1 disappearing. The vibrational modes of -CH2OH groups and the C-O
stretching vibration and C-O bending of the C-OH groups of carbohydrates are caused by the
band at 1045 cm-1 in the IR spectrum (Sindhu et al., 2014). To evaluate the functional group
alterations that happened after pretreatment, various absorption bands between 4000 and 600
cm-1 were seen. A modest absorbance band of about 901 cm1 in pretreated materials may be
caused by C-O-C stretching of the -(1,4) glycosidic linkage connecting the hemicellulose and
cellulose (Ramadoss and Muthukumar, 2016). The primary absorption band at 1038 cm-1
denotes the xylan region, and it can be attributed to the vibrational modes of the C-OH groups
of carbohydrates, particularly hemicellulose, that are -CH2OH and C-O stretching paired with
C-O bending (Parker, 2018). The distinctive band seen at 1236 corresponds to the respective
C=O vibration modes of the guaiacyl unit in lignin. The band at 1629 cm1 represents the bent
stretch of the absorbed water molecules (Su et al., 2015). After H2O2 treatments, there was a

of
decrease in band intensity (Fig. 4A and B), which demonstrated effective delignification. The
-CH2 vibrational mode is represented by the band at 1325 cm1, and the decrease in peak

ro
intensity during hydrolysis indicates that cellulose is effectively converted to monomeric
sugars (Moodley and Kana, 2017). The conspicuous band at 1420 cm-1 was attributed to
-p
cellulose's C-6 region's -CH2 bending and scissoring modes (Karatzos et al., 2012). The CH2
stretching of cellulose is responsible for the band at 2886 cm-1 in pretreated samples. The -CH
re
stretching, symmetric, and asymmetric -CH2 stretching vibrational modes are represented by a
number of minor bands between 2980 and 2835 cm-1. Three major biopolymers—cellulose,
lP

hemicellulose, and lignin have C-H methyl and methylene groups, which are represented by
the absorption peak at 2913 cm-1. The presence of a carbonyl group is indicated by the
a

absorption band at 2913 cm1. H-bound OH stretching was connected to the broad absorption
rn

band that occurred at 3318 cm-1 (Raghavi et al., 2016).

u
Jo
 A
Absorbance

Dry leaves

of
Green leaves
Tops

ro
4000 3600 3200 2800 2400 2000 1600 1200 800 400
Wave number (cm-1)

B
-p
re
lP
Absorbance

Jatropha shell
rn

Deoiled seed cake

u
Jo

3900 3400 2900 2400 1900 1400 900 400

Wavenumber (cm-1)

Fig. 3 Sodium hydroxide prtretment cellulose (A) sugarcane trash dry leaves, green leaves
and tops, (B) Jatropha (shell and deoiled seed cake)
 A

Absorbance

Dry leaves
Green leaves

of
Tops

ro
3900 3400 2900 2400 1900 1400 900 400
Wavenumber (cm-1)
B
-p
re
lP
Absorbance

Shell Jatopha
rn

Deoiled seed cake

u

3900 3400 2900 2400 1900 1400 900 400

wavenumber (cm-1)
Jo

Fig. 4 Hydrogen peroxide pretreatment cellulose (A) Dry leaves, Green leaves, Tops of
sugarcane, (B) Jatropha shell and Deoiled seed cake

A
 Absorbance

Dry leaves
Green leaves
Tops

3900 3400 2900 2400 1900 1400 900 400

Wavenumber (cm-1)

of
ro
B

-p
re
Absorbance

lP

Jatropha shell
a

Deoiled seed cake

rn

3900 3400 2900 2400 1900 1400 900 400

Wave number (cm-1)
u
Jo

Fig. 5 Bleached cellulose by sodium hypochroite (A) Dry leaves, Green leaves, Tops of
sugarcane, (B) Jatropha shell and Deoiled seed cake
3.7. Activity of comericial enzymes
Table 5. Determination of CMCase and Fpase activities of enzyme before application
DIULATION 6 CMC 6 Fpase 7 CMC 7 Fpase
(enzyme (ul): OD AFTER OD AFTER OD AFTER OD AFTER
dis water (ul) equation equation equation equation
U/ml/min U/ml/min U/ml/min U/ml/min
100 : 900 3.1043 64.28032 3.1370 64.95994 0.5759 11.73211 0.7393 16.02755
90:910 3.1016 64.22421 3.0843 63.86466 0.5380 10.94443 0.5627 12.13902
80:920 3.0976 64.14108 2.9505 61.08387 0.4441 8.992886 0.5188 11.17239
70:930 2.9584 61.24806 2.8976 59.98444 0.4136 8.358999 0.4415 9.470326
60:940 2.9262 60.57884 2.7530 56.97919 0.3690 7.432068 0.3935 8.413419
50:950 2.9222 60.49571 2.6698 55.25003 0.3463 6.96029 0.3177 6.744387
40:960 2.2833 47.21733 2.4794 51.29291 0.2972 5.939835 0.3233 6.867693
30:970 2.1989 45.46323 1.5224 31.4034 0.2426 4.805073 0.2210 4.61516

of
20:980 1.5587 32.15783 1.1945 24.58859 0.2163 4.258475 0.1068 2.100602
10:990 1.0628 21.85144 0.8754 17.95668 0.2030 3.982059 0.0747 1.393796
5:995 0.7327 14.99091 0.4204 8.500324 0.1081 2.009734 0.0308 0.427167

ro
In spite of the activity of enzyme is labeled on the imported container, the assay must be
-p
detected firstly to avoid unfavorable can be happened during shaping and secondly to the
proper dilution of enzyme from economically point data obtained refer to possibility to dilute
re
enzyme before application and this can make undifference in the cost of enzyme application
reflect on the bioethanol production costs, firstly to avoid unfavorable can be happened during
lP

shaping and secondly to the proper dilution of enzyme from economically point data obtained
refer to possibility to dilute enzyme before application and this can make undifference in the
a

cost of enzyme application reflect on the ethanol production costs.

rn

3.8. Scarification of pretreated agrowastes

Degrading and recycling the abundant cellulosic biomass in nature. From a
u

biotechnological standpoint, cellulases have a vital role to play in the generation of potentially
Jo

sustainable energy sources such as glucose and bioethanol (Zang and Lynd, 2005). Fig. 6 shows
the percent of hydrolyzing the five diferent agrowastes pretreated with H2O2 or bleached with
sodium hypochlorite comparable to native cellulose using different levels of cellulase.
However, pretreatment with H2O2 is more suitable than that applied with sodium hypochlorite.
The hydrolyzed percentages differed between wastes and the application of 30 IU/g substrate
was the best for all pretreated wastes. The more glucose was obtained from green leaves and
deoiled seed cake, followed by tops sugarcane and the lowest was from jatropha shell.
Pretreatment to remove lignins from lignocellulose and enhance the penetration of hydrolysis
agents is a vital step in the process of converting biomass by enzymes to bioethanol (Sheehan,
2001), Pretreatment of lignocellulosic biomass is applied to lignocellulose prior to hydrolysis
and fermentation in order to increase the amorphous regions. There are three categories in the
cellulase family: exo-1,4-glucanase (cellobiohydrolase or avicelase (EC 3.2.1.91), endo-1,4-
glucanase (EC 3.2.1.4), and glucosidase (EC 3.2.1.21). The cellulose chain is broken down into
individual sugars by their combined action (John et al., 2022). Enzymatic hydrolysis combined
with microbial fermentation is a more preferred technique with significantly improved
performance (Wyman, 1994). Lignocellulose is saccharified by hydrolyzing the
polysaccharide-rich material into single sugars (hexoses and pentoses) using enzymes after it
has undergone pretreatment. The commercially available cellulase, which is used to break
down cellulose and hemicellulose, is actually a blend of many enzymes that were taken from
microorganisms and given the generic name cellulase. These enzymes break down the

of
glycosidic bonds found in carbohydrates, usually via inverting or retaining processes, the latter
of which involves a two-step process that includes the production of a glycosyl-enzyme

ro
intermediate (Knott et al., 2014). Microorganism development during fermentation is
facilitated by enzymatic hydrolysis.
-p
Different cellulase concentration

85
re

84
10 20 30 40 50

78
76

76
74
74

74
74
72

72

72
71

70
69

68

68
68
68

lP 68

67
67
66

66

64
64

64
63
62

62
62

62
61
60

60

60
Hydrolysis (%)

58
58

58

58
56

56

56

54
53

52
51

50
48

48
46
a
44

42

42

36
u rn
Jo

GL* JS* TSC* DL* JDSC* TB DLB GLB JSB JDSCB CEL

pretreated substrates

Fig. 6 Scarification of pretreated substrates using different levels of cellulase at 50oC for24 hrs on a rotary shaker
100rpm. 1% substrate in citrate buffer 0.05M pH 4
N.B. Green leaves (GL*)- Jatropha shell (JS*)- Tops sugar cane (TSC*)- Dry leaves (DL*)- Jatropha deoiled seed
cake (JDSC*)- Tops bleached (TB)- Dry leaves bleached (DLB)- Green leaves bleached (GLB)- Jatropha shell
bleached (JSB)- Jatropha deoiled seed cake bleached (JDSCB)- Cellulose (CEL)

* means that pretreatment by H2O2-Without* means that bleached by sodium hypochlorite

3.9.Effect of substrate concentration on the substrate hydrolysis by cellulose

 The data presented in Fig. 7 was shown the effect of substrate percent in the reaction
mixture on the yield of glucose released by the action of cellulase on substrate. The
configuration of the substrate molecules affects the viscosity of the reaction mixture (Soni
et al., 2008), consequently affecting the movement of the enzyme to reach its active site.
However, the data obtained reported that the yield of glucose can be achieved at 3% (w/v)
for all tested substrates. Santos et al. (2011) used 1.5% of sugarcane bagasse for enzymatic
saccharication to obtain reducing sugars for bioethanol production.

Substrate concentrations

of
1 2 3 4 5

ro
85
84
78
78

78
76

76
74
74

74
74
72

72

72

72
71
71

-p
70

70
69

68

68

68

68

68
67
66

66

66

66
64

64

64

64

64

64

64
62

62

62

62
Hydrolysis (%)

60

60
58
56

56

56
54

52

52
51

48

48
re
42
37

a lP

GL* JS* TSC* DL* JDSC* TB DLB GLB JSB JDSCB CEL
rn

Substrate concentration (%)

Fig. 7 Effect of substrate concentration on the substrate hydrolysis bycellulase (30 IU /g

u

Substrate) at 50oC for 24 hr on a rotary shaker 100 rpm

*30 Iu/g substrate in citrate buffer 0.05M pH 4.8
Jo
 Incubation period

8 16 24 32 40

85
84
78
78

78
76

76
75

74
74
74

74
72
72

72
70
70
70

70
70

70
69
69
69

68
68

68
67
66
66

66
65

65

65
64
64

64

64

64

64
62

62

62
60
60

60
60
58

58
56

56

54

54
Hydrolysis (%)

52

52

of
GL* JS* TSC* DL* JDSC* TB DLB GLB JSB JDSCB CEL
pretreated substrate

ro
Fig. 8 Effect of incubation time on the substrate hydrolysis by cellulase (30 IU /g
Substrate) at 50oC for 24 hrs on a rotary shaker100 rpm. -p
re
The data presented in Table (6) clearly showed the bioethanol yield from the obtained
glucose from the enzymatic saccharification of different pretreated lignocellulosic wastes
lP

fermented by Saccharomyces cerevisiae F-307. The more bioethanol was obtained from green
leaves and deoiled seed cake, followed by tops sugarcane and the lowest was from jatropha
a

shell. In spit of the fact that the jatropha deoiled seed cake had the highest glucose content
rn

(85%) compared to the green leaves (40%), it superior to the jatropha deoiled seed cake in
bioethanol yield. This means that in the case of JDSC* most glucose was expend in the
u

Saccharomyces cerevisiae growth, while in the case of green leaves, it could use D-xylose or
Jo

fructose (Table 2). In general, as it was seen in (Table 5), (Table 6), there is a noticeable
difference in monosaccharides and metals between each of the different pretreated
lignocellulosic wastes. This led to variation in the growth of the yeast and also in enzymatic
production behavior; accordingly, the bioethanol production showed a clear variation
depending on the type of pretreated lignocellulosic waste. For instance, high concentrations
of Cl had an adverse impact on the growth and bioactivity of microorganisms, especially in the
presence of Na, and could affect the production of some important enzymes (Serrano, 1996).
The lowest Cl content, which was noticed in green leaves treated with H2O2 (0.05%), could
interpret the high bioethanol production. It is a favorable choice of yeast to ferment sugar
solutions into bioethanol (Table 3). Also,the EDXA table showed that the oxygen reduction
played a role in bioethanol yield; it was obvious that the GL*and JDSC* had low oxygen
compared to the DL*, TSC* and jatropha shell. Also, as it was seen in (Table 6), the high
content of silicon in green leaves might play a vital role in triggering important enzymes such
as invertase, which had a positive effect on ethanol fermentation (Karunakaran et al., 2013).
Also, the high sucrose content in the cultivation medium could activate the transferase
enzymes like levansucrase and dextransucrase (Ragab et al., 2019, Esawy et al., 2016) and
the hydrolysis enzymes such as levanase and invertase. Consequentially, the synergistic
effect between the transferase enzymes and the hydrolysis enzymes, such as invertase and
levanase, enriches the medium with monosaccharides, which might activate ethanol
production (Elgamily et al., 2019). Saccharomyces cerevisiae is well known as both the most

of
common and a classic yeast in the manufacture of bioethanol due to its tolerance to high ethanol
concentrations and th material's inhibitors. its resistance to high ethanol concentrations and the

ro
inhibitors of the substance (Sankh et al., 2011). An extra nutrient needs to be supplied in order
to offer an organic nitrogen source for the development of microorganisms throughout the
-p
fermentation process. Peptone, corn steep liquor (CSL), urea, and even the distillation waste
from the bioethanol manufacturing process are among the materials that are used and studied
re
(AbdElsalam et al., 2023). The cost-effectiveness of lignocellulosic ethanol production has not
lP

changed in an effort to increase the yield of bioethanol fermentation. Different lignocellulosic

wastes underwent varied physical pretreatments. Chemicals and biological processes were
followed by enzymatic hydrolysis to produce glucose, after which Saccharomyces cerevisiae
a

fermented the sugar syrup to produce bioethanol (AbdElsalam et al., 2023).

rn

Table 6. Ethanol yield from glucose obtained from substrate hydrolysis by cellulase
u

Sample Treatment Ethanol mg/g Biomass mg /g Fermentation

substrate substrate efficiency %
Jo

Green leaves H2O2 325.4 58 86

Jatopha shell H2O2 264.0 32 80
Tops sugarcane H2O2 282.9 37 82
Dry leaves H2O2 302.4 46 84
Jatropha deoiled seed H2O2 310.8 48 84
cake
Tops bleached bleached 290.5 43 83
Dry leaves bleached bleached 240. 0 35 80
Green leaves Bleached 262. 0 38 80
bleached
Jatropha shell bleached 310.8 51 81
bleached
Jatopha deoiled seed bleached 272.0 37 82
cake bleached
Cellulose ------------- 376. 0 64 88
N.B. Green leaves (GL*)- Jatropha shell (JS*)- Tops sugarcane (TSC*)- Dry leaves (DL*)- Jatropha deoiled seed
cake (JDSC*)- Tops bleached (TB)- Dry leaves bleached (DLB)- Green leaves bleached (GLB)- Jatropha Shell
bleached (JSB)- Jatropha deoiled seed cake bleached (JDSCB)- Cellulose (CEL)

* means that pretreatment by H2O2-Without* means that bleached by sodium hypochlorite

Conclusion
This work tried to discriminate the difference between the extraction of biopolymer
from sugarcane trash and jatropha by the using each of sodium hydroxide and H2O2. This idea
was implanted through comparative studies to characterize the difference between the two
methods. The results pointed to the priority of H2O2 in cellulose yield and showed that the
bioethanol yield was much better in samples treated with H2O2. The highest yield bioethanol

of
was obtained from green leaves and deoiled seed cake, followed by tops sugarcane and the
lowest was from jatropha shell.

ro
Data availability
-p
The data that support the findings of this study are available from the corresponding author
re
upon reasonable request.
Acknowledgments
lP

This work was supported by National Research Centre, Chemistry of Natural and Microbial
Products
a

Reference
rn

AOAC., 2000. Official methods of analysis of the Association of Official Analytical Chemistry. 12th.
ed.; AOAC: Washington.
u

Abdel-Salam, M. S., Hafez, S. S., Fadel, M., Mohamed, S. A., Hegazy, W. K., & Khalil, B. E., 2023.
Jo

Bio Ethanol Production from Rice Straw Saccharification via Avicelase Gene in E. coli
Recombinant Strain. Clean Technologies, 5(2), 451-465.

Attard, T. M., McElroy, C. R., Rezende, C. A., Polikarpov, I., Clark, J. H., & Hunt, A. J., 2015.
Sugarcane waste as a valuable source of lipophilic molecules. Industrial Crops and
Products, 76, 95-103.

Canilha, L., Chandel, A. K., Suzane dos Santos Milessi, T., Antunes, F. A. F., Luiz da Costa Freitas,
W., das Graças Almeida Felipe, M., & Da Silva, S. S., 2012. Bioconversion of sugarcane
biomass into ethanol: an overview about composition, pretreatment methods, detoxification of
hydrolysates, enzymatic saccharification, and ethanol fermentation. BioMed Research
International, 2012.
Chen, W. C., Lin, Y. C., Ciou, Y. L., Chu, I. M., Tsai, S. L., Lan, J. C. W., ... & Wei, Y. H., 2017.
Producing bioethanol from pretreated-wood dust by simultaneous saccharification and co-
fermentation process. Journal of the Taiwan Institute of Chemical Engineers, 79, 43-48.

Denk, W., & Horstmann, H., 2004. Serial block-face scanning electron microscopy to reconstruct three-
dimensional tissue nanostructure. PLoS biology, 2(11), e329.
dos Santos, R. S., de Macedo, A. L., de Araujo Pantoja, L., & dos Santos, A. S., 2014. Bioethanol from
Jatropha seed cakes produced by acid hydrolysis followed by fermentation with baker’s
yeast. International Journal of Applied, 4(4), 113.
DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. T., & Smith, F., 1956. Colorimetric method for

of
determination of sugars and related substances. Analytical chemistry, 28(3), 350-356.
Elgamily, H. M., Gamal, A. A., Saleh, S. A., Wahab, W. A. A., Hashem, A. M., & Esawy, M. A., 2019.

ro
Microbiological and environmental assessment of human oral dental plaque isolates. Microbial
pathogenesis, 135, 103626.
-p
Esawy, M. A., Gamal, A. A., Helal, M. M., Hassan, M. E., Hassanein, N. M., & Hashem, A. M., 2016.
Enzymatic synthesis using immobilized Enterococcus faecalis Esawy dextransucrase and some
re
applied studies. International journal of biological macromolecules, 92, 56-62.
Ewunie, G. A., Yigezu, Z. D., & Morken, J., 2021. Biochemical methane potential of Jatropha curcas
lP

fruit shell: comparative effect of mechanical, steam explosion and alkaline

pretreatments. Biomass Conversion and Biorefinery, 1-14.
a

Fadel, M., Keera, A. A., Mouafi, F. E., & Kahil, T., 2013. High level ethanol from sugarcane molasses
rn

by a new thermotolerant Saccharomyces cerevisiae strain in industrial scale. Biotechnology

research international, 2013.
u

Fadel, M., 2014. Production of Fuel Ethanol from Cane Molasses. In Microbes Process; Nova Science
Publisher: Hauppauge, NY, USA; pp. 93–117
Jo

Farid M.A., Shaker H.M., El-Diwany A.I., 1983. Effect of peracetic acid, sodium hydroxide and
phosphoric acid on cellulosic materials as a pretreatment for enzymatic hydrolysis.Enzyme and
Microbial Technology. 1983;5:421424

Fernandes, M.C., Ferro, M.D., Paulino, A.F., Mendes, J.A., Gravitis, J., Evtuguin, D.V., Xavier, A.M.,
2015. Enzymatic saccharification and bioethanol production from Cynara cardunculus
pretreated by steam explosion. Bioresource technology, 186, 309-315.

Franco, H.C. J., Pimenta, M. T. B., Carvalho, J. L. N., Magalhães, P. S. G., Rossell, C. E. V., Braunbeck,
O. A., ... & Rossi Neto, J., 2013. Assessment of sugarcane trash for agronomic and energy
purposes in Brazil. Scientia Agricola, 70, 305-312.
Fu, Y., Gao, H., Yu, H., Yang, Q. , Peng, H., Liu, P., Li, Y., Hu, Z., Zhang, R., Li, J., Qi, Z., Wang, L.,
Peng, L., Wang, Y., 2022. Specific lignin and cellulose depolymerization of sugarcane bagasse
for maximum bioethanol production under optimal chemical fertilizer pretreatment with
hemicellulose retention and liquid recycling. Renewable Energy, 200, 1371-1381.

García, A., Cara, C., Moya, M., Rapado, J., Puls, J., Castro, E., & Martín, C. ,2014. Dilute sulphuric
acid pretreatment and enzymatic hydrolysis of Jatropha curcas fruit shells for ethanol
production. Industrial Crops and Products, 53, 148-153.

Gómez, E. O., Torres, R., De Souza, G., & Jackson, G., 2014. Sugarcane trash as feedstock for second
generation processes. Sugarcane Bioethanol—R&D for Productivity and Sustainability; Cortez,

of
LAB, Ed, 637-660.
Hendry, G. A., Grime, J. P. (Eds.),1993. Methods in comparative plant ecology: a laboratory manual.

ro
Springer Science & Business Media.
Inekwe, U.V., Onyike, E., Odey, M.O., Agbaji, A.S., Joel, J.T., & Diafe, P., 2012. Comparative

-p
proximate composition of Jatropha curcas seed from India, Kaduna and Edo. International
Journal of Science and Technology, 2(6), 379-381.
re
Jayme, G., Knolle, H., 1956. Paper chromatography of sugar mixtures on glass fiber
papers. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 68(7), 243-246.
lP

John, A.J., Samuel, M.S., Govarthanan, M., Selvarajan, E., 2022. A comprehensive review on strategic
study of cellulase producing
marine actinobacteria for biofuel applications. Environ. Res. 2022, 214, 114018
a

Karatzos S.K., Edye L.A., Doherty W.O.S., 2012. Sugarcane bagasse pretreatment using three
rn

imidazolium-based ionic liquids; mass balances and enzyme kinetics. Biotechnol Biofuels 5:1–
12. https://doi. org/10.1186/1754-6834-5-62
u

Karunakaran, G., Suriyaprabha, R., Manivasakan, P., Yuvakkumar, R., Rajendran, V., Prabu, P., &
Jo

Kannan, N., 2013. Effect of nanosilica and silicon sources on plant growth promoting
rhizobacteria, soil nutrients and maize seed germination. IET nanobiotechnology, 7(3), 70-77.

Kratzeisen, M., & Müller, J. 2013. Suitability of Jatropha seed shells as fuel for small-scale combustion
units. Renewable Energy, 51, 46-52.
Knott, B. C., Haddad Momeni, M., Crowley, M. F., Mackenzie, L. F., Gotz, A. W., Sandgren, M., ... &
Beckham, G. T., 2014. The mechanism of cellulose hydrolysis by a two-step, retaining
cellobiohydrolase elucidated by structural and transition path sampling studies. Journal of the
American Chemical Society, 136(1), 321-329.
Martins, M. T. B., de Souza, W. R., da Cunha, B. A. D. B., Basso, M. F., de Oliveira, N. G., Vinecky,
F., ... & Molinari, H. B. C. ,2016. Characterization of sugarcane (Saccharum spp.) leaf
senescence: implications for biofuel production. Biotechnology for Biofuels, 9(1), 1-17.
Mandels, M.; Andreotti, R.E. and Roche, C. (1976). Measurement of saccharifyingcellulases.
Biotechnology and Bioengineering Symposium Journal, 6: 21-33.

Menandro, L. M. S., Cantarella, H., Franco, H. C. J., Kölln, O. T., Pimenta, M. T. B., Sanches, G. M.,
... & Carvalho, J. L. N., 2017. Comprehensive assessment of sugarcane straw: implications for
biomass and bioenergy production. Biofuels, Bioproducts and Biorefining, 11(3), 488-504.
Moodley, P., & Kana, E. G., 2017. Microwave-assisted inorganic salt pretreatment of sugarcane leaf
waste: Effect on physiochemical structure and enzymatic saccharification. Bioresource

of
technology, 235, 35-42. https://doi.org/10.1016/j.biortech.2017.03.031
Miller, G.L.,1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical

ro
Chemistry, 31: 426-428.

-p
Murata, K., Somwongsa, P., Larpkiattaworn, S., Liu, Y., Inaba, M., & Takahara, I., 2011. Analyses of
liquid products from catalytic pyrolysis of jatropha seed cakes. Energy & Fuels, 25(11), 5429-
re
5437.

Nazarpour F.L., Biological pretreatment of rubberwood with Ceriporiopsis subvermispora for

lP

enzymatic hydrolysis and bioethanol production. BioMed Research International. 2013;9:9

Niju, S., Nishanthini, T., & Balajii, M., 2020. Alkaline hydrogen peroxide-pretreated sugarcane tops
a

for bioethanol production—a process optimization study. Biomass Conversion and

rn

Biorefinery, 10(1), 149-165.

Odetoye, T.E., Afolabi, T. J., Abu Bakar, M.S., & Titiloye, J.O., 2018. Thermochemical
u

characterization of Nigerian Jatropha curcas fruit and seed residues for biofuel
Jo

production. Energy, Ecology and Environment, 3, 330-337.

Palmqvist, E., & Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: inhibitors
and mechanisms of inhibition. Bioresource technology, 74(1), 25-33.
Partridge, S.M., 1949. Aniline hydrogen phthalate as a spraying reagent for chromatography of
sugars. Nature, 164(4167), 443-443.
Patil, R.A., & Deshannavar, U. B. (2019, November). To Study the Effect of Pretreatment on Dry
Sugarcane Leaves. In IOP Conference Series: Materials Science and Engineering (Vol. 577,
No. 1, p. 012017). IOP Publishing.
Parker, F.S., 1971. Applications of infrared spectroscopy in biochemistry, biology, and medicine.
Plenum Press, New York https://link. springer.com/book/10.1007/978-1-4684-1872-9.
Accessed 30 July 2018 (1971) Applications of infrared spectroscopy in biochemistry, biology,
 and medicine. Plenum Press, New York https://link. springer.com/book/10.1007/978-1-4684-
1872-9. Accessed 30 July 2018
Pereira, S.C., Maehara, L., Machado, C. M. M., & Farinas, C. S., 2015. 2G ethanol from the whole
sugarcane lignocellulosic biomass. Biotechnology for biofuels, 8(1), 1-16.

Raghavi, S., Sindhu, R., Binod, P., Gnansounou, E., & Pandey, A., 2016. Development of a novel
sequential pretreatment strategy for the production of bioethanol from sugarcane trash. Bioresource
technology, 199, 202-210.
Ramadoss G, Muthukumar K (2016) Ultrasound assisted metal chloride treatment of sugarcane bagasse
for bioethanol production. Renew Energy 99:1092–1102. https://doi.org/10.1016/j.renene.
2016.08.003

of
Ragab, T. I., Amer, H., Wasfy, A. A. F., Hady, M. A., Mossa, A. T. H., & Liebner, F., 2014. Sulfated

ro
cellulose from agriculture wastes, anticoagulant, fibrinolytic and toxicological studies. Journal
of Environmental Science and Technology, 7(5), 266-280.

-p
Ragab, T.I.M, Abd Malek, R., Elsehemy, I.A., Farag, M.M.S., Salama, B.M. Abd EL-Baseer, M. A.,
Gamal-Eldeen, A.M., El Enshasy, H.A., Esawy, M.A., 2019 Scaling up of levan yield in
re
Bacillus subtilis M and cytotoxicity study on levan and its derivatives Journal of bioscience
and bioengineering 127 :655-662
lP

R. N. Singh, D. K. Vyas, N. S. L. Srivastava and M. Narra, Renew. Energ., 33, 1868 (2008).
Rolz, C., De Leon, R., De Arriola, M. C., & De Cabrera, S., 1986. Biodelignification of lemon grass
and citronella bagasse by white-rot fungi. Applied and environmental microbiology, 52(4), 607-
a

611.
rn

Santos, A.L.F., Kawase, K.Y.F., & Coelho, G.L.V., 2011. Enzymatic saccharification of lignocellulosic
materials after treatment with supercritical carbon dioxide. The Journal of Supercritical Fluids, 56(3),
u

277-282.
Jo

Sankh, S. N., Deshpande, P. S., & Arvindekar, A. U., 2011. Improvement of ethanol production using
Saccharomyces cerevisiae by enhancement of biomass and nutrient supplementation. Applied
biochemistry and biotechnology, 164, 1237-1245.

Sheehan, J., 2001. The road to bioethanol: a strategic perspective of the US Department of Energy's
national ethanol program.

Szczodrak, J., Llczuk, Z., Rogalski, J., & Leonowicz, A., 1986. Intensification of oak sawdust
enzymatic hydrolysis by chemical or hydrothermal pretreatment. Biotechnology and
bioengineering, 28(4), 504-510.
Shuhairi, N. M., Zahari, M. S. M., & Ismail, S., 2015. Lignocellulosic-based Jatropha seed pre-
treatment using ultrasonic reactive extraction for liquid biofuel production. Chemical
Engineering Transactions, 45, 1573-1578.
Sindhu, R., Kuttiraja, M., Binod, P., Sukumaran, R. K., & Pandey, A., 2014. Physicochemical
characterization of alkali pretreated sugarcane tops and optimization of enzymatic
saccharification using response surface methodology. Renewable Energy, 62, 362-368.
Serrano, R., 1996. Salt tolerance in plants and microorganisms: toxicity targets and defense
responses. International review of cytology, 165, 1-52.

Spiridon, I., Teaca, C. A., & Bodîrlău, R., 2011. Structural changes evidenced by FTIR spectroscopy in
cellulose materials after pre-treatment with ionic liquid and enzymatic

of
hydrolysis. BioResources, 6(1), 400-413.

ro
Su, Y., Du, R., Guo, H., Cao, M., Wu, Q., Su, R., ... & He, Z., 2015. Fractional pretreatment of
lignocellulose by alkaline hydrogen peroxide: Characterization of its major components. Food
and Bioproducts Processing, 94, 322-330.
-p
Wilson, E. O., 1959. Adaptive shift and dispersal in a tropical ant fauna. Evolution, 122-144.
re
Zhang, R., Hu, Z., Wang, Y., Hu, H., Li, F., Li, M., ... & Peng, L., 2023. Single-molecular insights
lP

into the breakpoint of cellulose nanofibers assembly during saccharification. Nature

Communications, 14(1), 1100.

Wang, Y., Liu, P., Zhang, G., Yang, Q., Lu, J., Xia, T., ... & Wang, Y., 2021. Cascading of engineered
a

bioenergy plants and fungi sustainable for low-cost bioethanol and high-value biomaterials
rn

under green-like biomass processing. Renewable and Sustainable Energy Reviews, 137,
110586.
u

Wyman, C. E., 1994. Ethanol from lignocellulosic biomass: technology, economics, and
Jo

opportunities. Bioresource Technology, 50(1), 3-15.

Xie, R., Tan, H., Huang, M., Zhou, L., Huan, J., Huang, X., ... & Wang, L., 2010. Study of plant
nutritional characteristics of high yield sugarcane. Southwest China Journal of Agricultural
Sciences, 23(3), 828-831.
Zhang, K., Pei, Z., & Wang, D., 2016. Organic solvent pretreatment of lignocellulosic biomass for
biofuels and biochemicals: A review. Bioresource technology, 199, 21-33.
Zhang, R., Gao, H., Wang, Y., He, B., Lu, J., Zhu, W., ... & Wang, Y., 2023. Challenges and
perspectives of green-like lignocellulose pretreatments selectable for low-cost biofuels and
high-value bioproduction. Bioresource Technology, 369, 128315.
Zhang, R., Hu, Z., Peng, H., Liu, P., Wang, Y., Li, J., ... & Peng, L., 2023. High density cellulose
nanofibril assembly leads to upgraded enzymatic and chemical catalysis of fermentable sugars,
 cellulose nanocrystals and cellulase production by precisely engineering cellulose synthase
complexes. Green Chemistry, 25(3), 1096-1106.
Zhang, Y. H. P., & Lynd, L. R., 2005. Determination of the number-average degree of polymerization
of cellodextrins and cellulose with application to enzymatic hydrolysis. Biomacromolecules, 6(3),
1510-1515.

Declaration of Competing Interest

N.B. Publication has been approved by all co-authors

of
N.B. The article must not be submitted to any elsewhere

ro
N.B. There is no conflict of interest

Highlights
-p
re
• The chemical composition of sugar cane trash was determined to be mainly cellulose,
lP

hemicellulose, and lignin.

a

• Hydrogen peroxide was the most suitable for the pretreatment of different cellulose
rn

types

• The pretreatment wastes were subjected to a fermentation procedure after being

u
Jo

saccharified by cellulase.

• All pretreated wastes recorded high ethanol yields to different degrees.