1. Elsevier Editorial System(tm) for Ecotoxicology and Environmental Safety
Manuscript Draft
Manuscript Number:
Title: UTILIZATION OF CELLULOSIC BIOMASS AS A SUBSTRATE FOR THE PRODUCTION OF
BIOETHANOL
Article Type: SI:Greener Eco-Environment
Section/Category: Environmental Safety
Keywords: Cotton wastes, Pretreatment, Enzyme, Purification
Corresponding Author: Ms. Rajalakshmi V, M.Phil
Corresponding Author's Institution: PSG College of Arts and Science
First Author: Rajalakshmi V, M.Phil
Order of Authors: Rajalakshmi V, M.Phil; Rajendran R, Ph.D; Radhai R, Ph.D
Abstract: Energy is considered a prime agent in the generation of wealth and a significant factor in
economic development. Recently there is renaissance in utilization of biomass for biofuel production
employing cellulases and hence forth in obtaining better yields and novel activities. The present study
deals with the bioconversion of cellulose from textile cotton waste into ethanol by using the methods
of physical (Steam explosion method) and chemical pretreatment (Acid and Alkali), optimization of
enzyme production and the ability to hydrolyze the cellulosic cotton biomass was also determined. The
results of physical and chemical pretreatment revealed that the chemical pretreated substrate
enriched the enzyme action, when compared to physical pretreatment method. The sugar analysis was
achieved by DNSA method and the cellulose estimation was performed using Anthrone method. The
enzyme production parameters such as temperature, pH, incubation time, inoculum concentration and
agitation were optimized. The produced enzyme was partially purified by Dialysis followed by
ammonium precipitation method and the ability to hydrolyze the cellulosic cotton biomass was also
determined. The conditions for enzymatic hydrolysis were also optimized. Our energy systems should
be renewable and sustainable, efficient and cost-effective, convenient and safe. These problems make it
urgent to develop an alternative energy resource that was both renewable and environmentally
friendly. The purpose of the contemporary investigation was to recover the solid waste, improve the
industrial application of cellulases and investigate the challenges in cellulase research exclusively in
the direction of enlightening the process economics of energy production.
Suggested Reviewers: Rajesh E M Ph.D
Associate Professor, Microbiology, PSG College of Arts and Science
emraajesh@gmail.com
He is a well known person in the field of microbial biotechnology.
Hasab Elrasool A.Bagi Muhammad Ahmed Ph.D
Professor, Textile Engineering, Sudan University
hasabotek@yahoo.com
Chandrasekar S Ph.D

2. Assistant Professor, Microbiology, Sri krishna College of Arts and Science
seker_biotech@gmail.com

3. RAJALAKSHMI V
Department of Microbiology
P.S.G. College of Arts and Science
Coimbatore,
TN India
Ph: 08220245259
Email: raji.ajjii@gmail.com
To
The Editor in Chief,
EES
Dear Editor,
Sub: - Submission of our original research paper for publication - regarding.
I am pleased to submit our original research article entitled “UTILIZATION OF CELLULOSIC
BIOMASS AS A SUBSTRATE FOR THE PRODUCTION OF BIOETHANOL” for your kind
perusal and publication. We strongly believe that the information provided in this manuscript
will be useful to the scientific community.
On behalf of all authors I certify that the article has not been published in another publication
and is not being submitted simultaneously to another journal. All authors are agreed to submit
the manuscript in your journal. I also certify that manuscript does not contain any animal and
human experiments. I look forward your feedback/comments regarding this manuscript.
With regards
RAJALAKSHMI V
Cover Letter

4. List of Suggesting Reviewers
1. First Name* : DR. Rajesh
Middle Initial :
Last Name* : E M
Academic Degree(s) : M.Sc., Ph.D
Position : Associate Professor
Department : Microbiology
Institution* : PSG College of Arts and Science, Coimbatore
E-mail Address* : emraajesh@gmail.com
2. First Name* : Dr. Chandrasekar
Middle Initial :
Last Name* : S
Academic Degree(s) : M.Sc., Ph.D
Position : Assistant Professor
Department : Microbiology
Institution* : Sri Krishna College of Arts and Science
E-mail Address* : seker_biotech@gmail.com
3. First Name* : Dr. Eng. Hasabelrasool A.
Middle Initial :
Last Name* : Bagi Muhammad Ahmed
Academic Degree(s) : Ph.D.
Position : Professor
Department : Textile Engineering
Institution* : College of Engineering, Sudan University
E-mail Address* : hasabotek@yahoo.com
*Reviewer Suggestions

5. UTILIZATION OF CELLULOSIC BIOMASS AS A SUBSTRATE FOR THE
PRODUCTION OF BIOETHANOL
Rajalakshmi V1
, Rajendran R1
, Radhai R1
1
PG and Research Department of Microbiology, PSG College of Arts and Science, Coimbatore,
Tamilnadu.
E mail id: raji.ajjii@gmail.com
Abstract
Energy is considered a prime agent in the generation of wealth and a significant factor in
economic development. Recently there is renaissance in utilization of biomass for biofuel production
employing cellulases and hence forth in obtaining better yields and novel activities. The present study
deals with the bioconversion of cellulose from textile cotton waste into ethanol by using the methods
of physical (Steam explosion method) and chemical pretreatment (Acid and Alkali), optimization of
enzyme production and the ability to hydrolyze the cellulosic cotton biomass was also determined. The
results of physical and chemical pretreatment revealed that the chemical pretreated substrate enriched
the enzyme action, when compared to physical pretreatment method. The sugar analysis was achieved
by DNSA method and the cellulose estimation was performed using Anthrone method. The enzyme
production parameters such as temperature, pH, incubation time, inoculum concentration and agitation
were optimized. The produced enzyme was partially purified by Dialysis followed by ammonium
precipitation method and the ability to hydrolyze the cellulosic cotton biomass was also determined.
The conditions for enzymatic hydrolysis were also optimized. Our energy systems should be
renewable and sustainable, efficient and cost-effective, convenient and safe. These problems make it
urgent to develop an alternative energy resource that was both renewable and environmentally
friendly. The purpose of the contemporary investigation was to recover the solid waste, improve the
industrial application of cellulases and investigate the challenges in cellulase research exclusively in
the direction of enlightening the process economics of energy production.
Keywords: Cotton wastes, Pretreatment, Enzyme, Purification.
Introduction
Through the eternally increasing demand for energy and the fast diminishing petroleum
resources, globally there is a better interest in substitute fuels, particularly liquid transportation
energies (Wyman, 2007; Lynd et al., 2008). Energy crisis is one of the most serious threats towards the
sustainability of human kind and civilization. Generally, bio-ethanol converted from edible source is
called first-generation bio-ethanol (FGB). The most common disposal methods of the above waste
include direct land application, composting and combustion. Waste textiles are mainly composed of
*Manuscript
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6. cotton and viscose fibers, and holds, thanks to their cellulose content, a significant potential for
production of different biofuels, such as biogas. The polymers of cellulose and hemicelluloses should
be first released from the fibrils in ‘‘pretreatment” in order to have an effective hydrolysis (Zhang et al,
2011). Lignocellulose provides a cheap and abundant raw material, part of which can be converted to
fermentable sugars through hydrolysis.
In developing countries, where adequate disposable technology is not much available, the
cotton waste generated is mostly disposed off by scorching. Waste management is one of the biggest
problems faced by textile industry. The wastes containing minute fibers which cause serious lung
infection once it gets spread through air. They are often dumped as such or incinerated. A solution to
this problem will be bioconversion of these wastes into ethanol using microorganisms present in the
environment. Zhang et al., established a procedure for generating stimulated amorphous cellulose by
acid pretreatment method. Silverstein et al 2007 compared the effectiveness of sulphuric acid, sodium
hydroxide and hydroden peroxide pretreatments for enzyme conversion of cotton stalks. When
compared to the starch or sugar based resources, conversion of lignocelluloses is much more intricate
due to their conflict to enzymatic attacks. Therefore, pretreatment is an important stage for cellulose
bioconversion processes which helps to separate lignin and hemicellulose from cellulose, reduce the
crystallinity of cellulose, and enhance the porosity of the materials. These process inturn supports the
further enzymatic degradation (Vahid jafari 2011).
The present study deals with the preliminary steps for the bioconversion of cotton wastes into
bioethanol using pretreatment, production of enzyme, purification of produced enzyme and the
determination of enzymatic hydrolysis.
Materials and Methods
a) Physical pretreatment
(i) Steam Explosion method
The steam explosion is typically initiated at a temperature of 160- 260 °C (corresponding
pressure, 0.69 4.83 MPa) for several seconds to a few minutes before the material is exposed to
atmospheric pressure. The biomass/steam mixture is held for a period of time to promote hemicellulose
hydrolysis, and the process is terminated by an explosive decompression. The biomass/steam mixture
is held for a period of time to promote hemicellulose hydrolysis, and the process is terminated by an
explosive decompression. The process causes hemicellulose degradation and lignin transformation due
to high temperature, thus increasing the potential of cellulose hydrolysis. Hemicellulose is thought to
be hydrolysed by acetic and other acids released during steam-explosion pretreatment. The
compositional analysis was done after physio chemical pretreatment using standard ASTM (1995)
procedures.

7. b) Chemical pretreatment
(i) Acid and alkali Pretreatment
About 50 ml of dilute sulphuric acid was prepared in concentration range of 0 – 5.0 % at 0.5 %
interval. The flasks added with 3 g of processed cotton wastes were separately autoclaved at 121 °C for
30 min. The flasks containing the pretreated waste were then neutralized with distilled water. The
samples were then estimated for the amount of glucose released according to DNSA method after
pretreatment. The same procedure was carried out for alkali pretreatment. In order to dilute sulphuric
acid, sodium hydroxide solution was used in alkali pretreatment.
(i) Fungal and Enzymatic pretreatment
For fungal hydrolysis, formerly known strains for cellulose breakdown (Trichoderma ressei- MTCC
No 164) was obtained from Microbial Type Culture Collection Center, Chandigarh. The medium
selection was carried out for cellulase enzyme production.
Selection and standardization of medium for cellulase enzyme production
The fungal organism was grown in three different medium (Czapek Dox broth medium,
Reese and Mandels Mineral Salts Medium and Production medium) to determine the preeminent
composition for cellulase enzyme production. The fungal isolate (T.ressei) was grown in all the above
mentioned medium and incubated at 27 ºC for 5 days with constant shaking at 150rpm. After
incubation, the culture flasks were retrieved and the culture broth was transferred to sterile centrifuge
tubes and centrifuged at 8000 rpm for 15 mins. The centrifugation continues until the cells get settled.
After that, the supernatant was filtered through a sterile nylon cloth and stored at 4 ºC as cellulase
enzyme source. Extraction was done under sterile conditions to prevent any microbial contamination.
The preeminent medium was selected based on the cellulase enzyme production and its activity. The
cellulase enzyme activity was assayed by determining the glucose released according to Anthrone
method.
Optimization of the Cellulase Enzyme production parameters
Effect of incubation time on production of cellulase enzyme
Cellulase enzyme production broth was prepared, inoculated with T.ressei culture and
incubated for 0 – 240 hrs. Culture flasks were retrieved after every 24 hrs and the cellulase production
was assayed according to Anthrone method.
Effect of pH on production of cellulase enzyme
The optimum pH for cellulase enzyme production was demonstrated by preparing the
production medium with pH values ranging from 4.0 to 8.5 and incubated for 120 hrs. The flasks were
inoculated with T.ressei and then incubated. After incubation, the culture filtrates were assayed for
cellulase production according to Anthrone method.

8. Effect of temperature on cellulase enzyme production
The temperature optimum was studied by incubating the production medium (pH 7) inoculated
with T.ressei at varying incubation temperature such as 20 – 65 °C for 120 h. The culture filtrates were
retrieved and assayed for cellulase production.
Effect of substrate concentration on cellulase production
The effect of substrate concentration on enzyme production was determined by incubating the
inoculated culture vessels with the production medium containing varied substrate (Cellulose)
concentration from 0.5% to 5.0% at an interval of 0.5 % (separately) for 120 h and assaying the cell
free supernatant at the end of fermentation. After incubation, the optimum substrate concentration for
cellulase production was determined.
Effect of agitation condition on cellulase enzyme production
To determine the effect of agitation on cellulase production, the production medium was kept
under different shaking condition (0, 50, 100 and 150 rpm). After incubation the culture flasks were
estimated for cellulase activity.
Purification of enzyme
After optimizing the cellulase enzyme production system, the next step was to purify the
enzyme to determine the characteristics of the enzyme. The crude enzyme synthesized under
standardized conditions was extracted by centrifugation at 3000 rpm for 15 min followed by filtration
through sterile nylon cloth.
Partial purification by ammonium sulphate precipitation
The enzyme in the crude preparation was precipitated by the addition of ammonium sulphate to
80% saturation, followed by adding solid ammonium sulphate to 80% saturation (Iqbal et al., 2011).
The mixture was left overnight at 4 ºC in a magnetic stirrer and centrifuged at 4000 rpm in a
refrigerated centrifuge at 4 ºC for 30 min. The precipitate was redissolved in 10 ml of 0.02 M sodium
phosphate buffer (pH 7.0). The partially purified enzyme was taken for dialysis. The dialysis bag was
cut to the required length and allowed to boil for 10 min in 2% sodium bicarbonate and 1 mM EDTA
(pH 8). The activated dialysis bag was used for the dialysis of the enzyme collected. About 10 ml of
the partially purified enzyme obtained after the ammonium sulphate precipitation was dialyzed against
30 mM phosphate buffer (pH 7.4) at 4 ºC with three changes of buffer. The partially purified sample
was assayed for enzyme activity.
Determination of Rate of Enzymatic Hydrolysis for cotton wastes
The reaction was carried out in 0.05M Sodium Acetate buffer (pH 5). About 0.5g of pooled
cotton waste sample was taken in 100ml flasks. About 20ml of sterile buffer and 0.2ml of the enzyme
extract were added to the flask. An enzyme blank was also prepared without adding substrate and

9. flasks were incubated at 50°C for 24 hours. The flasks were checked for the sugar content at 0th
hour
and after 24 hours by means of DNSA method as mentioned in table1.
Table 1. Experimental Plan for Enzymatic Hydrolysis of Pooled Cotton Waste
S.No Buffer (ml) Sample Enzyme Extract (ml)
1 20 0 grams (A) 0.2
2 20
0.5 grams pooled cotton
waste sample (B)
0.2
3 20
0.5 grams pooled cotton
waste sample (C)
0.2 (Commercially
available Cellulase
enzyme)
Results
Compositional analysis of untreated cotton waste
The raw cotton wastes collected from textile mills were pooled together and their chemical
composition were analyzed. Various components were analyzed in the cotton wastes such as moisture
content, total sugars, acid insoluble residues, ash content and ethanol extractives. The compositions
were mentioned in table 2.
Table 2 Compositional analysis of Cotton waste before pretreatment
From the table no , it was observed that the moisture content in the cotton wastes was predominant
(66%), followed by total sugars in the cotton wastes was crucial (55.8%) and the acid insoluble
residues (29.3%) respectively. The above results clearly indicate that the cotton waste with essential
amount of sugars can be used as substrate for bioethanol production. The results obtained in this study
were supported by Mahalakshmi et al., (2011) who similarly stated cotton wastes contained maximum
total sugar concentration, followed by glucan and acid insoluble residues. The main composition of
Compositional analysis Before
pretreatment
%
Moisture 66
Ash 12.8
Acid insoluble residue 29.3
Total sugars 55.8
Ethanol Extractives 7.91

10. cotton gin waste is as follows: 22.40% of acid soluble lignin, 26.80 % of cellulose, 32.10 % of
Hemicellulose, 10.2 % of ash content & 9.9 % of moisture content (Gupta 2009).
Compositional analysis of pretreated of Cotton waste
The different cotton wastes used in the present study were processed mechanically and the chemical
composition of the pooled cotton waste was determined according to the standard methods and the
results were compared to the contents released before pretreatment and mentioned in table 3. The
physiochemical pretreated samples exhibited 38.23 and 42.08 percentage of total sugars in
compositional analysis. Steam explosion method i.e., destruction of a portion of the xylan fraction,
incomplete disruption of the lignin carbohydrate matrix and generation of compounds inhibitory to
microorganisms.
Table 3 . Compositional analysis of Cotton waste after pretreatment
The result showed that acid pre-treated cotton waste was found to possess relatively higher
percentage of total sugars (59.3%) compared to alkali pre-treated cotton wastes (46.80%).These
observations agree with similar results obtained in previous works done by Mahalakshmi et al., (2011),
Jiacheng Shen and Foster A. Agblevor (2011). Similarly, the percentage concentration of moisture and
acid insoluble residues was also found to be relatively higher for acid pre-treated (77.80% and 30.31%)
than for alkali pre-treated cotton wastes (72.2% and 24.86%). The correlation coefficient between the
acid and alkali pre-treated cotton waste was found to be with high degree of positive correlation
(r=0.9967) thereby confirming that there was not much difference in the acid and alkali pretreatments.
Compositional analysis After pretreatment %
Acid
Pretreatment
Alkali
Pretreatment
Physio
chemical
Pretreatment
Moisture 77.8 72.2 56.2
Ash 3.96 3.37 8.4
Acid insoluble residue 30.31 24.86 24.2
Total sugars 59.3 46.8 42.8
Ethanol Extractives 7.56 9.90 7.0

11. 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Cellulase production
Cellulaseactivity(IU/ml)
pH
Effect of pH on cellulase production by Trichoderma.reesei
Thousands of microorganisms have the ability to grow on cellulose. Many of them grow quite
rapidly, but only few produce extracellular cellulase that is capable of converting the native crystalline
cellulose to sugars in vitro (Mandels, M.; Weber, J, 1969). Trichoderma reesei (MTCC 164) are the
excellent sources of cellulase suitable for practical applications. Cellulase is an inducible enzyme in
Trichoderma, with highest yields obtained when the fungus is grown on cellulose rich medium. The
shake flasks, with nutrients and inoculum, were adjusted and controlled at different pHs (3.5, 4.0,
4.5,5.0, 5.5,6.0 and 6.5) and incubated for 6 days. During incubation, samples were withdrawn for
every 24 h and analyzed for the enzyme levels.
.
Fig 1. Effect of initial pH on cellulase production by Trichoderma
reesei
Maximum activity was reached at pH 4.5 (Fig.1). The Filter paper (FP) activity increased with
increasing incubation time. Enzyme activities were found to be higher with the mycelial inoculum
compared to the spore inoculum and the absorbance were observed in UV-Vis Spectrophotometer.
Inoculum age was also found to be important. The yield of cellulase in a cellulose culture is reduced
unless a second more readily metabolized substrate is added.
Effect of incubation time on cellulase production by Trichoderma. reesei
The effect of time on cellulase production was studied by incubating the production media for 7
days in orbital shaking incubator and determined for optimum time for maximum cellulase production.
The extracellular enzyme produced in the media was determined by estimating the cellulase activity at
different time duration (1, 2, 3, 4, 5, 6, and 7 days) and cellulase activities of 0.2, 1.02, 4.3, 6.4, 7.1,
7.8 and 6.6 IU/ml were obtained, respectively (Fig 2). The time of fermentation had a great effect on
enzyme production as the maximum cellulase activity was found as 7.8 U/ml on 6th
day. The highest

12. cellulase level of 1.88, 1.53 and 2.40 IU/mL of cellulase activity was achieved on the 4th day of the
fermentation period by Trichoderma harzianam and Phanerochaete chrysosporium respectively (Khan
et al., 2007). Ojumu et al. (2003) found that the highest level of cellulase activity occurred at the 12th
hr of fermentation by Aspergillus flavus. It was perceived that a high concentration of reducing sugar
was released on the 4th day of the fermentation (Khan et al., 2007). Similar drift was also reported in
cellulase production using Trichoderma sp. Appropriate cultivation time was significant for growth
and production (Liu and Yang, 2007).
Fig 2. Effect of incubation time on cellulase production by Trichoderma reesei
The time of fermentation had a great effect on enzyme production, as the cellulase activity of
7.8 IU/ml was obtained after 6 days of fermentation. However, further increase in the incubation time,
reduced the enzymes production. It might be due to the depletion of macro- and micronutrients in the
fermentation medium with the lapse in time, which stressed the fungal physiology resulting in the
inactivation of secretory machinery of the enzymes. In addition, the substances were initially more
susceptible, making a rapid rise in enzymes biosynthesis. But with the prolongation of cultural time,
the susceptible portions were completely hydrolyzed by microorganisms, which inhibited the enzyme
secretion pathways (Nochure et al., 1993).
Effect of temperature on cellulase production by Trichoderma reesei
Incubation temperature plays an important role in the metabolic activities of microorganism.
Temperature optimization was carried out by incubating the fermentation flask at 20°C, 25°C, 30°C,
35°C, 40°C, 45°C, 50°C, 55°C, 60°C and 65°C and the cellulase activities were found to be 6.2, 7.3,
6.9, 5.2, 2.9, 2.2, 1.6, 0.7, 0.1 and 0.0 IU/ml, respectively. (Fig 3) Thus, as shown in figure 12,
maximum cellulase production was observed at 25°C, as the temperature increased the cellulase
1 2 3 4 5 6 7
0.0
2.0
4.0
6.0
8.0
Cellulase production
Cellulaseactivity(IU/ml)
(IU/ml)
Incubation time (days)

13. 15 20 25 30 35 40 45 50 55 60 65 70
0.0
2.0
4.0
6.0
8.0
Cellulase production
Cellulaseactivity(IU/ml)
(IU/ml)
Temperature (°C)
production gradually reduced, finally there was no enzyme production seen after 60°C. Any change,
either increase or decrease in temperature resulted in the gradual decrease in protein production
(Ikram-ul-Haq et al., 2006). A higher temperature alters the cell membrane composition and stimulates
protein catabolism, thus causing cell death. The incubation temperature is a factor regulating the
enzyme synthesis (Liu and Yang, 2007). Similarly Liu and Yang (2007) reported that maximum
cellulase production of Trichoderma koningii was observed in the temperature range of 27-33°C.
Fig 3. Effect of temperature on cellulase production by Trichoderma reesei
Effect of static and agitated condition on cellulase production by Trichoderma. reesei
To determine the effect of agitation on cellulase production, the production media were kept
under different shaking condition (0, 50, 100 and 150 rpm). After incubation the culture extract from
all flasks were estimated for cellulase activity and the results were shown in fig 4. There was a strong
influence on agitation on cellulase production; the cellulase production was high in shaking condition
then in static condition.
0
50
100
150
0.0 2.0 4.0 6.0 8.0
Agitation(rpm)
Cellulase activity (IU/ml)
Cellulase production
200

14. Fig 4. Effect of static and agitated conditions on cellulase production by Trichoderma reesei
The maximum cellulase yield of about 8.33IU/ml was obtained in the flask kept at 150 rpm.
The enzyme production in the flask kept at shaking condition of 0, 50 and 100 was 7.47 IU/ml, 7.70
IU/ml and 8.00 IU/ml respectively. When the shaking speed was increased beyond 150 rpm , a slight
drop in enzyme production was observed, which could be due to the fact that the increase in the rpm
level has resulted in the coagulation of the organisms to form lumps and decrease in rate of mass
transfer. Similar result was reported by Lejeune and Baron, (1995) that the enzyme production of T.
reesei was strongly affected by the agitation and at the higher agitation rates the enzyme production
was dropped. On the other hand, lower agitation speed of less than 130 rpm resulted in low growth,
which thus resulted in low enzyme production. This could be due to low amount of dissolved oxygen
in the cultivation medium.
Purification of cellulase enzyme
The harvested enzyme was then purified to study its characteristics of the enzyme. Crude
enzyme produced under standardized conditions was centrifuged to extract the enzyme.
Partial purification by ammonium sulphate precipitation
Ammonium sulphate precipitation was carried out at 80% saturation. Precipitate obtained was
redissolved in sodium phosphate buffer. The precipitated enzyme was dialyzed against 30 mM
phosphate buffer with three changes of buffer. Cellulase enzyme activity was calculated according to
DNSA method and enzyme activity was found to be 1.18 IU/ml/min. (Iqbal et al., 2011).
Determining the rate of Enzyme Hydrolysis
The Rate of enzymatic hydrolysis of cotton waste samples were computed from concentration
of glucose released per hydrolysis time:
Where, v = enzyme hydrolysis rate (mg/mL glucose per hour)
Glut = Concentration of glucose at time, t (mg/mL),
Glu0 = Initial glucose concentration at time = 0 h (mg/mL),
t = hydrolysis time (h), and
to = time = 0 hour (h).

15. The rate and fidelity of the indigenous enzyme on the pooled cotton waste samples were calculated
based on the sugar released with the formula mentioned and the results are mentioned in Table 4. The
results revealed that the sample B and C exhibited similar glucose reaction rate.
Table 4. Determination of rate of enzymatic hydrolysis
Conclusion
India is a fast growing economy with an inherent increase in demand for energy. The country
with a positive outlook towards renewable energy technologies and committed to the use of renewable
sources to supplement its energy requirements. The country is one among the few nations to have a
separate ministry for renewable energy which address the development of biofuels along with other
renewable energy sources. The solution to renewable transportation fuels may not exclusively be ‘‘bio-
ethanol”, but it will definitely play a significant part. The country lacks mature technologies for ethanol
production from lignocellulosic biomass and though biomass itself is cheap, the costs of its processing
are relatively higher. Various magnum stanches in such technologies comprises the pretreatment of
biomass, enzymatic saccharification of the pretreated biomass, and fermentation of the hexose and
pentose sugars released by the hydrolysis and saccharification. One of the major difficulties that would
be faced by bio-ethanol technology developers as well as future entrepreneurs will be the choice of
feedstock. Though India generates a huge amount of biomass residues as agro, solid waste and forest
residues, the only feasible feedstock among these would be the solid waste predominantly cotton
wastes residues due to problems in pooling and logistics. The main objective of this research was to
break the sugars, which is present in the solid waste i.e., cotton waste using pretreatment, Enzymatic
saccharification and Microbial Fermentation. In this present study, the major strides of bioethanol
production were carried out. The possibility of converting cotton wastes to bioethanol via pretreatment
and enzymatic hydrolysis. However, they need a pretreatment for the better yield of sugar release
which would help to provide more yield of bioethanol on further fermentation. It must also be
accentuated that the use of textile cotton waste as a secondary fuels are essential to fulfil the energy
requirements of the biorefinery with hydrolysis processes.
S No Sample
Reaction Rate (moles
glucose released per hour)
1 Sample A 0.0002
2 Sample B 0.1646
3 Sample C 0.1638

16. Acknowledgement
The Author would like to thank Department of Biotechnology (DBT). This work was
financially supported by the Department of Biotechnology (DBT), Government of India.
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