Bioethanol production from cotton wastes using fusarium sp. was discussed in the slide. This slide is for promotional purpose of our publication. This slide is based on our publication “Venkatramanan.V et al /Int.J. ChemTech Res.2014,6(9),pp 4061-4069”. Please cite this article if used in research.

1. Presented by,
V.Venkatramanan, S.Aravinth, C.Santhosh prabhu
Department of Biotechnology
K.S.Rangasamy college of technology
Tiruchengode
Bioethanol production from cotton waste
using cellulase extracted from Fusarium
species
This slide is based on our publication “Venkatramanan.V et al
/Int.J. ChemTech Res.2014,6(9),pp 4061-4069”. Please cite
this article if used in research.

2. CONTENT
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1.INTRODUCTION
1.1 TYPES OF CELLULASE
1.2 COTTON - RAW MATERIAL FOR PRODUCTION OF BIOFUEL
2.OBJECTIVES
3.METHODS AND MATERIALS
3.1 PRODUCTION OF CELLULASE ENZYME
3.2 ESTIMATION OF REDUCED SUGAR BY DNS METHOD
3.3 COLLECTION AND PRETREATMENT OF SAMPLE
3.4 CULTIVATION OF YEAST
3.5 SIMULTANEOUS SACCHARIFICATION ANDFERMENTATION
3.6 EXTRACTION OF ETHANOL BY DISTILLATION METHOD
3.7 CONFORMATION AND ESTIMATION OF ETHANOL
4. RESULTS AND DISCUSSION
5. CONCLUSION
6. REFERENCES

3. 1.INTRODUCTION
CELLULASE ENZYME
 Cellulolytic enzymes are synthesized by a number of microorganisms.
Fungi and bacteria are the main natural agents of cellulose degradation.
 Cellulases are inducible enzymes synthesized by microorganisms during
their growth on cellulosic materials .
 Cellulases have been used for several years in food processing, feed
preparation, waste-water treatment, detergent formulation, textile
production and in other areas
Fig 1: Various Applications of cellulase enzyme

4. 1.1 TYPES OF CELLULASE
 The complete enzymatic hydrolysis of cellulosic materials needs
different types of cellulase; endoglucanase (1,4-β-d- glucan- 4-
glucanohydrolase),exocellobiohydrolase (1,4-β-d-glucan
glucohydrolase) and β-glucosidase (β-d-glucoside
glucohydrolase).
 The endoglucanase randomly hydrolyzes the β-1,4 bonds in the
cellulose molecules and the exocellobiohydrolases in most cases
release a cellobiose unit showing a recurrent reaction from chain
extremity. Lastly, the cellobiose is converted to glucose by β-
glucosidase.
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5. Continued…
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6. 1.2 COTTON- RAW MATERIAL FOR
PRODUCTION OF BIOFUEL
 Cotton is typically composed of 88–96% cellulose, the remainder being protein, pectin
materials and wax. It is therefore possible to hydrolyze the cotton by enzyme or acids to
glucose and then ferment it to ethanol.
 Globally, 236 million tons of cotton wastes are produced every year and it can be
converted into useful products that could be sold for more than $ 27.8 million.
 The increasing demand for ethanol for various industrial purposes such as alternative
source of energy, industrial solvents, cleansing agents and preservatives has
necessitated increased production of this alcohol.
 Ethanol can be used as a gasoline fuel additive and transportation fuel.
11/13/2015 7:02 AM 6Fig 2: cotton industry waste

7. 2. OBJECTIVES
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Production of cellulase using Fusarium
species.
Pretreatment of cotton waste using acid and
alkali..
Simultaneous saccharification and fermentation for
the production of Bioethanol.
Extraction and estimation of ethanol

8. 3. MATERIALS AND METHODS
3.1 PRODUCTION OF CELLULASE ENZYME
The flask containing the production media was inoculated with a 3% (v/v) of the fungal spore suspension grown
in potato dextrose medium isolated from degrading paper and identified using lacto phenol cotton blue staining.
Content Amount
KH2PO4 2 g
(NH4)2PO4 1.4 g
Urea 0.3 g
Cacl2.2H2O 0.3 g
MgSo4.7H2O 0.3 g
Peptone 1 g
Tween 80 0.2 % (v/v)
FeSo4.7H2O 5 mg
MnSo4.2H2O 1.6 mg
ZnSo4.7H2O 1.4 mg
CoCl2.6H2O 2 mg
Carboxy methyl cellulose 10 g
Distilled water 1000 Ml
pH 5.5
MEDIA COMPOSITION

9. 3.2 ESTIMATION OF REDUCED SUGAR BY
DNS METHOD
 Cell suspension was centrifuged at 3000 rev/min for 20 mins. Supernatant was used as a
crude extract and it was used for further analysis.
Preparation of standard graph
 An aqueous solution of glucose was prepared at a concentration of 1mg/ml. To a series of 10 test
tubes, a glucose stock solution corresponding to the required sugar concentration (0.1-1ml) was
added. The volume was made up to 3ml using double distilled water (use distilled water as a
blank). 2ml of DNS was added and heated in boiling water bath at 80°c for 15 minutes and cooled.
The absorbance was read at 580nm. The values were plotted on a graph.
ESTIMATION OF SAMPLE
 0.05ml of supernatant was added to a test tube and the volume was made up to 3ml
using distilled water.
 To this 2ml of DNS was added and heated in boiling water bath at 80°c for 15 minutes
and cooled.
 The absorbance was read at 580nm.
 The concentration of reducing sugar/ml is determined using the standard graph.
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10. 3.3 COLLECTION AND PRETREATMENT
OF SAMPLE
 The cotton sample was collected from the nearby area of Devanankurichi
(Tiruchengode) for the production of bioethanol.
Pretreatment of cotton waste
Acid pretreatment
 About 200 ml of dilute sulphuric acid was prepared with a concentration range
of 1.0% up to 5.0% in separate 500ml Erlenmeyer flasks.
 The flasks were added with 3g of processed cotton waste and autoclaved at
121°C for 30 minutes.
 The flasks containing the cotton waste were then neutralized by washing with
distilled water.
 The acid pre-treated samples were dried separately for further analysis.
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11. CONTINUED…
Alkaline pretreatment
 About 200 ml of dilute sodium hydroxide was prepared with a
concentration range of 1.0% up to 5.0% in separate 500 ml
Erlenmeyer flasks.
 The flasks were added with 3g of processed cotton waste and
autoclaved at 121°C for 30 minutes.
 The flasks containing the cotton waste were then neutralized by
washing with distilled water.
 The alkaline pre-treated samples were dried separately for further
analysis.
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12. 3.4 CULTIVATION OF YEAST
 Dried-form of industrial Saccharomyces cerevisiae yeast was used in this
research.
For inoculum, 100ml of distilled water was heated to 40°c in a shake flask. After
that, 0.5% (w/v) of S. cerevisiae (yeast) was added into the warmed water to
activate the yeast.
The mixture was left for 15 min at 150 rpm.The Saccharomyces cerevisiae was
enriched in Saboraud’s Dextrose Broth.
Media composition
Dextrose - 4g
Mycological peptone - 1g
Agar - 1.5g
Distilled water - 100ml
pH - 5.6±0.2 at 25oC
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13. 3.5 SIMULTANEOUS SACCHARIFICATION
AND FERMENTATION
 The SSF experiments were carried out in 250ml conical flasks
added with 3% (w/v) solid substrates containing 50mM sodium
citrate buffer (pH 4.8).
The media was sterilized an autoclave at 121oC. After that 2.0 ml
of the crude enzyme were added for enzymatic hydrolysis at 45oC
for 5days.
After 5 days of saccharification, 10% of S. cerevesiae culture were
added to all the flasks and incubated at 35oC for six days.
During the fermentation process every 24 hours samples were
taken for the estimation of bioethanol.
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14. 3.6 EXTRACTION OF ETHANOL BY
DISTILLATION METHOD
 Distillation apparatus was set up.
 The fermented mash was centrifuged at 3000 rpm for 10 mins and then the supernatant
was transferred into the distillation apparatus.
 The first 10% of the liquid was distilled into the graduated cylinder( It was not distilled
to dryness because ethanol evaporate first with the first 5ml that are distilled).
 The distillate was collected and used for further analysis.
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Fig 3: Distillation apparatus

15. 3.7 CONFORMATION AND ESTIMATION
OF ETHANOL
IODOFORM TEST
 10 drops of distillate and 25 drops of iodine along with 10 drops of NaOH was added in
the test tube.
 After few minutes cloudy formation in the test tube gives the conformation of the
presence of ethanol and it also gives yellow precipitate and antiseptic smell.
ESTIMATION OF ETHANOL
 Prepared different concentration of alcohol 1-10% in double distilled water. Starting
with 1% of the alcoholic solution added 24 ml of distilled water in the conical flask.
Poured 25ml of sample to distillation flask. Distilled the contents. Collected 10-15 ml
of distillate in beaker containing 25 ml of 3.4% chromic acid. Make up the volume to
50 ml using double distilled water and mixed thoroughly.Heated the contents up to 80°c
for 15 minutes. The absorbance was readed at 580nm. Plotted the values and prepared
the standard graph. Similarly the samples are read at 580nm.
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16. 4.Results and discussion
IDENTIFICATION OF CULTURE
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Fig 4. Fusarium species
PRODUCTION OF CELLULASE ENZYME
Fig 5. cellulase enzyme production

17. ESTIMATION OF REDUCED SUGAR (DNS
METHOD)
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
Reducingsugar(mg/ml)
Fermentation period ( days)
Fig 6: Reduced sugar content

18. EFFECT OF PRETREATMENT ON
THE COTTON WASTE
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Acid / Alkaline
treatment (%)
Sugar released upon
enzymatic hydrolysis (mg/ml)
Acid pretreatment Alkaline
pretreatment
1 60 20
2 72 30
3 79 42
4 83 55
5 90 63
Table 1: Efficiency of pretreatment on enzymatic hydrolysis of cotton waste

19. Continued…
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0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5
Sugarreleaseduponenzymatic
hydrolysis(mg/ml)
Severity of acid/ alkaline pretreatment (%)
alkaline
acid
Fig 6: Efficiency of pretreatment on enzymatic hydrolysis of cotton waste

20. IODOFORM TEST
Fig 7: Iodoform test (cloudy formation)
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A B
A - Alkaline treated
B - Acid treated

21. FERMENTATION OF RELEASED
SUGAR TO BIOETHANOL
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0
2
4
6
8
10
12
14
1 2 3 4 5
alcoholproduction(mg/ml)
severity of acid/ alkaline pretreatment (%)
alkaline
acid
Fig 8: Alcohol production from pretreated and hydrolyzed cotton waste

22. 5. CONCLUSION
 In this present study cotton waste from textile mill was collected, processed
and used a cheaper substrate for the production of Bioethanol.
 The enzyme cellulase obtained in this study was used for enzymatic hydrolysis
of the cheaper substrate cotton by simultaneous saccharification and
fermentation.
 The result showed that acid pretreated cotton waste yields more sugar when
compared to alkaline pretreated cotton waste and hence the yield of ethanol
was high in acid pretreated cotton compared with alkaline pretreated cotton
waste.
 The bioethanol extracted could be used for various applications such as
solvent, anti septic, transportation fuel etc.
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23. 6. REFERENCES
 Azam, J., Mohammad J. Taherzadeh, 2009. Ethanol production from cotton- based waste textiles.
Bioresource technology. 100: 1007-1010.
 Abdelnasser, S. and Ahmed I EI-dewany, 2007. Isolation and Identification of new cellulases
producing thermophilic bacteria from an Egyptian hot spring and some properties of the crude
enzume. Australian Journal of Basic and Applied Science. 1 (4): 473-478.
 Alexander, M. 1961. Microbiology of cellulase. In: Introduction to Soil Microbiology (2nd Ed.).
Johnwiley and Son, Inc. New Yark and London.
 Aswini, K., Anjali, S., Umesh, C., Indu, S., 2011. Biodegradation and delignification of sugar cane
bagasse of pulp and paper mill effluent by Cryptococcus albidus for production of bioethanol.
Research article, Biotechnol. Bioinf. Boieng. 1(3): 387-399.
 Chandrashekhar, B., Mohit S. Mishra, Kavitha, S and Sushma, D., 2011. Bio-Ethanol production
from textile cotton waste via dilute acid hydrolysis and fermentation by Saccharomyces
cerevisiae. Journal of ecobiotechnology. 3(4): 06-09.
 Charilaos, X. and Paul, C., 2009. Enhanced ethanol production from brewer’s spent grain by a
Fusarium oxysporum consolidated system. Biotechnology for biofuel, 2:4: 1-12.
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24. THANK YOU
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