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Conversion of cassava waste into sugar using Aspergillus niger and Trichoderma reesei for ethanol production

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Cassava waste has the potential to be utilized to produce ethanol due to its containing cellulose, hemi-cellulose and starch at levels of 24.99, 6.67 and 61% (w/w), respectively. The bioconversion of cassava waste into sugar by using microbial enzyme was carried out. The experiment using of two fungal strains, Aspergillus niger TISTR 3352 which produce alpha-amylase for conversion of starch into sugar and Trichoderma reesei TISTR 3080 which produce cellulase for conversion of cellulose into sugar. The conversion of cassava waste starch by A. niger was conducted in submerge cultivation. A. niger at 10% (v/v) inoculum (10 7 spore/ml) was inoculated in cultivation medium containing of 45% (w/v) cassava waste, supplemented with 0.02% (w/v) ammonium sulfate, adjusted initial pH to 5.5. After cultivation at 30 o C with shaking speed of 200 rpm for 72 hours, the maximum yield of reducing sugar was 56.2 g/L. In the same way, conversion of cassava waste-lignocellulose into sugar by T. reesei was conducted in similar system. T. reesei at 10% (v/v) inoculum (10 8 spore/ml) was inoculated in cultivation medium containing of 15% (w/v) lignocellulose, supplemented with 0.02% (w/v) ammonium sulfate, 0.04% (w/v) potassium dihydrogen phosphate, 0.025% (w/v) magnesium sulfate and 0.05% (w/v) yeast extract, adjusted initial pH to 6.0. After cultivation at 35 o C with shaking speed of 150 rpm for 72 hours, the maximum yield of cellulase was 0.3 U/mL. The fungal cellulase properties were investigated. The optimum temperature, pH and conversion period for bioconversion were 60 o C, 6.0, and 6 hrs, respectively.
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by adeboye oladayo on September 03rd, 2010 at 12:33 pm
a brilliant work.am a chemical engineering student working on same topic.wish to hear from you guys.2347060897975.thanks
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Conversion of cassava waste into sugar using Aspergillus niger
and Trichoderma reesei for ethanol production


Bundit FUNGSIN*, Suthkamol SUTTIKUL, Ancharida AKARACHARANY, and
Teerapatr SRINORAKUTARA

Thailand Institute of Scientific and Technological Research (TISTR)
Technopolis 35 M. 3 Klong 5, Klong Luang, Pathumthani 12120
Tel: +662-5779062, E-mail: bundit@tistr.or.th

Abstract

Cassava waste has the potential to be utilized to produce ethanol due to its containing
cellulose, hemi-cellulose and starch at levels of 24.99, 6.67 and 61% (w/w), respectively.
The bioconversion of cassava waste into sugar by using microbial enzyme was carried
out. The experiment using of two fungal strains, Aspergillus niger TISTR 3352 which
produce alpha-amylase for conversion of starch into sugar and Trichoderma reesei
TISTR 3080 which produce cellulase for conversion of cellulose into sugar. The
conversion of cassava waste starch by A. niger was conducted in submerge cultivation.
A. niger at 10% (v/v) inoculum (107 spore/ml) was inoculated in cultivation medium
containing of 45% (w/v) cassava waste, supplemented with 0.02% (w/v) ammonium
sulfate, adjusted initial pH to 5.5. After cultivation at 30 oC with shaking speed of 200
rpm for 72 hours, the maximum yield of reducing sugar was 56.2 g/L. In the same way,
conversion of cassava waste-lignocellulose into sugar by T. reesei was conducted in
similar system. T. reesei at 10% (v/v) inoculum (108 spore/ml) was inoculated in
cultivation medium containing of 15% (w/v) lignocellulose, supplemented with 0.02%
(w/v) ammonium sulfate, 0.04% (w/v) potassium dihydrogen phosphate, 0.025% (w/v)
magnesium sulfate and 0.05% (w/v) yeast extract, adjusted initial pH to 6.0. After
cultivation at 35 oC with shaking speed of 150 rpm for 72 hours, the maximum yield of
cellulase was 0.3 U/mL. The fungal cellulase properties were investigated. The
optimum temperature, pH and conversion period for bioconversion were 60 oC, 6.0, and
6 hrs, respectively.

Keywords: Ethanol, Cassava waste, Cellulose, Cellulolytic enzyme

Conversion of cassava waste into sugar using Aspergillus niger
and Trichoderma reesei for ethanol production
Suthkamol Suttikul1, Ancharida Akaracharany2, Bundit Fungsin1 and Teerapatr Srinorakutara1
1Biotechnology Department, Thailand Institute of Scientific and Technological Research (TISTR) 35 Moo 3 Technopolis, Klong 5, Klong Loung, Pathumthani 12120, Thailand. 2Department of Microbiology, Faculty of Science,
TISTR
Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
Abstract
Cassava waste has the potential to be utilized to produce ethanol due to its containing cellulose, hemi-cellulose and starch at levels of 24.99, 6.67 and 61% (w/w), respectively. The bioconversion of cassava waste into sugar by using microbial
enzyme was carried out. The experiment using of two fungal strains, Aspergillus niger TISTR 3352 which produce alpha-amylase for conversion of starch into sugar and Trichoderma reesei TISTR 3080 which produce cellulase for conversion of cellulose
into sugar. The conversion of cassava waste starch by A. niger was conducted in submerge cultivation. A. niger at 10% (v/v) inoculum (107 spore/ml) was inoculated in cultivation medium containing of 45% (w/v) cassava waste, supplemented with 0.02%
(w/v) ammonium sulfate, adjusted initial pH to 5.5. After cultivation at 30 oC with shaking speed of 200 rpm for 72 hours, the maximum yield of reducing sugar was 56.2 g/L. In the same way, conversion of cassava waste-lignocellulose into sugar by T.
reesei
was conducted in similar system. T. reesei at 10% (v/v) inoculum (108 spore/ml) was inoculated in cultivation medium containing of 15% (w/v) lignocellulose, supplemented with 0.02% (w/v) ammonium sulfate, 0.04% (w/v) potassium dihydrogen
phosphate, 0.025% (w/v) magnesium sulfate and 0.05% (w/v) yeast extract, adjusted initial pH to 6.0. After cultivation at 35 oC with shaking speed of 150 rpm for 72 hours, the maximum yield of cellulase was 0.3 U/mL. The fungal cellulase properties
were investigated. The optimum temperature, pH and conversion period for bioconversion were 60 oC, 6.0, and 6 hrs, respectively.
Introduction
Cassava (Manihot esculenta Crantz) is one of the most potential economic plant of Thailand besides of rice and rubber tree. It has played an important role as a major raw material in many industries in Thailand. Namely, production of cassava flour, MSG
(monosodium glutamate) and other amino acids, sweetener, ethanol. The cassava wastes left over from these production process. Particularly in cassava flour industry, there are abundant cassava waste left over, which can cause of the environment
pollution. Cassava waste has the potential to be utilized to produce ethanol due to its containing cellulose, hemi-cellulose and starch at levels of 24.99, 6.67 and 61% (w/w), respectively. Cellulose and starch are hydrolysable carbohydrates that can be
conversed into fermentable sugar. At the present, two technologies used to convert cellulose and hemicellulose to ethanol are acid and enzymatic hydrolyses. Acid hydrolysis is an effective method used for raw cellulosic materials to ethanol (Badger,
2002;Yu & Zhang, 2003; 2004), however, it is a strong reaction that cause of erosive, hazardous and affect to the environment. Enzymatic hydrolysis method (Yang et al., 2006) is a mild reaction which gentle and friendly to the environment. There are
several microbial strains that can produce cellulolytic enzymes, particularly, fungi which has the high potential in the enzyme production. In this experiment, we aim to study on enzymatic hydrolysis by using fungal enzymes to convert cassava waste into
fermentable sugar.
Methods
Optimization on the conversion of cassava waste-starch by Aspergillus niger
TISTR 3352.
The investigation of cassava waste starch conversion by A. niger TISTR 3352 was carried out in submerge
cultivation. The cultivation medium containing of cassava waste supplemented with nitrogen source and adjust initial pH
to 5.5. The fungal spore suspension was inoculated into the medium and cultivate by shaking flask at 200 rpm, 30 oC
for 72 or 96 hours. After cultivation, the reducing sugar was measured to indicate of the conversion efficiency. For
optimization of the conversion, the following parameters were investigated.
To find an optimum cassava waste content in the cultivation medium. The amount of cassava waste was varying,
15, 30, 45, 60 and 70% (w/v) (wet weight) in the cultivation medium.
To find an optimum fungal inoculum size. 10% (v/v) of the various concentration of the fungal spore suspension,
106, 107, and 108 spore/ml were inoculated into the cultivation medium containing of 45% (w/v) (wet weight) cassava
waste.
To find an optimum amount of ammonium sulfate, the nitrogen source supplemented in the cultivation medium.
The various concentration of ammonium sulfate, 0.2, 0.4, and 0.8% (w/v) were supplemented in the cultivation medium
containing of 45% (w/v) (wet weight) cassava waste.
A
B
Analysis of reducing sugar
The reducing sugar obtained from the conversion process was analyzed according to Nelson-Somogyi method
(Somogyi, 1952).
The study on conversion of cassava waste-lignocellulose by cellulase from Trichoderma reesei TISTR 3080.
Optimization for the appropriate condition of cellulase production by Trichoderma reesei TISTR 3080 by using
cassava waste lignocellulose as substrate had done. Cassava waste-lignocellulose was obtained by squeezing out the
soluble starch from the fibrous residue of the hydrolysis of cassava waste by using A. niger (Figure 1). After obtaining
the appropriate condition for cellulase production, the study on the properties of the fungal enzyme was conducted as the
following.
To find an optimum pH for conversion of cellulose by the fungal enzyme. The various pH, 4.5, 5.0, 5.5, 6.0, 6.5
and 7.0 in the hydrolysis reaction were investigated in enzyme activity of the fungal enzyme. The hydrolysis was
performed by using 15% (w/v) (wet weight) cassava waste-lignocellulose in liquid medium and shaking at 150 rpm at 30
oC.
To find an optimum temperature for conversion of cellulose by using the fungal cellulase enzyme. The various
temperature, 30, 35, 40, 45, 50, 55, 60, 65 and 70 oC were used to incubate for the hydrolysis reactions.
To find an optimum incubation time for conversion of cassava waste-lignocellulose by the fungal enzyme. The
reaction of hydrolysis of cellulose by the fungal enzyme was incubated in the various period of time 0, 3, 6, 9,12, and 15
C
D
hours.
Result and Discussion
Fig. 1 A: Cassava waste B: Cassava waste-ligonocellulose C: A. niger TISTR 3352 (left) T. reesei TISTR 3080 (right) D: Hydrolysed cassava waste by the
The reducing sugar was analyzed at intervals of time in all experiments to indicate the efficiency of conversion and
fungi
th
O e
p ta
i c
mtiiv
z i
a ttyi oof
n tohne tfu
hen g
c a
o ln e
v n
e z
r y
si m
o e
n . of cassava waste-starch by Aspergillus niger TISTR 3352.
The optimization on cassava waste content in the cultivation medium for conversion by A. niger. After cultivation
The study on conversion of cassava waste-lignocellulose by cellulase from Trichoderma reesei TISTR 3080.
by shaking flask at 200 rpm at 30 oC for 72 hours, the maximum reducing sugar were obtained in closed values among the
The optimum pH for conversion of lignocellulose by cellulase from T. reesei. The various pH, 4.5, 5.0, 5.5, 6.0, 6.5 and
experiments cassava waste content of 45, 60 and 70% (w/v) which were 44.91, 45.50 and 42.87 g/L, respectively (Figure
7.0 were adjusted in the hydrolysis reaction. After incubation by shaking flask at 150 rpm at 30 oC for 72 hours, the maximum
2). According to the result, the cassava content of 45%(w/v) (wet weight was selected as an appropriate cassava content in
enzyme activity was obtained in the hydrolysis condition of pH to 6.0 which was 0.3 unit/mL (Figure 5).
the cultivation medium.
The optimum temperature for conversion of lignocellulose by using the fungal cellulase enzyme. The various
The optimization of fungal inoculum size to inoculate in the cultivation medium for conversion of cassava waste.
temperature 30, 35, 40, 45, 50, 55, 60, 65 and 70 oC were used to incubate for the hydrolysis reactions. After incubation by
After cultivation by shaking flask at 200 rpm at 30 oC for 96 hours, the maximum reducing sugar obtained from the
shaking flask at 150 rpm for 72 hours, the maximum enzyme activity was observed in the hydrolysis condition of temperature
cultivation medium using of inoculation of 10% (v/v) fungal spore at concentration 107 spore/mL which was reached to
60oC which was 0.3 unit/mL (Figure 6).
52.60 g/L at 72 hours. Therefore, inoculum size of 10% (v/v) fungal spore concentration 107 spore/ml was selected as an
The optimum incubation time for conversion of cassava waste-lignocellulose the fungal enzyme, after incubation by
appropriate fungal spore inoculum size (Figure 3).
shaking flask at 150 rpm at 60 oC for the various time, 0, 3, 6, 9,12, and 15 hours. The maximum enzyme activity was
The optimization of ammonium sulfate, the nitrogen source supplemented in the cultivation medium. After
observed at the incubation at time 6 hrs and carry on at the steady value to 15 hrs (Figure 7).
cultivation by shaking flask at 200 rpm at 30 oC for 96 hours, the maximum reducing sugar was observed from the
cultivation medium supplemented with ammonium sulfate 0.2% (w/v) which was 52.60 g/l at 72 hours (Figure 4).
Fig. 2 Reducing sugar at the various cassava waste content hydrolysed by A. niger TISTR
Fig. 3 Reducing sugar at various spore inoculum site of A. niger TISTR 3352
Fig. 4 Reducing sugar at various amount of ammonium sulfate hydrolysed by A. niger TISTR
3352
3352
Fig. 5 Effect of pH to cellulase enzyme activity produced by T. reesei TISTR 3080
Fig. 6 Effect of temperature to cellulase enzyme activity produced by T. reesei TISTR 3080
Fig. 7 Reducing sugar from the various time for hydrolysis lignocellulose by cellulase
enzyme produced by T. reesei TISTR 3080
Conclusion
The optimum condition for the conversion of cassava waste starch by A. niger TISTR 3352 was obtained by using of spore suspension of A. niger at 10% (v/v) inoculum (107 spore/ml) to inoculate in cultivation medium containing of 45%
(w/v) cassava waste. The cultivation medium was supplemented with 0.02% (w/v) ammonium sulfate, adjusted initial pH to 5.5. After cultivation at 30 oC with shaking speed of 200 rpm for 72 hours, the maximum yield of reducing sugar was
56.2 g/l. The cellulase enzyme was produced from T. reesei TISTR 3080 by using the cassava waste-cellulose as the substrate in submerge cultivation. The fungal cellulase properties were investigated in the conversion activity of the
lignocellulose. The optimum temperature, pH and conversion period for bioconversion by the fungal enzyme were 60 oC, 6.0, and 6 hrs, respectively.
References
Badger, P. C. (2002). “Ethanol production from cellulose: A general review. In: J. Janick and A. Whipkey (eds.), Trends
Yu, Z. and Zhang, H. (2003). “Pretreatment of cellulose pyrolysate for ethanol production by
in new crops and new uses” ASHS Press, Alexandria, VA. 17-21.
Saccharomyces cerevisiae, Pichia sp. YZ-1 and Zymomonas mobilis” Biomass and Bioenergy 24, 257-262.
Somogyi, M. (1952). “Notes on sugar determination” The Journal of Biological Chemistry, Baltimore, 195: 19-23.
Yu, Z. and Zhang, H. (2004). “Ethanol fermentation of acid hydrolyzed cellulosic pyrolysate with
Yang, B, D.M. Willies, and C.E. Wyman (2006). “ Changes in the enzymatic hydrolysis rate of avicel cellulose with the
Saccharomyces cerevisiae” Bioresource Technology 93, 199-204.
conversion” Biotechnology and Bioengineering, 64:1122-1128.
Acknowledgement : The authors are grateful to Associate Professor Wichien Kitpreechavanit, Faculty of Science, Kasetsart University, Bangkok, Thailand, for valuable discussion. We also thank to
The Royal Thai Military Energy Department and Loxley Trading Co., Ltd. for supporting the research fund.

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