Production of Crude Enzyme Cellulases from Cassava Waste by Trichoderma viride.
Production of Crude Enzyme Cellulases from Cassava Waste
by Trichoderma viride
I Wayan Arnata 1, Dwi Setyaningsih2, Nur Richana3
1
2
Agroindustrial Technology, Udayana University, Bali, Indonesia
Agroindustrial Technology, Bogor Agricultural University, Bogor, Indonesia
3
Research Institute for Food Crops Biotechnology, Bogor, Indonesia
Correspondence author: yan_kadir@yahoo.com
Abstract
Cellulases are a complex enzymes system, commercially produced by filamentous
fungi and bacteria. It has wide applicability in food and beverage industry for effective
saccharification process. In this study, the crude enzyme cellulase was produced from
cassava waste by Trichoderma viride. The experiments was carried out in two phases.
The first phase was the cultivation of Trichoderma viride to determine the stationary
phase of maximum spore yield. The second phase was the determination of the produce
time needed that gave maximum crude enzyme activity. Enzyme assay was conducted
by measuring the FPase and CMCase activities. The results showed that the stationary
phase of maximum spore yield occurred after 6 days of cultivation Trichoderma viride
with maximum spore number of 1.6 x 109 spores mL-1. Optimum activity of the crude
enzyme obtained after 7 days of fermentation with the FPase activities of 4.77±0.72
UmL-1 and the CMCase activities of 5.05± 0.42 UmL-1.
Key words : cassava waste, cellulase, Trichoderma viride.
INTRODUCTION
Cellulose being an abundant and renewable resource, are the largest fraction of
the plant cell wall of agricultural residues such as straw from wheat, corn, rice soy and
cotton, sugarcane bagasse, and cassava waste. In Indonesia, cassava waste was called
“onggok”. Onggok is one of the agro-industrial by products that it was obtained from
processing of cassava tubers for tapioca production. Onggok generally in solid formed
and discarded to the environment without any treatment, so that it would coused
problems to environmental polution, espesially in arounded of factory areas (Arnata et
al. 2011).
The dry of onggok has a composition of starch 56-60%, cellulose 15-18%,
hemicellulose 4-5%, lignin 2-3%, protein 1.5-2.0%, pentosans 2%, and reducing sugars
0.4-0.5% (Wongskeo et al. 2012), while, Susijahadi et al. (1997); Yuwono and Sutopo
(2008) reported that onggok was contained high carbohydrates such as 10 % crude
fiber and 50-70 % starch. Utilization of “onggok” for
material in bioprocesses
product is one way to given value added in cassava tuber, in the other hand, it was
helped to solve pollution problem. The “onggok” can be used as substrate in ethanol
production (Susijahadi et al. 1997) and lactic acid production (Yuwono and Sutopo,
2008). Various product of starch derived such as maltose, maltodextrine and glucose
syrup was also made from onggok. In the fermentation process, onggok was used as
substrate for fermentation of citic acid and fumaric acid (Panday et al., 2000),
amyloglucosidase enzyme (Arnata et al. 2011)
One of the alternative to give value edded onggok was used as a substrate for
fermentation of cellulase enzyme.
Cellulase is a complex enzyme system,
commercially produced by filamentous fungi under solid-state and submerged
cultivation. It has wide applicability in textile, food and beverage industry for effective
saccharification process (Chellapandi and Jani, 2008) . Commercial cellulase generally
obtained from filamentous fungi (Stoilova et al. 2005). One of filamentous fungi that it
known able to produce cellulose is Trichoderma viride. The production of cellulase is
highly influenced by several factor i.a. component of the growth medium, especially
carbon sources and time of fermentation process. In this study was conducted to
determine the effect of time of fermentation process with T. viride as an inoculum and
onggok as an inducer on crude enzyme cellulose.
MATERIALS AND METHODS
Raw material
The “onggok” (by-product tapioca factory) was obtained from Bogor West Java.
It was sun dried and milled to uniform size (40 mesh). T. viride was obtained from
microbiology laboratorium PAU IPB. Onggok was analysed to determine chemical
compotition before it is used to fermentation process. Analysed that is moisture, fat,
proteine, carbohydrate and crude fiber contents.
Inoculum culture Trichoderma viride
The strains T. viride were used in this work. Stock cultures were maintained on
potato dextrose agar (PDA) and cultivation for 5 days at 30 oC. Properly sporulated
cultured were used for inoculation.
Cellulase Production
Fermentation process to cellulase production was conducted in batch system at
250 mL erlenmeyer flask with work volume 100 ml. The composition of medium for
cellulase production by T. viride was 14 ml (NH4) 2SO4 10%, 15 ml 1M KH2PO4, 3 ml
of 10% urea, 3 ml CaCl2 10%, 3 ml MgSO4. 7H2O 10%, 1 ml of trace elements stock
and 2 ml Tween 80, then made 1 liter. Trace elements composed of 495 ml of distilled
water, 5 ml of concentrated HCl, 2.5 g FeSO4, 0.89 g MnCl2. 4H2O, 1.76 g ZnSO4.H2O,
1.25 g Co (NO3) 2. 6H2O. For the production of cellulase added 5-10 g cassava
wate/”onggok” and 0.5 to 1.0 g polipepton to 1 liter of medium. The production process
is carried out at a temperature of 30°C and pH 4.0. The ability to produce cellulase
enzyme was observed by measuring activity of FPase and CMCase every 24 hours for
7 days. Enzyme activity curves were made and used to see the achievement of
maximum activity time T. viride to produce cellulase.
Isolation of cellulase
Samples were collected from each periodic time of fermentation were added 1%
tween 80 and agitated thoroughly on orbital shaker at 100 rpm for 30 min. The crude
enzyme was filtered through Whatman # 42 filter paper. The filtrates were centrifuged
at 10000 rpm for 30 min at 4oC to remove the suspended particles.
Analysis methods
The crude enzym cellulase was determined by Pothiraj et al. (2006) method.
The celluase activity was assayed using filter paper (FPase) and carboxymethyl
cellulase (CMCase). Reducing sugars released were determined by the dinitrosalicylic
acid method (Miller, 1959). One unit of enzyme activity is defined as 1 μmol glucose
released/min/ml of culture supernatant. The pH of substrate was determined by pHmeter.
RESULTS AND DISCUSSION
The cultivation
ultivation of T. viride was carried out for 7 days by calculating the
number of spores were formed every day. At the beginning of inoculation are the
average number of spores 7.08 x 107/ml. At the end of the first da
day, the number of
spores decreased significantly due to spore germination. The next day
day, began to form
spores are white with an average number of 1.02 x 108/ml. T. viride produces spores
quickly from the first day until the third day. After
fter the third da
day, T. viride started
showing relatively slow phase of spore formation. The color change
hange of the mycelium
and spores from white to green was formed after cultivation for 6 days
days. The maximum
9
number of spores with an average of 1.58 x 10 /ml spores occurred on the sixth day.
Growth curve T. viride is presented in Figure 1.
Figure 1. The growth curve of T. viride during fermentation
process
In this study the production of cellulase enzyme using a modified Andreoti
Media. Modifications done by replacing the pure cellulose as an induction of cellulase
using cassava waste.. Cassava can be used as an material inducement because it contains
crude fiber is mainly fraction of cellulose that could be used as a carbon source for
microbial growth. In addition, cellulose is also a compound of synthesis cellulase
enzyme. Crude fiber content of cassava on average 6.58 ± 0.08
0.08% (w/w). The
concentration of cassava waste used in this study was 10 g/L. According Richana et al.
(2004), crude fiber content of cassava was 9.7%, consisting of 1.3% lignin, 5.8% xylan
and cellulose 2.61%. Arnata et al. (2011) also reported that the contents of onggok were
8.65 ± 0.10 % moisture, 2.55 ± 0.14 % ash, 6.54 ± 0.02 % fat, 1.81 ± 0.03 % proteine,
2.69 ± 0.04 % crude fiber and 62.54 ± 0.00 % starch. Crude fiber content 69.98 %
hemicellulose and 13,44 cellulose.
cellulose Padonou et al. (2005) was reported that cassava flour
content 0,56 % fat (wb),
(wb) while according to Pothiraj and Eyini (2007) Cassava waste
was found to have by dry weight 55.8% starch, 14.5% cellulose, 1.21% free reducing
sugars and 3.13% protein.
In Figure 2 shows that at the initial time of fermentation was decreased CMCase
activity until the fourth day.
day After that, CMCase enzyme activity tend to have increased
and the maximal activity of 5.05 ± 0.42 U/ml was obtained after fermentation for 7 days
or a week. It is also shown on the amount of FPase activity, which at the initial time of
fermentation was decreased until the third day and on the next day was increased.
Maximal activity of 4.77 ± 0.72 U / ml was also obtained after fermentation for 7 days.
Figure 2. The change of pH and cellulase
llulase activity
during fermentation process
The increased activity of the enzyme cellulase (CMCase and FPase) showed that
T. viride have to degradation of the cellulose fraction contained in the substarat to
produce glucose to be used for cell metabolism. Suhartono (19
(1989) reported that the
synthesis of extracellular enzymes in the greatest number, normally occurred in the
time before sporulation,
sporulation ie, at the end of the exponential phase and early stationary
phase. The condition is expected because in the transition of exponential phase
followed by decrease
ease in the number of carbon sources in the medium, so the synthesis
of cellulase enzymes started to increase.
The
he increased activity of the enzyme in the fermentation process thought to be
caused by the change of pH from pH 4.0 to 3.28 at the beginning of the seventh day.
Enari (1983) mentioned that the optimal pH for growth of Trichoderma about 4.0,
whereas for cellulase production approaching 3.0 . During the production of the
enzyme, the pH should be maintained in the range from 3.0 to 4.0 due to inactivation of
the enzyme would occur if the pH is below 2.0. Decrease in pH that occurs in cellulase
production is directly related to the consumption of carbohydrates contained in the
cassava. The pattern of change in pH during fermentation for the production of cellulase
enzymes is presented in Figure 2.
CONCLUSION
The stationary phase of maximum spore yield
ield occurred after 6 days of
cultivation T. viride with maximum spore number of 1.5 x 109spores m/L. Optimum
activity of the crude enzyme
enz
obtained after 7 days of fermentation with the FP-ase
activities of 4.77±0.72
72 U m/L and CMC-ase activities of 5.05±0.42
42 U m/L
m/L.
REFERENCES
Arnata I W, Setyaningsih D, Richana N. 2011. Production of Crude Enzyme
Amyloglucosidase From “Onggok” by Aspergillus niger. Proceeding International
Seminar of Indonesian Society for Microbiology ang IUMS-IMS
IMS Outrech Program
in Food Safety,, Bali: 236-240.
236
Chellapandi P, Jani HM. 2008. Production Of Endoglucanase By The Native Strains of
Streptomyces Isolates in Submerged Fermentation. Journal of Microbiology
39:122-127
Enari, TM. 1983. Microbial Cellulose. In W.M. Fogarty (ed.) Microbial Enz
Enzyme and
Biotechnology Applied Science Published, New York.
Pandey, A., C.R. Soccol, P. Nigarm, V.T. Soccol, L.P.S. Vandenberghe and R. Mohan,
2000. Biotechnological potential of agro-industries residue II. Cassava bagasse.
Bioresour. Technol., 74: 81-87.
Pothiraj C., Balaji P. Eyini M. 2006. Enhanced Production Of Cellulases By Various
Fungal Cultures in Solid State Fermentation of Cassava Waste. African Journal of
Biotechnology Vol. 5 (20): 1882-1885.
Pothiraj C, Eyini M. 2007. Enzyme Activities and Substrate Degradation by Fungal
Isolates on Cassava Waste During Solid State Fermentation. Mycobiology 35(4):
196-204.
Richana N, Lestina P, Irawadi TT. 2004. Karakterisasi Lignoselulosa dari Limbah
Tanaman Pangan dan Pemanfaatannya Untuk Pertumbuhan Bakteri RXA III-5
Penghasil xilanase. Penelitian pertanian Tanaman Pangan 23 (3) : 171-176.
Stoilova IS, Gargova SA, Krastanov AI. 2005. Production of Enzymes by Mixed
Culture From Micelial Fungi in Solid State Fermentation. J. Biotechnol and 19 :
103-107.
Suhartono MT. 1989. Enzim dan Bioteknologi. Departemen Pendidikan dan
Kebudayaan Direktorat Pendidikan Tinggi Antar Universitas Bioteknologi. IPB.
Bogor.
Susijahadi, Neran, Kurniawan MF. 1997. Fermentation Control With Settings Sugar
Concentration Cassava Starch Hydrolysis to Produce Alcohol. Proceedings of the
seminar of Food Technology, Jember : 45-54.
Yuwono D. Sutopo H. 2008. Production of Lactic Acid from Onggok and Tofu Liquid
Waste with Concentrate Maguro Waste Supplement by Streptococcus bovis.
Australian Journal of Basic and Applied Sciences, 2(4): 939-942.
Wongskeo P. Pramoch R. Sumaeth C. 2012. Production of Glucose from the
Hydrolysis of Cassava Residue using Bacteria Isolates from Thai Higher Termites. J
Chemical and Biological Engineering 6: 277-280.
by Trichoderma viride
I Wayan Arnata 1, Dwi Setyaningsih2, Nur Richana3
1
2
Agroindustrial Technology, Udayana University, Bali, Indonesia
Agroindustrial Technology, Bogor Agricultural University, Bogor, Indonesia
3
Research Institute for Food Crops Biotechnology, Bogor, Indonesia
Correspondence author: yan_kadir@yahoo.com
Abstract
Cellulases are a complex enzymes system, commercially produced by filamentous
fungi and bacteria. It has wide applicability in food and beverage industry for effective
saccharification process. In this study, the crude enzyme cellulase was produced from
cassava waste by Trichoderma viride. The experiments was carried out in two phases.
The first phase was the cultivation of Trichoderma viride to determine the stationary
phase of maximum spore yield. The second phase was the determination of the produce
time needed that gave maximum crude enzyme activity. Enzyme assay was conducted
by measuring the FPase and CMCase activities. The results showed that the stationary
phase of maximum spore yield occurred after 6 days of cultivation Trichoderma viride
with maximum spore number of 1.6 x 109 spores mL-1. Optimum activity of the crude
enzyme obtained after 7 days of fermentation with the FPase activities of 4.77±0.72
UmL-1 and the CMCase activities of 5.05± 0.42 UmL-1.
Key words : cassava waste, cellulase, Trichoderma viride.
INTRODUCTION
Cellulose being an abundant and renewable resource, are the largest fraction of
the plant cell wall of agricultural residues such as straw from wheat, corn, rice soy and
cotton, sugarcane bagasse, and cassava waste. In Indonesia, cassava waste was called
“onggok”. Onggok is one of the agro-industrial by products that it was obtained from
processing of cassava tubers for tapioca production. Onggok generally in solid formed
and discarded to the environment without any treatment, so that it would coused
problems to environmental polution, espesially in arounded of factory areas (Arnata et
al. 2011).
The dry of onggok has a composition of starch 56-60%, cellulose 15-18%,
hemicellulose 4-5%, lignin 2-3%, protein 1.5-2.0%, pentosans 2%, and reducing sugars
0.4-0.5% (Wongskeo et al. 2012), while, Susijahadi et al. (1997); Yuwono and Sutopo
(2008) reported that onggok was contained high carbohydrates such as 10 % crude
fiber and 50-70 % starch. Utilization of “onggok” for
material in bioprocesses
product is one way to given value added in cassava tuber, in the other hand, it was
helped to solve pollution problem. The “onggok” can be used as substrate in ethanol
production (Susijahadi et al. 1997) and lactic acid production (Yuwono and Sutopo,
2008). Various product of starch derived such as maltose, maltodextrine and glucose
syrup was also made from onggok. In the fermentation process, onggok was used as
substrate for fermentation of citic acid and fumaric acid (Panday et al., 2000),
amyloglucosidase enzyme (Arnata et al. 2011)
One of the alternative to give value edded onggok was used as a substrate for
fermentation of cellulase enzyme.
Cellulase is a complex enzyme system,
commercially produced by filamentous fungi under solid-state and submerged
cultivation. It has wide applicability in textile, food and beverage industry for effective
saccharification process (Chellapandi and Jani, 2008) . Commercial cellulase generally
obtained from filamentous fungi (Stoilova et al. 2005). One of filamentous fungi that it
known able to produce cellulose is Trichoderma viride. The production of cellulase is
highly influenced by several factor i.a. component of the growth medium, especially
carbon sources and time of fermentation process. In this study was conducted to
determine the effect of time of fermentation process with T. viride as an inoculum and
onggok as an inducer on crude enzyme cellulose.
MATERIALS AND METHODS
Raw material
The “onggok” (by-product tapioca factory) was obtained from Bogor West Java.
It was sun dried and milled to uniform size (40 mesh). T. viride was obtained from
microbiology laboratorium PAU IPB. Onggok was analysed to determine chemical
compotition before it is used to fermentation process. Analysed that is moisture, fat,
proteine, carbohydrate and crude fiber contents.
Inoculum culture Trichoderma viride
The strains T. viride were used in this work. Stock cultures were maintained on
potato dextrose agar (PDA) and cultivation for 5 days at 30 oC. Properly sporulated
cultured were used for inoculation.
Cellulase Production
Fermentation process to cellulase production was conducted in batch system at
250 mL erlenmeyer flask with work volume 100 ml. The composition of medium for
cellulase production by T. viride was 14 ml (NH4) 2SO4 10%, 15 ml 1M KH2PO4, 3 ml
of 10% urea, 3 ml CaCl2 10%, 3 ml MgSO4. 7H2O 10%, 1 ml of trace elements stock
and 2 ml Tween 80, then made 1 liter. Trace elements composed of 495 ml of distilled
water, 5 ml of concentrated HCl, 2.5 g FeSO4, 0.89 g MnCl2. 4H2O, 1.76 g ZnSO4.H2O,
1.25 g Co (NO3) 2. 6H2O. For the production of cellulase added 5-10 g cassava
wate/”onggok” and 0.5 to 1.0 g polipepton to 1 liter of medium. The production process
is carried out at a temperature of 30°C and pH 4.0. The ability to produce cellulase
enzyme was observed by measuring activity of FPase and CMCase every 24 hours for
7 days. Enzyme activity curves were made and used to see the achievement of
maximum activity time T. viride to produce cellulase.
Isolation of cellulase
Samples were collected from each periodic time of fermentation were added 1%
tween 80 and agitated thoroughly on orbital shaker at 100 rpm for 30 min. The crude
enzyme was filtered through Whatman # 42 filter paper. The filtrates were centrifuged
at 10000 rpm for 30 min at 4oC to remove the suspended particles.
Analysis methods
The crude enzym cellulase was determined by Pothiraj et al. (2006) method.
The celluase activity was assayed using filter paper (FPase) and carboxymethyl
cellulase (CMCase). Reducing sugars released were determined by the dinitrosalicylic
acid method (Miller, 1959). One unit of enzyme activity is defined as 1 μmol glucose
released/min/ml of culture supernatant. The pH of substrate was determined by pHmeter.
RESULTS AND DISCUSSION
The cultivation
ultivation of T. viride was carried out for 7 days by calculating the
number of spores were formed every day. At the beginning of inoculation are the
average number of spores 7.08 x 107/ml. At the end of the first da
day, the number of
spores decreased significantly due to spore germination. The next day
day, began to form
spores are white with an average number of 1.02 x 108/ml. T. viride produces spores
quickly from the first day until the third day. After
fter the third da
day, T. viride started
showing relatively slow phase of spore formation. The color change
hange of the mycelium
and spores from white to green was formed after cultivation for 6 days
days. The maximum
9
number of spores with an average of 1.58 x 10 /ml spores occurred on the sixth day.
Growth curve T. viride is presented in Figure 1.
Figure 1. The growth curve of T. viride during fermentation
process
In this study the production of cellulase enzyme using a modified Andreoti
Media. Modifications done by replacing the pure cellulose as an induction of cellulase
using cassava waste.. Cassava can be used as an material inducement because it contains
crude fiber is mainly fraction of cellulose that could be used as a carbon source for
microbial growth. In addition, cellulose is also a compound of synthesis cellulase
enzyme. Crude fiber content of cassava on average 6.58 ± 0.08
0.08% (w/w). The
concentration of cassava waste used in this study was 10 g/L. According Richana et al.
(2004), crude fiber content of cassava was 9.7%, consisting of 1.3% lignin, 5.8% xylan
and cellulose 2.61%. Arnata et al. (2011) also reported that the contents of onggok were
8.65 ± 0.10 % moisture, 2.55 ± 0.14 % ash, 6.54 ± 0.02 % fat, 1.81 ± 0.03 % proteine,
2.69 ± 0.04 % crude fiber and 62.54 ± 0.00 % starch. Crude fiber content 69.98 %
hemicellulose and 13,44 cellulose.
cellulose Padonou et al. (2005) was reported that cassava flour
content 0,56 % fat (wb),
(wb) while according to Pothiraj and Eyini (2007) Cassava waste
was found to have by dry weight 55.8% starch, 14.5% cellulose, 1.21% free reducing
sugars and 3.13% protein.
In Figure 2 shows that at the initial time of fermentation was decreased CMCase
activity until the fourth day.
day After that, CMCase enzyme activity tend to have increased
and the maximal activity of 5.05 ± 0.42 U/ml was obtained after fermentation for 7 days
or a week. It is also shown on the amount of FPase activity, which at the initial time of
fermentation was decreased until the third day and on the next day was increased.
Maximal activity of 4.77 ± 0.72 U / ml was also obtained after fermentation for 7 days.
Figure 2. The change of pH and cellulase
llulase activity
during fermentation process
The increased activity of the enzyme cellulase (CMCase and FPase) showed that
T. viride have to degradation of the cellulose fraction contained in the substarat to
produce glucose to be used for cell metabolism. Suhartono (19
(1989) reported that the
synthesis of extracellular enzymes in the greatest number, normally occurred in the
time before sporulation,
sporulation ie, at the end of the exponential phase and early stationary
phase. The condition is expected because in the transition of exponential phase
followed by decrease
ease in the number of carbon sources in the medium, so the synthesis
of cellulase enzymes started to increase.
The
he increased activity of the enzyme in the fermentation process thought to be
caused by the change of pH from pH 4.0 to 3.28 at the beginning of the seventh day.
Enari (1983) mentioned that the optimal pH for growth of Trichoderma about 4.0,
whereas for cellulase production approaching 3.0 . During the production of the
enzyme, the pH should be maintained in the range from 3.0 to 4.0 due to inactivation of
the enzyme would occur if the pH is below 2.0. Decrease in pH that occurs in cellulase
production is directly related to the consumption of carbohydrates contained in the
cassava. The pattern of change in pH during fermentation for the production of cellulase
enzymes is presented in Figure 2.
CONCLUSION
The stationary phase of maximum spore yield
ield occurred after 6 days of
cultivation T. viride with maximum spore number of 1.5 x 109spores m/L. Optimum
activity of the crude enzyme
enz
obtained after 7 days of fermentation with the FP-ase
activities of 4.77±0.72
72 U m/L and CMC-ase activities of 5.05±0.42
42 U m/L
m/L.
REFERENCES
Arnata I W, Setyaningsih D, Richana N. 2011. Production of Crude Enzyme
Amyloglucosidase From “Onggok” by Aspergillus niger. Proceeding International
Seminar of Indonesian Society for Microbiology ang IUMS-IMS
IMS Outrech Program
in Food Safety,, Bali: 236-240.
236
Chellapandi P, Jani HM. 2008. Production Of Endoglucanase By The Native Strains of
Streptomyces Isolates in Submerged Fermentation. Journal of Microbiology
39:122-127
Enari, TM. 1983. Microbial Cellulose. In W.M. Fogarty (ed.) Microbial Enz
Enzyme and
Biotechnology Applied Science Published, New York.
Pandey, A., C.R. Soccol, P. Nigarm, V.T. Soccol, L.P.S. Vandenberghe and R. Mohan,
2000. Biotechnological potential of agro-industries residue II. Cassava bagasse.
Bioresour. Technol., 74: 81-87.
Pothiraj C., Balaji P. Eyini M. 2006. Enhanced Production Of Cellulases By Various
Fungal Cultures in Solid State Fermentation of Cassava Waste. African Journal of
Biotechnology Vol. 5 (20): 1882-1885.
Pothiraj C, Eyini M. 2007. Enzyme Activities and Substrate Degradation by Fungal
Isolates on Cassava Waste During Solid State Fermentation. Mycobiology 35(4):
196-204.
Richana N, Lestina P, Irawadi TT. 2004. Karakterisasi Lignoselulosa dari Limbah
Tanaman Pangan dan Pemanfaatannya Untuk Pertumbuhan Bakteri RXA III-5
Penghasil xilanase. Penelitian pertanian Tanaman Pangan 23 (3) : 171-176.
Stoilova IS, Gargova SA, Krastanov AI. 2005. Production of Enzymes by Mixed
Culture From Micelial Fungi in Solid State Fermentation. J. Biotechnol and 19 :
103-107.
Suhartono MT. 1989. Enzim dan Bioteknologi. Departemen Pendidikan dan
Kebudayaan Direktorat Pendidikan Tinggi Antar Universitas Bioteknologi. IPB.
Bogor.
Susijahadi, Neran, Kurniawan MF. 1997. Fermentation Control With Settings Sugar
Concentration Cassava Starch Hydrolysis to Produce Alcohol. Proceedings of the
seminar of Food Technology, Jember : 45-54.
Yuwono D. Sutopo H. 2008. Production of Lactic Acid from Onggok and Tofu Liquid
Waste with Concentrate Maguro Waste Supplement by Streptococcus bovis.
Australian Journal of Basic and Applied Sciences, 2(4): 939-942.
Wongskeo P. Pramoch R. Sumaeth C. 2012. Production of Glucose from the
Hydrolysis of Cassava Residue using Bacteria Isolates from Thai Higher Termites. J
Chemical and Biological Engineering 6: 277-280.