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Hydrolysis of tuber peels and sorghum chaff by cellulolytic culture filtrates of aspergillus niger ac4 isolated from agricultural waste dumpsites
1. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
Hydrolysis of Tuber Peels
Culture Filtrates
Agri
1. Biological Sciences Department, Oduduwa University, Ile
2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.
*E –
Abstract
Six of the twenty strains of Aspergillus niger
two agricultural waste dumpsites show strong capacity for producing cellulase enzyme us
supplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production was
detected by staining the plates with 2% congo red solution and then measuring the zone of clearance around the
fungal colonies on the plates. Aspergillus niger
congo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected for
further studies. Culture filtrates of the
4% and 5.0 respectively. Culture filtrates of
peel, sweet potato peel, sorghum chaff and carboxylmethylcellulase (CMC) in
significantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantly
in the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded th
cassava peel might be a better substrate for the production of cellulase by
carboxymethlcellulase (CMC).
Key words: Tuber Peels, Sorghum Chaff, cellulolytic filtrate and hydrolysis.
1.0 Introduction
The recent thrust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive
studies on cellulolytic enzymes produced by fungi and bacteria. Cellulose is a potentially valuable resource for fibre,
fuel and feed. Investigations into ability of microbes to degrade native and modified cellulose so far have revealed
that only a few fungi possess ability to degrade native cellulose (Moo
can however degrade modified cellulose. The cost of carboh
fermentation processes, hence the cost play a decisive role in future and scope of industries employing fermentation
processes (Sani et.al.,1992; Doppelbauer
important staple foods and they are source of food for about 200
1986).The first step in the processing of these tubers is the removal of the peels which are the two coverings of the
tubers. These peels have been found to be very rich in cellulose.The possibility of high cellulase production from
selected tubers and sorghum chaff by
carbon substrate for the production of cellulase for industrial uses.
2.0 Materials and Methods
2.1 Sample treatment
Cassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agricultural
waste dumpsites in Ilorin metropolis, Kwara state. They
very fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60
proximate analyses determined by standard methods. There was no significant differenc
samples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein was
from sorghum chaff and it is significantly different from the others with sweet potato having the lowest value o
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
66
f Tuber Peels and Sorghum Chaff b
Culture Filtrates of Aspergillus Niger AC4 Isolated
Agricultural Waste Dumpsites
Adeyemo I.A1*
Sani A2
nces Department, Oduduwa University, Ile – Ife , Osun State.Nigeria.
2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.
mail of corresponding author: yemitv@yahoo.com
Aspergillus niger isolated from tuber peels and sorghum chaff collected from twenty
two agricultural waste dumpsites show strong capacity for producing cellulase enzyme us
supplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production was
detected by staining the plates with 2% congo red solution and then measuring the zone of clearance around the
Aspergillus niger AC4 produced the largest zone of clearance (3.63cm) on the PDA
congo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected for
further studies. Culture filtrates of the Aspergillus nigerAC4 had optima temperature, concentration and pH at 35
4% and 5.0 respectively. Culture filtrates of Aspergillus nigerAC4 was also used to hydrolyze yam peel, cocoyam
peel, sweet potato peel, sorghum chaff and carboxylmethylcellulase (CMC) in comparison. Hydrolysis was
significantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantly
in the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded th
cassava peel might be a better substrate for the production of cellulase by Aspergillus niger
Tuber Peels, Sorghum Chaff, cellulolytic filtrate and hydrolysis.
ust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive
studies on cellulolytic enzymes produced by fungi and bacteria. Cellulose is a potentially valuable resource for fibre,
ability of microbes to degrade native and modified cellulose so far have revealed
that only a few fungi possess ability to degrade native cellulose (Moo- young et.al.,1987). A majority of microbes
can however degrade modified cellulose. The cost of carbohydrate raw material influences the economy of many
fermentation processes, hence the cost play a decisive role in future and scope of industries employing fermentation
.,1992; Doppelbauer et al.,1987). Cassava, yam, sweet potatoes, coc
important staple foods and they are source of food for about 200 – 300 million people in tropical areas (FAO,
1986).The first step in the processing of these tubers is the removal of the peels which are the two coverings of the
These peels have been found to be very rich in cellulose.The possibility of high cellulase production from
selected tubers and sorghum chaff by Aspergillus nigeris investigated in this study with a view to obtain a cheaper
ion of cellulase for industrial uses.
Cassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agricultural
waste dumpsites in Ilorin metropolis, Kwara state. They were then dried in an oven at 80
very fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60
proximate analyses determined by standard methods. There was no significant difference in the dry matter of all the
samples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein was
from sorghum chaff and it is significantly different from the others with sweet potato having the lowest value o
www.iiste.org
by Cellulolytic
Isolated from
Ife , Osun State.Nigeria.
2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.
isolated from tuber peels and sorghum chaff collected from twenty
two agricultural waste dumpsites show strong capacity for producing cellulase enzyme using PDA plate
supplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production was
detected by staining the plates with 2% congo red solution and then measuring the zone of clearance around the
AC4 produced the largest zone of clearance (3.63cm) on the PDA
congo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected for
AC4 had optima temperature, concentration and pH at 35o
C,
AC4 was also used to hydrolyze yam peel, cocoyam
comparison. Hydrolysis was
significantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantly
in the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded that
Aspergillus niger than commercial
ust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive
studies on cellulolytic enzymes produced by fungi and bacteria. Cellulose is a potentially valuable resource for fibre,
ability of microbes to degrade native and modified cellulose so far have revealed
young et.al.,1987). A majority of microbes
ydrate raw material influences the economy of many
fermentation processes, hence the cost play a decisive role in future and scope of industries employing fermentation
.,1987). Cassava, yam, sweet potatoes, cocoyam and sorghum are
300 million people in tropical areas (FAO,
1986).The first step in the processing of these tubers is the removal of the peels which are the two coverings of the
These peels have been found to be very rich in cellulose.The possibility of high cellulase production from
Aspergillus nigeris investigated in this study with a view to obtain a cheaper
Cassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agricultural
were then dried in an oven at 80o
C and then ground into
very fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60o
C and their
e in the dry matter of all the
samples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein was
from sorghum chaff and it is significantly different from the others with sweet potato having the lowest value of
2. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
crude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide was
absent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored in
different desiccators until required.
2.2 Source and Maintenance of Organisms
Five grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which they
were collected from source) into sterile universal bottles containing 100ml of distilled water
The settled supernatants were decanted off into separate test tubes serving as stock solutions. Serial dilutions of the
stock were made with 0.1ml of the aliquot spread on Potato Dextrose Agar (PDA) plates containing 1%
streptomycin.(to inhibit bacterial growth).The plates were incubated at room temperature (27
(Berghem, 1976; Chahal, 1992; Lawal
each plate and purified on fresh PDA
temperature for 48 hours and stored at 4
2.3 Screening for Cellulase Production
The method of Bisaria and Ghose,(1987 ) was used. Point inoculations of the spores of the isolates were grown on
PDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at room
temperature (270
C- 32) for 72 hours after which it was stained
dye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellular
cellulase by the organism was indicated by a zone of clearance around the fungal colonies on th
clearance was measured on each plate and the average determined.
of clearance was selected for further studies.
2.4 Production of Crude Cellulase
Mineral salts media (MSM) for cultivatio
KH2PO 4 ,10g;(NH4)2SO4 ,10.5 g ; MgSO
ZnSO4.7H2O 0.04; Yeast extract 0.5g; Carbon source (40g). The carbon sources were
carboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask and
sterilized in the autoclave at 1210
C for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 M
NaOH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures of
were harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension was
used to inoculate 150ml of each of the prepared medium.
The culture media were incubated at room temperature (27
100rpm for seven days.
Hydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tub
sorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghum
chaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction culture
filtrate was determined colorimetrically by measuring the increase in reducing groups by the hydrolysis of
Carboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Ali
samples were filtered through whatmann filter paper to remov
to assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) of
the crabon sources were incubated at a temperature of 35
2.0ml of DNS Reagent materials ( 1.0g of 3,5
tartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. The
color intensity was determined at 560nm using a spectrophotometer (Jenwey 6405 UV/visible).
The effect of hydrolysis was also studied alongside time of incubation.One unit of cellulase activity was defined as
the amount of enzyme which will release 1.0
sugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as the
amount of glucose produced per ml in the reaction mixture per unit time
3.0 Results
From the result (Table 1), Aspergillus niger
clearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantly
different from the others ( P< 0.05 ) and produced the largest zone of clearance hence it was selected for further
studies.
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
67
crude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide was
absent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored in
ired.
2.2 Source and Maintenance of Organisms
Five grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which they
were collected from source) into sterile universal bottles containing 100ml of distilled water
The settled supernatants were decanted off into separate test tubes serving as stock solutions. Serial dilutions of the
stock were made with 0.1ml of the aliquot spread on Potato Dextrose Agar (PDA) plates containing 1%
n.(to inhibit bacterial growth).The plates were incubated at room temperature (27
(Berghem, 1976; Chahal, 1992; Lawal et al.,2005). After incubation, representative fungal colonies were picked from
each plate and purified on fresh PDA plates. The purified isolates were transferred to PDA slants incubated at room
temperature for 48 hours and stored at 40
C. The isolates were identified according to the scheme of McGinnis (1980).
2.3 Screening for Cellulase Production
(1987 ) was used. Point inoculations of the spores of the isolates were grown on
PDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at room
32) for 72 hours after which it was stained with 2% Congo red solution for 15 minutes. Excess
dye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellular
cellulase by the organism was indicated by a zone of clearance around the fungal colonies on th
clearance was measured on each plate and the average determined. Aspergillus niger AC4 showing the biggest zone
of clearance was selected for further studies.
Mineral salts media (MSM) for cultivation of fungal isolates was prepared with compositions as shown below {g/l}.
SO4 ,10.5 g ; MgSO4.7H2O , 0.3g; CaCl2, 0.5 g; FeSo4, 0.013g ; MnSO
O 0.04; Yeast extract 0.5g; Carbon source (40g). The carbon sources were the delignified peels, chaff and
carboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask and
C for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 M
OH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures of
were harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension was
of the prepared medium.
The culture media were incubated at room temperature (27-320
C) in an orbital shaker (Gallenhamp, England ) at
Hydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tub
sorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghum
chaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction culture
ed colorimetrically by measuring the increase in reducing groups by the hydrolysis of
Carboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Ali
samples were filtered through whatmann filter paper to remove the mycelia and other particle.The filtrates were used
to assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) of
the crabon sources were incubated at a temperature of 35o
C for 20 minutes and the rea
2.0ml of DNS Reagent materials ( 1.0g of 3,5-dinitrosalicylic acid, 20ml of NaoH, 30 g of sodium potassium
tartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. The
tensity was determined at 560nm using a spectrophotometer (Jenwey 6405 UV/visible).
The effect of hydrolysis was also studied alongside time of incubation.One unit of cellulase activity was defined as
the amount of enzyme which will release 1.0µmole or one unit of enzyme activity (U) of D
sugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as the
amount of glucose produced per ml in the reaction mixture per unit time
Aspergillus niger AC4 was considered to be the best cellulase producer with zone of
clearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantly
0.05 ) and produced the largest zone of clearance hence it was selected for further
www.iiste.org
crude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide was
absent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored in
Five grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which they
were collected from source) into sterile universal bottles containing 100ml of distilled water and shaken thoroughly.
The settled supernatants were decanted off into separate test tubes serving as stock solutions. Serial dilutions of the
stock were made with 0.1ml of the aliquot spread on Potato Dextrose Agar (PDA) plates containing 1%
n.(to inhibit bacterial growth).The plates were incubated at room temperature (27 – 310
C) for 48 hours
l.,2005). After incubation, representative fungal colonies were picked from
plates. The purified isolates were transferred to PDA slants incubated at room
C. The isolates were identified according to the scheme of McGinnis (1980).
(1987 ) was used. Point inoculations of the spores of the isolates were grown on
PDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at room
with 2% Congo red solution for 15 minutes. Excess
dye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellular
cellulase by the organism was indicated by a zone of clearance around the fungal colonies on the plate. The zone of
AC4 showing the biggest zone
n of fungal isolates was prepared with compositions as shown below {g/l}.
, 0.013g ; MnSO4.H2O 0.04;
the delignified peels, chaff and
carboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask and
C for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 M
OH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures of Aspergillus nigerAC4
were harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension was
C) in an orbital shaker (Gallenhamp, England ) at
Hydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tuber peels and
sorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghum
chaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction culture
ed colorimetrically by measuring the increase in reducing groups by the hydrolysis of
Carboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Ali et al., 1991). Cultured
e the mycelia and other particle.The filtrates were used
to assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) of
C for 20 minutes and the reactions stopped by adding
dinitrosalicylic acid, 20ml of NaoH, 30 g of sodium potassium
tartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. The
tensity was determined at 560nm using a spectrophotometer (Jenwey 6405 UV/visible).
The effect of hydrolysis was also studied alongside time of incubation.One unit of cellulase activity was defined as
e unit of enzyme activity (U) of D- glucose or reducing
sugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as the
AC4 was considered to be the best cellulase producer with zone of
clearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantly
0.05 ) and produced the largest zone of clearance hence it was selected for further
3. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
3.1 Effect of temperature cellulase Activities
Cultures of Aspergillus niger AC4 were incubated at different temperature ranges of 25
temperature regulated orbital shaker at 5
Cellulase production increased progressively from 25
in cellulase production as shown in figure 1.
3.2 Effect of pH on Cellulase production
The effect of pH (2.0 -8.0) on cellulase production of
hydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH
1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweet
potato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there were
decrease in cellulase activities as shown in the figure 2
3.3 Effect of concentration cellulase production
Different concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of the
hydrolyzing medium with concentration unit increments of one. c
4% where it is maximum above which cellulase activities values started decreasing as shown in the figure 3.
The various optimal conditions for cellulase production were combined and the effect of time of
hydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by the
figure 4.
4.0 Discussion
The ability of only six out of the twenty strains of
confirm the earlier reports of Doppelbauer
2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDA
stained plates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaff
hence the use of PDA-Congo red stained plates present a simple and easy way of screening for cellulolytic microbes.
The study also showed that the susceptibility of the raw tuber peels and sorghum chaff to the crude enzyme of
Aspergillus niger AC4 was significantly dependent on the carbon source and time of incubation. Cultures of
Aspergillus niger AC4 grown on 2% (w/v) PDA carboxylmethycellulase
cellulase production increased to the optimum at 35
Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero a
probably because the enzymes are being denatured as temperature increases. Initial experiments by Sani
and Omojasola et.al.,2009 had indicated the sixth and seventh day as the ideal period to harvest for temperature
dependent growth but this work is at variance with their reports indicating the fourth day for yam, cassava, sweet
potato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest of
the growth. When the determined optima conditio
culture,cellulase production increased significantly and this agrees with the earlier reports of Ali
1994 that the extracellular production of microbial cellulase depend on a
size,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aeration
and growth time and that if these conditions are combined optimally, cellulase production will be enhanced
The ability of the crude cellulase of
peels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirty
million tonnes of these tubers are lost as wastes yearly after harvesting due to poor storage (FAO,1986).
Conversion of these tuber peels by this enzyme means that these wastes can be used by industries employing
hydrolysis processes for value added products.( Sani
In conclusion, it has been shown in this study that cassava peel may be a better carbon substrate than commercial
carboxylmethycellulase for cellulase production by the
wastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the most
economical carbon substrate for the production of cellulase for industrial use.
References
Ali, S., Sayed, A., Sarker, R.T., Alam, R. (1991).F
water hyacinth. World journal of microbiology and biotechnology,
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
68
3.1 Effect of temperature cellulase Activities
AC4 were incubated at different temperature ranges of 25
regulated orbital shaker at 5O
C interval and cellulase production monitored in shake flask culture.
Cellulase production increased progressively from 25o
C to the maximum at 35o
C above which there was decrease
in cellulase production as shown in figure 1.
3.2 Effect of pH on Cellulase production
8.0) on cellulase production of Aspergillus niger AC4 was studied by adjusting pH of the
hydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH
1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweet
potato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there were
tivities as shown in the figure 2
3.3 Effect of concentration cellulase production
Different concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of the
hydrolyzing medium with concentration unit increments of one. cellulase activities was found to be increasing up to
4% where it is maximum above which cellulase activities values started decreasing as shown in the figure 3.
The various optimal conditions for cellulase production were combined and the effect of time of
hydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by the
The ability of only six out of the twenty strains of Aspergillus niger isolated from agricultural
confirm the earlier reports of Doppelbauer et.al., 1987; Moo-young et.al., 1987; Raji et.al
2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDA
plates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaff
Congo red stained plates present a simple and easy way of screening for cellulolytic microbes.
e susceptibility of the raw tuber peels and sorghum chaff to the crude enzyme of
AC4 was significantly dependent on the carbon source and time of incubation. Cultures of
AC4 grown on 2% (w/v) PDA carboxylmethycellulase produced low level of cellulase at 25
cellulase production increased to the optimum at 35o
C for cassava and other carbon sources and 40
Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero a
probably because the enzymes are being denatured as temperature increases. Initial experiments by Sani
.,2009 had indicated the sixth and seventh day as the ideal period to harvest for temperature
this work is at variance with their reports indicating the fourth day for yam, cassava, sweet
potato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest of
the growth. When the determined optima conditions for cellulase production were combined in the submerged
culture,cellulase production increased significantly and this agrees with the earlier reports of Ali
1994 that the extracellular production of microbial cellulase depend on a number of factor such as inoculums
size,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aeration
and growth time and that if these conditions are combined optimally, cellulase production will be enhanced
The ability of the crude cellulase of Aspergillus niger AC4 to hydrolyze the tuber peels especially cassava and tuber
peels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirty
hese tubers are lost as wastes yearly after harvesting due to poor storage (FAO,1986).
Conversion of these tuber peels by this enzyme means that these wastes can be used by industries employing
hydrolysis processes for value added products.( Sani et.al.,1992; Doppelbauer et al.,1987).
In conclusion, it has been shown in this study that cassava peel may be a better carbon substrate than commercial
carboxylmethycellulase for cellulase production by the Aspergillus niger AC4 investigated. Other materials and
wastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the most
economical carbon substrate for the production of cellulase for industrial use.
Ali, S., Sayed, A., Sarker, R.T., Alam, R. (1991).Factors affecting cellulase production by
World journal of microbiology and biotechnology, 7: 62-66.
www.iiste.org
AC4 were incubated at different temperature ranges of 250
C to 700
C using
C interval and cellulase production monitored in shake flask culture.
C above which there was decrease
AC4 was studied by adjusting pH of the
hydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH meter at
1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweet
potato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there were
Different concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of the
ellulase activities was found to be increasing up to
4% where it is maximum above which cellulase activities values started decreasing as shown in the figure 3.
The various optimal conditions for cellulase production were combined and the effect of time of incubation on the
hydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by the
isolated from agricultural waste dumpsites
et.al., 1988; Desvaux et.al.,
2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDA-congo red
plates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaff
Congo red stained plates present a simple and easy way of screening for cellulolytic microbes.
e susceptibility of the raw tuber peels and sorghum chaff to the crude enzyme of
AC4 was significantly dependent on the carbon source and time of incubation. Cultures of
produced low level of cellulase at 25o
C but
C for cassava and other carbon sources and 40o
C yam peel.
Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero at 55o
C
probably because the enzymes are being denatured as temperature increases. Initial experiments by Saniet.al., 1992
.,2009 had indicated the sixth and seventh day as the ideal period to harvest for temperature
this work is at variance with their reports indicating the fourth day for yam, cassava, sweet
potato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest of
ns for cellulase production were combined in the submerged
culture,cellulase production increased significantly and this agrees with the earlier reports of Ali et.al.,1991; Davies,
number of factor such as inoculums
size,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aeration
and growth time and that if these conditions are combined optimally, cellulase production will be enhanced.
AC4 to hydrolyze the tuber peels especially cassava and tuber
peels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirty
hese tubers are lost as wastes yearly after harvesting due to poor storage (FAO,1986).
Conversion of these tuber peels by this enzyme means that these wastes can be used by industries employing
.,1987).
In conclusion, it has been shown in this study that cassava peel may be a better carbon substrate than commercial
AC4 investigated. Other materials and
wastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the most
actors affecting cellulase production by Aspergillu sterres using
4. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
Berghem, L.E.R., Petterson, L.G and Axiofredrikson U.B (1976): The mechanism of enzymatic cellulase degradation.
Isolation and some properties of a
Biochemistry,46: 295-300.
Bisaria, V.S and Ghose, T.K (1987).Biodegradation of cellulosic materials substrates, microorganisms, enzymes and
products. Enzymes and Microbial Technology
Chahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin in
Lignocellulosics: Science Technology Development and Use
Davies, R .W .(1994). Heterologous gene expression and protein secretion in
Microbioliology.29 :527 -560
Desvaux, M., Guedon, E., and Petitdemange,H.(2000). Cellulose catabolism by
in batch culture on defined medium.
Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastes
for production of cellulase by Trichoderma reesei
FAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. Report
series NO. 52, pp210 .
Lawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod with
some isolated tropical fungi : Growth resources and carcass quality of broilers finisher birds.
Annual Conf., Anim. Sci. Ass of Nig.
McGinnins, M.R. (1980). Laboratory handbook of medical mycology.
Moo- young, M., Lamptey, J., Glick, B and Bungay, H. (1987).
Oxford.
Omojasola P.F, Jilani, O.P and Ibiyemi S.A.(2009). Cellulase production by some cultured on pineapple waste.
Nature and Science, 6(2), Pp 64
Panda .T. (1989). Simulation of shake flask conditions in a bioreactor for the biosynthesis of cellulases and xylanases
by a mixed culture of Trichoderma reesi
767-772.
Raji, A.I., Ameh. J.B. and Ndukwe., M. (1988). Production of cellulase enzyme by
delignified wheat straw and rice husk substrates..
Sani , A., Awe, F.A and Akinyanju J.A. (1992). Amylase synthesis in
on cassava peel. Journal of Industrial Microbio
Table 1: Cellulase production capacities of
Isolate code
AC4
AC2
AY14
AY19
AC 12
AP20
a,b,c,d,e
Means on the same column with different superscripts differ significantly. (P<0.05)
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
69
Berghem, L.E.R., Petterson, L.G and Axiofredrikson U.B (1976): The mechanism of enzymatic cellulase degradation.
olation and some properties of a β –glucosidase from trichodermaviride.
Bisaria, V.S and Ghose, T.K (1987).Biodegradation of cellulosic materials substrates, microorganisms, enzymes and
crobial Technology. 3: 90-140.
Chahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin in
Science Technology Development and Use. Ellis Horwood Ltd., England. Pp 83
W .(1994). Heterologous gene expression and protein secretion in Aspergillus
Desvaux, M., Guedon, E., and Petitdemange,H.(2000). Cellulose catabolism by clostridium cellulolyticum
n defined medium. Applied and Environmental Microbiology,66:2461
Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastes
Trichoderma reesei. Applied Microbiology and Biotechnology
FAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. Report
Lawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod with
some isolated tropical fungi : Growth resources and carcass quality of broilers finisher birds.
Annual Conf., Anim. Sci. Ass of Nig. (ASAN) 109 -112.
Laboratory handbook of medical mycology. Academic press, London.pp 80
young, M., Lamptey, J., Glick, B and Bungay, H. (1987). Biomass conversion technology
Omojasola P.F, Jilani, O.P and Ibiyemi S.A.(2009). Cellulase production by some cultured on pineapple waste.
Nature and Science, 6(2), Pp 64 -79.
Panda .T. (1989). Simulation of shake flask conditions in a bioreactor for the biosynthesis of cellulases and xylanases
Trichoderma reesi D 1-6 and Aspergillus wentil pt- 2804 process Biochemistr
Raji, A.I., Ameh. J.B. and Ndukwe., M. (1988). Production of cellulase enzyme by Aspergillus niger
delignified wheat straw and rice husk substrates..Nigerain Journal of Technical Education
ju J.A. (1992). Amylase synthesis in Aspergillus niger and
Journal of Industrial Microbiology 10: 55-59.
Table 1: Cellulase production capacities of A.niger strains
Means of clear zones Cellulase Act
3.36 240a
2.27 161b
2.00 138c
1.82 130c
1.80 129d
1.53 109e
Means on the same column with different superscripts differ significantly. (P<0.05)
www.iiste.org
Berghem, L.E.R., Petterson, L.G and Axiofredrikson U.B (1976): The mechanism of enzymatic cellulase degradation.
glucosidase from trichodermaviride. Europian Journal Of
Bisaria, V.S and Ghose, T.K (1987).Biodegradation of cellulosic materials substrates, microorganisms, enzymes and
Chahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin in
. Ellis Horwood Ltd., England. Pp 83- 93.
Aspergillus Program. Industrial
clostridium cellulolyticum growing
,66:2461-2468.
Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastes
ogy and Biotechnology, 26: 485-495
FAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. Report
Lawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod with extracted enzymes from
some isolated tropical fungi : Growth resources and carcass quality of broilers finisher birds. Proceedings of
demic press, London.pp 80-84
Biomass conversion technology. Pergamon press,
Omojasola P.F, Jilani, O.P and Ibiyemi S.A.(2009). Cellulase production by some cultured on pineapple waste.
Panda .T. (1989). Simulation of shake flask conditions in a bioreactor for the biosynthesis of cellulases and xylanases
2804 process Biochemistry, 5:
Aspergillus niger– CS14 from
Nigerain Journal of Technical Education.15: 57-63
and Aspergillus flavus grown
Cellulase Activity (U/ml)
5. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
Figure 1: Effect of varying substrate concentratio
Figure 2: Effect of varying pH on cellulase activities of
0
50
100
150
200
250
25 30 35 40
CellulaseActivity(U/ml)
Temperature (
0
20
40
60
80
100
120
140
160
180
200
2 3 4
CellulaseActivity(U/ml)
pH of the growth medium
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
70
Figure 1: Effect of varying substrate concentration on cellulose activities of A.niger AC4
Figure 2: Effect of varying pH on cellulase activities of A.niger AC4 in shake flask culture
45 50 55 60 65 70
Temperature (oC)
cassava peel
yam peel
S.potato peel
Sorghum chaff
cocoyam peel
CMC
5 6 7 8 9 10
pH of the growth medium
cassava peel
yam peel
S.potato peels
Cocoyam peel
Sorghum chaff
CMC
www.iiste.org
AC4 in shake flask culture
S.potato peel
Sorghum chaff
cocoyam peel
cassava peel
yam peel
S.potato peels
Cocoyam peel
Sorghum chaff
CMC
6. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225
Vol.3, No.5, 2013
Figure 3: Effects of varying substrate concentrations on cellulase activities of
Figure 4: Enzyme activities of Aspergillus niger
for other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperature
40o
C for cocoyam and 35o
C for other carbon sources.
0
20
40
60
80
100
120
140
160
180
200
1 2 3 4
cellulaseactivity(U/ml)
Substrate concentration of the growth medium
0
50
100
150
200
250
300
1 2 3
CellulaseActivity(U/ml)
Incubation Period (Days)
Journal of Biology, Agriculture and Healthcare
208 (Paper) ISSN 2225-093X (Online)
71
Figure 3: Effects of varying substrate concentrations on cellulase activities of A.niger AC4 in shake flask culture.
Aspergillus niger under optimal conditions of pH 4.0 for sweet potato peel and 5.0
for other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperature
C for other carbon sources.
5 6 7 8 9
Substrate concentration of the growth medium
cassava peel
yam peel
cocoyam peel
Sorghum chaff
S.potato peel
CMC
3 4 5 6 7
Incubation Period (Days)
www.iiste.org
AC4 in shake flask culture.
under optimal conditions of pH 4.0 for sweet potato peel and 5.0
for other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperature
cocoyam peel
Sorghum chaff
S.potato peel
cassava peel
yam peel
Sorghum chaff
cocoyam peel
CMC
S.potato peel
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