Hydrolysis of  tuber peels and sorghum chaff  by cellulolytic culture filtrates of aspergillus niger  ac4 isolated from agricultural waste dumpsites
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Hydrolysis of tuber peels and sorghum chaff by cellulolytic culture filtrates of aspergillus niger ac4 isolated from agricultural waste dumpsites

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Hydrolysis of  tuber peels and sorghum chaff  by cellulolytic culture filtrates of aspergillus niger  ac4 isolated from agricultural waste dumpsites Hydrolysis of tuber peels and sorghum chaff by cellulolytic culture filtrates of aspergillus niger ac4 isolated from agricultural waste dumpsites Document Transcript

  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 2013Hydrolysis of Tuber PeelsCulture FiltratesAgri1. Biological Sciences Department, Oduduwa University, Ile2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.*E –AbstractSix of the twenty strains of Aspergillus nigertwo agricultural waste dumpsites show strong capacity for producing cellulase enzyme ussupplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production wasdetected by staining the plates with 2% congo red solution and then measuring the zone of clearance around thefungal colonies on the plates. Aspergillus nigercongo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected forfurther studies. Culture filtrates of the4% and 5.0 respectively. Culture filtrates ofpeel, sweet potato peel, sorghum chaff and carboxylmethylcellulase (CMC) insignificantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantlyin the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded thcassava peel might be a better substrate for the production of cellulase bycarboxymethlcellulase (CMC).Key words: Tuber Peels, Sorghum Chaff, cellulolytic filtrate and hydrolysis.1.0 IntroductionThe recent thrust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensivestudies 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 revealedthat only a few fungi possess ability to degrade native cellulose (Moocan however degrade modified cellulose. The cost of carbohfermentation processes, hence the cost play a decisive role in future and scope of industries employing fermentationprocesses (Sani et.al.,1992; Doppelbauerimportant staple foods and they are source of food for about 2001986).The first step in the processing of these tubers is the removal of the peels which are the two coverings of thetubers. These peels have been found to be very rich in cellulose.The possibility of high cellulase production fromselected tubers and sorghum chaff bycarbon substrate for the production of cellulase for industrial uses.2.0 Materials and Methods2.1 Sample treatmentCassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agriculturalwaste dumpsites in Ilorin metropolis, Kwara state. Theyvery fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60proximate analyses determined by standard methods. There was no significant differencsamples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein wasfrom sorghum chaff and it is significantly different from the others with sweet potato having the lowest value oJournal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)66f Tuber Peels and Sorghum Chaff bCulture Filtrates of Aspergillus Niger AC4 IsolatedAgricultural Waste DumpsitesAdeyemo I.A1*Sani A2nces Department, Oduduwa University, Ile – Ife , Osun State.Nigeria.2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.mail of corresponding author: yemitv@yahoo.comAspergillus niger isolated from tuber peels and sorghum chaff collected from twentytwo agricultural waste dumpsites show strong capacity for producing cellulase enzyme ussupplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production wasdetected by staining the plates with 2% congo red solution and then measuring the zone of clearance around theAspergillus niger AC4 produced the largest zone of clearance (3.63cm) on the PDAcongo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected forfurther studies. Culture filtrates of the Aspergillus nigerAC4 had optima temperature, concentration and pH at 354% and 5.0 respectively. Culture filtrates of Aspergillus nigerAC4 was also used to hydrolyze yam peel, cocoyampeel, sweet potato peel, sorghum chaff and carboxylmethylcellulase (CMC) in comparison. Hydrolysis wassignificantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantlyin the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded thcassava peel might be a better substrate for the production of cellulase by Aspergillus nigerTuber Peels, Sorghum Chaff, cellulolytic filtrate and hydrolysis.ust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensivestudies 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 revealedthat only a few fungi possess ability to degrade native cellulose (Moo- young et.al.,1987). A majority of microbescan however degrade modified cellulose. The cost of carbohydrate raw material influences the economy of manyfermentation 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, cocimportant 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 theThese peels have been found to be very rich in cellulose.The possibility of high cellulase production fromselected tubers and sorghum chaff by Aspergillus nigeris investigated in this study with a view to obtain a cheaperion of cellulase for industrial uses.Cassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agriculturalwaste dumpsites in Ilorin metropolis, Kwara state. They were then dried in an oven at 80very fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60proximate analyses determined by standard methods. There was no significant difference in the dry matter of all thesamples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein wasfrom sorghum chaff and it is significantly different from the others with sweet potato having the lowest value owww.iiste.orgby CellulolyticIsolated fromIfe , Osun State.Nigeria.2. Microbiology Department, University of Ilorin, Kwara state, Nigeria.isolated from tuber peels and sorghum chaff collected from twentytwo agricultural waste dumpsites show strong capacity for producing cellulase enzyme using PDA platesupplemented with 2% (w/v) carboxylmethylcellulase incubated at room temperature. Cellulase production wasdetected by staining the plates with 2% congo red solution and then measuring the zone of clearance around theAC4 produced the largest zone of clearance (3.63cm) on the PDAcongo red stained plate and also gave the highest cellulase activity (240U/ml) on hydrolysis hence it was selected forAC4 had optima temperature, concentration and pH at 35oC,AC4 was also used to hydrolyze yam peel, cocoyamcomparison. Hydrolysis wassignificantly dependent on cellulose source and length of incubation (P<0.05). cellulose activities differ significantlyin the cellulose sources ranging from 240U/ml in cassava peel to 54U/ml in sorghum chaff. It is concluded thatAspergillus niger than commercialust in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensivestudies 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 revealedyoung et.al.,1987). A majority of microbesydrate raw material influences the economy of manyfermentation processes, hence the cost play a decisive role in future and scope of industries employing fermentation.,1987). Cassava, yam, sweet potatoes, cocoyam and sorghum are300 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 theThese peels have been found to be very rich in cellulose.The possibility of high cellulase production fromAspergillus nigeris investigated in this study with a view to obtain a cheaperCassava, yam, cocoyam and sweet potato peels as well as sorghum chaff were collected from twenty two agriculturalwere then dried in an oven at 80oC and then ground intovery fine powder separately. Each of the ground powder was dried into constant weight in an oven at 60oC and theire in the dry matter of all thesamples with cocoyam having the highest dry matter content and cassava with the least. Highest crude protein wasfrom sorghum chaff and it is significantly different from the others with sweet potato having the lowest value of
  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 2013crude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide wasabsent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored indifferent desiccators until required.2.2 Source and Maintenance of OrganismsFive grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which theywere collected from source) into sterile universal bottles containing 100ml of distilled waterThe settled supernatants were decanted off into separate test tubes serving as stock solutions. Serial dilutions of thestock 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; Lawaleach plate and purified on fresh PDAtemperature for 48 hours and stored at 42.3 Screening for Cellulase ProductionThe method of Bisaria and Ghose,(1987 ) was used. Point inoculations of the spores of the isolates were grown onPDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at roomtemperature (270C- 32) for 72 hours after which it was staineddye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellularcellulase by the organism was indicated by a zone of clearance around the fungal colonies on thclearance was measured on each plate and the average determined.of clearance was selected for further studies.2.4 Production of Crude CellulaseMineral salts media (MSM) for cultivatioKH2PO 4 ,10g;(NH4)2SO4 ,10.5 g ; MgSOZnSO4.7H2O 0.04; Yeast extract 0.5g; Carbon source (40g). The carbon sources werecarboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask andsterilized in the autoclave at 1210C for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 MNaOH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures ofwere harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension wasused to inoculate 150ml of each of the prepared medium.The culture media were incubated at room temperature (27100rpm for seven days.Hydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tubsorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghumchaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction culturefiltrate was determined colorimetrically by measuring the increase in reducing groups by the hydrolysis ofCarboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Alisamples were filtered through whatmann filter paper to removto assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) ofthe crabon sources were incubated at a temperature of 352.0ml of DNS Reagent materials ( 1.0g of 3,5tartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. Thecolor 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 asthe amount of enzyme which will release 1.0sugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as theamount of glucose produced per ml in the reaction mixture per unit time3.0 ResultsFrom the result (Table 1), Aspergillus nigerclearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantlydifferent from the others ( P< 0.05 ) and produced the largest zone of clearance hence it was selected for furtherstudies.Journal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)67crude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide wasabsent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored inired.2.2 Source and Maintenance of OrganismsFive grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which theywere collected from source) into sterile universal bottles containing 100ml of distilled waterThe settled supernatants were decanted off into separate test tubes serving as stock solutions. Serial dilutions of thestock 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 fromeach plate and purified on fresh PDA plates. The purified isolates were transferred to PDA slants incubated at roomtemperature for 48 hours and stored at 40C. 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 onPDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at room32) for 72 hours after which it was stained with 2% Congo red solution for 15 minutes. Excessdye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellularcellulase by the organism was indicated by a zone of clearance around the fungal colonies on thclearance was measured on each plate and the average determined. Aspergillus niger AC4 showing the biggest zoneof 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 ; MnSOO 0.04; Yeast extract 0.5g; Carbon source (40g). The carbon sources were the delignified peels, chaff andcarboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask andC for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 MOH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures ofwere harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension wasof the prepared medium.The culture media were incubated at room temperature (27-320C) in an orbital shaker (Gallenhamp, England ) atHydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tubsorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghumchaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction cultureed colorimetrically by measuring the increase in reducing groups by the hydrolysis ofCarboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Alisamples were filtered through whatmann filter paper to remove the mycelia and other particle.The filtrates were usedto assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) ofthe crabon sources were incubated at a temperature of 35oC for 20 minutes and the rea2.0ml of DNS Reagent materials ( 1.0g of 3,5-dinitrosalicylic acid, 20ml of NaoH, 30 g of sodium potassiumtartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. Thetensity 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 asthe amount of enzyme which will release 1.0µmole or one unit of enzyme activity (U) of Dsugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as theamount of glucose produced per ml in the reaction mixture per unit timeAspergillus niger AC4 was considered to be the best cellulase producer with zone ofclearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantly0.05 ) and produced the largest zone of clearance hence it was selected for furtherwww.iiste.orgcrude protein. Cassava peel has the highest carbohydrate content with the least from sorghum chaff. Cyanide wasabsent from all the samples except cassava peels with 1.74mg/ 100ml of the cassava peel. They were stored inFive grams each of the freshly obtained peels and chaff was taken from the sealed cellophane bags (with which theywere 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 thestock 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 – 310C) for 48 hoursl.,2005). After incubation, representative fungal colonies were picked fromplates. The purified isolates were transferred to PDA slants incubated at roomC. The isolates were identified according to the scheme of McGinnis (1980).(1987 ) was used. Point inoculations of the spores of the isolates were grown onPDA supplemented with Carboxylmethylcellulase (CMC,2% W/V) medium. The plates were incubated at roomwith 2% Congo red solution for 15 minutes. Excessdye was removed by washing with 1M NaCl and the plates were fixed with 1M HCl. The production of extracellularcellulase by the organism was indicated by a zone of clearance around the fungal colonies on the plate. The zone ofAC4 showing the biggest zonen of fungal isolates was prepared with compositions as shown below {g/l}., 0.013g ; MnSO4.H2O 0.04;the delignified peels, chaff andcarboxylmethylcellulase. One hundred and fifty milliliter of each medium was dispersed into conical flask andC for 15 minutes. The final pHs of the medium was adjusted to 5.0with 0.1 MOH and 0.1M HCl using a pH meter ( pye unicam pH). Spores of 72 hour old cultures of Aspergillus nigerAC4were harvested by washing slants with 10ml of sterile distilled water. An aliquot of 5ml of the spore suspension wasC) in an orbital shaker (Gallenhamp, England ) atHydrolysis of tuber peels and sorghum chaff. The ability of the crude enzyme to hydrolyze the tuber peels andsorghum chaff was studied using the grounded cassava,yam,sweet potato and cocoyam peels as well as sorghumchaff. Commercial carboxymethylcellulase (CMC) was used as the standard. Cellulase activity of the suction cultureed colorimetrically by measuring the increase in reducing groups by the hydrolysis ofCarboxylmethylcellulase (CMC) substrate and the other carbon sources (Panda 1989; Ali et al., 1991). Culturede the mycelia and other particle.The filtrates were usedto assay enzymatic activity. The reaction mixture containing 0.5ml of the enzyme solutions and 1ml of 2% (w/v) ofC for 20 minutes and the reactions stopped by addingdinitrosalicylic acid, 20ml of NaoH, 30 g of sodium potassiumtartarate in 100ml). The total mixture was then heated for 5 minutes, cooled and 20ml of distilled water added. Thetensity 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 ase unit of enzyme activity (U) of D- glucose or reducingsugar as glucose per millimeter of digest in 20 minutes under the specified conditions. It is also defined as theAC4 was considered to be the best cellulase producer with zone ofclearance 3.36cm and cellualse activity of 240U/ml. It has the highest cellualse activity which is significantly0.05 ) and produced the largest zone of clearance hence it was selected for further
  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 20133.1 Effect of temperature cellulase ActivitiesCultures of Aspergillus niger AC4 were incubated at different temperature ranges of 25temperature regulated orbital shaker at 5Cellulase production increased progressively from 25in cellulase production as shown in figure 1.3.2 Effect of pH on Cellulase productionThe effect of pH (2.0 -8.0) on cellulase production ofhydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweetpotato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there weredecrease in cellulase activities as shown in the figure 23.3 Effect of concentration cellulase productionDifferent concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of thehydrolyzing medium with concentration unit increments of one. c4% 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 ofhydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by thefigure 4.4.0 DiscussionThe ability of only six out of the twenty strains ofconfirm the earlier reports of Doppelbauer2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDAstained plates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaffhence 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 ofAspergillus niger AC4 was significantly dependent on the carbon source and time of incubation. Cultures ofAspergillus niger AC4 grown on 2% (w/v) PDA carboxylmethycellulasecellulase production increased to the optimum at 35Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero aprobably because the enzymes are being denatured as temperature increases. Initial experiments by Saniand Omojasola et.al.,2009 had indicated the sixth and seventh day as the ideal period to harvest for temperaturedependent growth but this work is at variance with their reports indicating the fourth day for yam, cassava, sweetpotato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest ofthe growth. When the determined optima conditioculture,cellulase production increased significantly and this agrees with the earlier reports of Ali1994 that the extracellular production of microbial cellulase depend on asize,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aerationand growth time and that if these conditions are combined optimally, cellulase production will be enhancedThe ability of the crude cellulase ofpeels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirtymillion 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 employinghydrolysis processes for value added products.( SaniIn conclusion, it has been shown in this study that cassava peel may be a better carbon substrate than commercialcarboxylmethycellulase for cellulase production by thewastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the mosteconomical carbon substrate for the production of cellulase for industrial use.ReferencesAli, S., Sayed, A., Sarker, R.T., Alam, R. (1991).Fwater hyacinth. World journal of microbiology and biotechnology,Journal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)683.1 Effect of temperature cellulase ActivitiesAC4 were incubated at different temperature ranges of 25regulated orbital shaker at 5OC interval and cellulase production monitored in shake flask culture.Cellulase production increased progressively from 25oC to the maximum at 35oC above which there was decreasein cellulase production as shown in figure 1.3.2 Effect of pH on Cellulase production8.0) on cellulase production of Aspergillus niger AC4 was studied by adjusting pH of thehydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweetpotato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there weretivities as shown in the figure 23.3 Effect of concentration cellulase productionDifferent concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of thehydrolyzing medium with concentration unit increments of one. cellulase activities was found to be increasing up to4% 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 ofhydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by theThe ability of only six out of the twenty strains of Aspergillus niger isolated from agriculturalconfirm the earlier reports of Doppelbauer et.al., 1987; Moo-young et.al., 1987; Raji et.al2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDAplates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaffCongo 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 ofAC4 was significantly dependent on the carbon source and time of incubation. Cultures ofAC4 grown on 2% (w/v) PDA carboxylmethycellulase produced low level of cellulase at 25cellulase production increased to the optimum at 35oC for cassava and other carbon sources and 40Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero aprobably 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 temperaturethis work is at variance with their reports indicating the fourth day for yam, cassava, sweetpotato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest ofthe growth. When the determined optima conditions for cellulase production were combined in the submergedculture,cellulase production increased significantly and this agrees with the earlier reports of Ali1994 that the extracellular production of microbial cellulase depend on a number of factor such as inoculumssize,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aerationand growth time and that if these conditions are combined optimally, cellulase production will be enhancedThe ability of the crude cellulase of Aspergillus niger AC4 to hydrolyze the tuber peels especially cassava and tuberpeels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirtyhese 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 employinghydrolysis 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 commercialcarboxylmethycellulase for cellulase production by the Aspergillus niger AC4 investigated. Other materials andwastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the mosteconomical carbon substrate for the production of cellulase for industrial use.Ali, S., Sayed, A., Sarker, R.T., Alam, R. (1991).Factors affecting cellulase production byWorld journal of microbiology and biotechnology, 7: 62-66.www.iiste.orgAC4 were incubated at different temperature ranges of 250C to 700C usingC interval and cellulase production monitored in shake flask culture.C above which there was decreaseAC4 was studied by adjusting pH of thehydrolyzing medium to various values ranging from 2 to 10 with 0.1 M NaOH and 0.1M HCl using a pH meter at1.0 pH unit increments and cellulose activity monitored. Highest cellulase activity was obtained at pH 4.0 for sweetpotato peel and 5.0 for cassava, yam, cocoyam peels, CMC and sorghum chaffand above these pHs, there wereDifferent concentrations of the substrates ranging from 1% to 9 % (w/v) were used in the preparation of theellulase activities was found to be increasing up to4% 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 thehydrolysis of the different carbon sources were studied under the determined optimal conditions as shown by theisolated from agricultural waste dumpsiteset.al., 1988; Desvaux et.al.,2000; that only a few fungi possess ability to degrade cellulose. All the isolates selected from the PDA-congo redplates produced detectable quantities of cellulase during hydrolysis of the raw tuber peels and sorghum chaffCongo 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 ofAC4 was significantly dependent on the carbon source and time of incubation. Cultures ofproduced low level of cellulase at 25oC butC for cassava and other carbon sources and 40oC yam peel.Beyond these optimal temperatures, cellulase activities started reducing and eventually became zero at 55oCprobably 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 temperaturethis work is at variance with their reports indicating the fourth day for yam, cassava, sweetpotato peels and sorghum chaff while cocoyam peel and CMC had the fifth day as the ideal period for the harvest ofns for cellulase production were combined in the submergedculture,cellulase production increased significantly and this agrees with the earlier reports of Ali et.al.,1991; Davies,number of factor such as inoculumssize,carbon source, pH, temperature, presence of inducers and or inhibitors, medium additives,batch size, aerationand growth time and that if these conditions are combined optimally, cellulase production will be enhanced.AC4 to hydrolyze the tuber peels especially cassava and tuberpeels presents a remarkable property since these tuber peels are abundantly available in the tropics and over thirtyhese 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 commercialAC4 investigated. Other materials andwastes are also being tried with some other cellulase producing microorganisms with a view to obtaining the mostactors affecting cellulase production by Aspergillu sterres using
  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 2013Berghem, L.E.R., Petterson, L.G and Axiofredrikson U.B (1976): The mechanism of enzymatic cellulase degradation.Isolation and some properties of aBiochemistry,46: 295-300.Bisaria, V.S and Ghose, T.K (1987).Biodegradation of cellulosic materials substrates, microorganisms, enzymes andproducts. Enzymes and Microbial TechnologyChahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin inLignocellulosics: Science Technology Development and UseDavies, R .W .(1994). Heterologous gene expression and protein secretion inMicrobioliology.29 :527 -560Desvaux, M., Guedon, E., and Petitdemange,H.(2000). Cellulose catabolism byin batch culture on defined medium.Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastesfor production of cellulase by Trichoderma reeseiFAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. Reportseries NO. 52, pp210 .Lawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod withsome 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 64Panda .T. (1989). Simulation of shake flask conditions in a bioreactor for the biosynthesis of cellulases and xylanasesby a mixed culture of Trichoderma reesi767-772.Raji, A.I., Ameh. J.B. and Ndukwe., M. (1988). Production of cellulase enzyme bydelignified wheat straw and rice husk substrates..Sani , A., Awe, F.A and Akinyanju J.A. (1992). Amylase synthesis inon cassava peel. Journal of Industrial MicrobioTable 1: Cellulase production capacities ofIsolate codeAC4AC2AY14AY19AC 12AP20a,b,c,d,eMeans on the same column with different superscripts differ significantly. (P<0.05)Journal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)69Berghem, 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 andcrobial Technology. 3: 90-140.Chahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin inScience Technology Development and Use. Ellis Horwood Ltd., England. Pp 83W .(1994). Heterologous gene expression and protein secretion in AspergillusDesvaux, M., Guedon, E., and Petitdemange,H.(2000). Cellulose catabolism by clostridium cellulolyticumn defined medium. Applied and Environmental Microbiology,66:2461Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastesTrichoderma reesei. Applied Microbiology and BiotechnologyFAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. ReportLawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod withsome 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 80young, M., Lamptey, J., Glick, B and Bungay, H. (1987). Biomass conversion technologyOmojasola 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 xylanasesTrichoderma reesi D 1-6 and Aspergillus wentil pt- 2804 process BiochemistrRaji, A.I., Ameh. J.B. and Ndukwe., M. (1988). Production of cellulase enzyme by Aspergillus nigerdelignified wheat straw and rice husk substrates..Nigerain Journal of Technical Educationju J.A. (1992). Amylase synthesis in Aspergillus niger andJournal of Industrial Microbiology 10: 55-59.Table 1: Cellulase production capacities of A.niger strainsMeans of clear zones Cellulase Act3.36 240a2.27 161b2.00 138c1.82 130c1.80 129d1.53 109eMeans on the same column with different superscripts differ significantly. (P<0.05)www.iiste.orgBerghem, L.E.R., Petterson, L.G and Axiofredrikson U.B (1976): The mechanism of enzymatic cellulase degradation.glucosidase from trichodermaviride. Europian Journal OfBisaria, V.S and Ghose, T.K (1987).Biodegradation of cellulosic materials substrates, microorganisms, enzymes andChahal, D.S. (1992). Bioconversion of polysaccharides of lignocelluloses and simultaneous degradation of lignin in. Ellis Horwood Ltd., England. Pp 83- 93.Aspergillus Program. Industrialclostridium cellulolyticum growing,66:2461-2468.Doppelbauer, R., Estabauer, H., Steiner, W., Lafferty, R.M and Steimuller,H.(1987).The use of lignocellulosic wastesogy and Biotechnology, 26: 485-495FAO (1986). Food and Agricultural Organization.World food production, Geneva FAO nutritional meeting. ReportLawal, T.E; Iyayi, E .A. and Aderemi, F. A. (2005). Biodegradation of groundnut pod with extracted enzymes fromsome isolated tropical fungi : Growth resources and carcass quality of broilers finisher birds. Proceedings ofdemic press, London.pp 80-84Biomass 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 xylanases2804 process Biochemistry, 5:Aspergillus niger– CS14 fromNigerain Journal of Technical Education.15: 57-63and Aspergillus flavus grownCellulase Activity (U/ml)
  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 2013Figure 1: Effect of varying substrate concentratioFigure 2: Effect of varying pH on cellulase activities of05010015020025025 30 35 40CellulaseActivity(U/ml)Temperature (0204060801001201401601802002 3 4CellulaseActivity(U/ml)pH of the growth mediumJournal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)70Figure 1: Effect of varying substrate concentration on cellulose activities of A.niger AC4Figure 2: Effect of varying pH on cellulase activities of A.niger AC4 in shake flask culture45 50 55 60 65 70Temperature (oC)cassava peelyam peelS.potato peelSorghum chaffcocoyam peelCMC5 6 7 8 9 10pH of the growth mediumcassava peelyam peelS.potato peelsCocoyam peelSorghum chaffCMCwww.iiste.orgAC4 in shake flask cultureS.potato peelSorghum chaffcocoyam peelcassava peelyam peelS.potato peelsCocoyam peelSorghum chaffCMC
  • Journal of Biology, Agriculture and HealthcareISSN 2224-3208 (Paper) ISSN 2225Vol.3, No.5, 2013Figure 3: Effects of varying substrate concentrations on cellulase activities ofFigure 4: Enzyme activities of Aspergillus nigerfor other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperature40oC for cocoyam and 35oC for other carbon sources.0204060801001201401601802001 2 3 4cellulaseactivity(U/ml)Substrate concentration of the growth medium0501001502002503001 2 3CellulaseActivity(U/ml)Incubation Period (Days)Journal of Biology, Agriculture and Healthcare208 (Paper) ISSN 2225-093X (Online)71Figure 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 5.0for other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperatureC for other carbon sources.5 6 7 8 9Substrate concentration of the growth mediumcassava peelyam peelcocoyam peelSorghum chaffS.potato peelCMC3 4 5 6 7Incubation Period (Days)www.iiste.orgAC4 in shake flask culture.under optimal conditions of pH 4.0 for sweet potato peel and 5.0for other carbon sources; substrate concentrations of 3% for CMC and 4% for other carbon sources and temperaturecocoyam peelSorghum chaffS.potato peelcassava peelyam peelSorghum chaffcocoyam peelCMCS.potato peel
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