Research Journal of Agriculture and Forestry Sciences __________________________________ ISSN 2320-6063 Vol. 1(7), 18-20, August (2013) Res. J. Agriculture and Forestry Sci. International Science Congress Association        
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Short Communication Optimization and Production of Cellulase from Agricultural WasteSharma Harendra K.1*, Burnwal Pappu Kumar, Dubey Laxmi and Rao R.J.1 Indira Gandhi Academy of Environmental Education, Research and Ecoplanning, Jiwaji University, Gwalior-474011, MP, INDIA Department of Chemical Engineering, Institute of Engineering, Jiwaji University, Gwalior-474011, INDIADepartment of Botany, Government SMS Science College, Gwalior, MP, INDIA Available online at: 
www.isca.in
Received 22nd July 2013, revised 31st July 2013, accepted 6th August 2013Abstract This work represents the optimization of agricultural waste the production of cellulase using filamentous fungi Myrothecium verrucaria has been adopted. Solid sate fermentation a potential technology for production of cellulase utilizing agricultural residues. All experiments were carried out in triplicate and mean values are reported. The appropriate incubation period is one of the important parameters for cellulase production in solid state fermentation and is governed by microorganism. The result shows that optimum incubation period is important factor for production of cellulase from agriculturalwaste. The maximum cellulase activity has been recorded after 3 days of fermentation. Keywords: Cellulase, fungus, fermentation, incubation period. Introduction Cellulase is a hydrolase enzyme which is highly applicable enzyme in textile industry, laundry detergents, pulp and paper industry for various purposes, and even used for pharmaceutical applications. The production of cellulase has been widely studied  in  submerged  fermentation,  but  the  high  cost  of enzyme  production  is typical in  the  industrial  application of cellulose bio-conversion. Cellulase is an important enzyme for conversion of lignocellulosic biomass to bio-ethanol. Solid state fermentation is a potential technology for production of cellulases utilizing agro-industrial residues as solid  substrate due to low capital investment, low  energy  requirement, eco-friendly  operation, and higher  yield compared to submerged fermentation and lower  chance  of  contamination  due  to  low  moisture  level Cellulolytic enzymes were separated by microbial degradation of banana waste under solid state bioprocessing using two lignocellulolytic fungiPothyraj et alhave enhanced production of cellulase from cassava waste from various fungal cultures. Large numbers of microorganism are capable for producing of celulase but few of them produces significant quantities of enzymes. Many microorganisms have the capacity to degrade these cellulosic wastes. Myrothecium verrucaria is a plant pathogen and it is common throughout the world, often found on materials such as paper, textiles, canvas and cotton. Literature reveals that there is less work done on cellulase production from solid state fermentation of agricultural waste usingM. verrucaria. Present research is based on   production of cellulase using M. verrucaria from solid state fermentation of various agro waste. In present investigation filamentous fungi like Myrothecium verrucaria have been used for production of cellulase from solid agricultural waste. Present manuscript records optimization and production of cellulase from agricultural waste. Present manuscript records production of cellulase from agricultural waste and optimization of process parameters like temperature, incubation period, hydration, pH and weight of substrate. Material and Methods Spores of M.  verrucaria   have been cultivated  on  2%  potato  dextrose  agar  (PDA)  and  incubated  at 30°C  for  7  days.  The isolated form of white colonies containing sporodochia with a flattened or convex spore mass and one celled conidia on cylindrical, bundled phialides. Solid substrate used: Agricultural solid waste is most cellulose abundant in nature. The agro solid waste like sugarcane bagasse, cotton seeds, cabbage, cauliflower, wheat bran and dry coconut leaves are obtained from local market of Gwalior city in month of April for the present research work of cellulose production by solid state fermentation process using micro organism M. verrucaria. Fermentation process and fungal spore suspensions were made using sterile water and were added to the sterile solid substrates. Flasks were incubated for 5 days in the B.O.D incubator at 30C. Extraction process: Cellulose extracted by adding 50mL of double distilled water to fermentation broth and kept it for overnight at 4C in incubator. After incubation contents were filtered through cheese clothes and centrifuged at 10,000 rpm for 30minutes. The cellulose contents supernatant was kept at C before used. Analysis method: Filter paper activity for total cellulase present in the culture supernatant was determined according to the method recommended by Ghose.  Appropriately  diluted  culture  supernatant was added to 4 ml sodium acetate buffer (pH 4) containing 100 mg of  Whatman  No.1  filter  paper  
Research Journal of Agriculture and Forestry Sciences _______________________________________________ ISSN 2320-6063Vol. 1(7), 18-20, August (2013)                     Res. J. Agriculture and Forestry Sci. International Science Congress Association 
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(Sigma–Aldrich,  St.  Louis, MO) strips.  After  incubation  for  1  h  the  reducing  sugar  released  was estimated by the dinitrosalicylic  method. 1 unit (U) of filter paper activity was defined as the amount of enzyme releasing 1 mol of reducing sugar from the filter paper/min/g dry solid. Results and Discussion Cellulase production: M. verrucaria was initially grown on the surface of PDA slants at 30°C for 7 days. The fermentation process was carried out in 250- ml conical flasks. Separate flask was used for all six substrates. Each flask was filled with 5 g of solid substrate followed by the addition of 3 ml of water.  Then the flasks were plugged with cotton and autoclaved at 121°C for 15 min at 15 psi. Under aseptic conditions fungal spores were transferred from culture slants to the solid substrate and mixed thoroughly.  Then the flasks were incubated in incubator maintained at 30°C. The extra- cellular  enzyme  was  extracted  by  soaking  the  fermented  solid material with 50 ml of sterile water overnight at 4°C and filtering through  muslin  cloth. All six filtrates were centrifuged at 10,000 rpm for 30 min and temperature has been maintained 4°C. The supernatant was used to measure the amount of cellulase produced.Optimization of incubation periodinfluencing cellulase yield. An experiment with different incubation periods was executed in 250ml conical flasks at 30°C. All experiments were carried out in triplicate and the mean values are reported. Assay of cellulase activity: Filter paper activity (FPA) for total cellulase activity in the cultural filtrate was determined according to the method of Mandels et al. Appropriately diluted culture filtrate as enzyme source was added to Whatman No.1 filter paper strip (1 X 6 cm; 50mg) immersed in one milliliter of 0.05 M sodium citrate buffer of pH 4.0. After incubation at 50°C for 1 hour, the reducing sugar released was estimated by dinitrosalicylic acid (DNS) method. Statistical analysis: Optimization of incubation period on sugarcane bagasse and wheat bran as a substrate showing maximum activity after 3 days. Activity decreases if kept for long time. Figure-1 and figure-2 showing activity of different day period and agricultural substrates. Maximum crude cellulase activity was recorded after 3 days of fermentation.
Figure-1  Activity on different days 
Figure-2 Screening of agricultural substrates
 
Research Journal of Agriculture and Forestry Sciences _______________________________________________ ISSN 2320-6063Vol. 1(7), 18-20, August (2013)                     Res. J. Agriculture and Forestry Sci. International Science Congress Association 
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Enzyme production depends on various factors. The proper incubation period is most favorable parameter of solid state fermentation for producing cellulase. The result shows optimum incubation period for production of cellulose. The maximum cellulose activity of wheat bran after 3 days of fermentation was 0.23 U/g whereas for sugar bagasse it was 0.229 U/g. Conclusion The present work concluded that the incubation period is most important factor for growth of microorganism as well as the level of production of cellulose enzymes. The plant pathogen M. verrucaria  is capable to producing significant yield of cellulase from wheat bran and  sugar bagasse containing cellulose. Cellulase activity was increased when the incubation period increased to 3 days. AcknowledgmentAuthors are thankful to Indira Gandhi Academy of Environmental Education, Research and Ecoplanning (IGAEERE), Jiwaji University, Gwalior, (M.P.) India for providing experimental facilities during the work. References 1.Varga E., Scengyel Z. and K. Recaey, Chemical pretreatments of corn stover for enhancing enzymatic digestibility, Appl. Biochem. Biotechnol.,98–100(1-9),  73–87 (2002)2.Pandey A., Solidstate fermentation, Biochemical Engineering Journal,13(2), 8184 (2003)3.Maulin P. Shah, Reddy G.V.,
 
Banerjee R., Ravindra Babu 
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Biotechnology,5 (20), (1882-1885) 5.Kumara Suman, Shahana J. and Khan, Optimization of medium for cellulase production by Aspergillus niger using wheat bran, Bionano Frontier,5(2), 210-12 (2012)6.Murakami M., Kaneko Y. and Kadokawa J., Preparation of cellulose-polymerized ionic liquid composite by in-situ polymerization of polymerizable ionic liquid in cellulose-dissolving solution, Carbohydrate Polymers, 69(2), 378–381 (2007)7.Ghose T.K., Measurement of cellulose activities, Pure & Appl.Chem., 59, 257-268 (1987)8.Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugars,  Analytical Chemistry,31, 426–8 (1959)9.Mandels M., Andreotti R. and Roche C., Measurment of saccharifying cellulose, Biotechnol Bioeng Symp., 6, 21-33 (1976)