PRETREATMENT OF AGRICULTURE WASTE USING Aspergillus flavus FOR THE PRODUCTION OF ETHANOL BY Saccharomyceses cerviceae K. S.Thippareddy* and Pushpa Agrawal Department of Biotechnology, R.V.College of Engineering, Bangalore (INDIA) Received June 27, 2010 Accepted October 10, 2010 ABSTRACT` The agricultural crop like Maize is the most abundant cereal crop grown in India. It also produces the agro wastes like corncobs. It is used for the production of bio ethanol. Aspergillus flavus isolated locally was used to hydrolyze the corncobs into simple sugars (5%). Thus pretreated simple sugars are filtered and the filtrate was inoculated with Saccharomyces cerviceae for further at different ph and temperature. The rate of degradation effect of Aspergillus flavus and Saccharomyces cerviceae was enumerated using various parameters viz., temperature, ph, etc. The preliminary results using corn cob as biomass show the production of 3.2 % ethanol at ph 5.0 and temperature 30 o C. Key Words : Saccharomyces cerevisiae, Fermentation, Lignin, Aspergillus flavus,ethanol INTRODUCTION The burning fossil fuel, at the current rate, is likely to create an environmental crisis globally. Use of fossil fuel generates carbon dioxide, methane and a significant quantity of nitrous oxide. Most of these harmful gases are formed due to incomplete combustion of fossil fuel; since ethanol contains 35% oxygen that may result in a more complete combustion of fuel and thus reduces tailpipe emissions. Moreover, biomass energy can play an important role in reducing green house gas emissions. Ethanol production process only uses energy from renewable energy sources. Hence no net carbon dioxide * Author for correspondence is added to the atmosphere, making ethanol an environmentally beneficial energy source 1-2. Furthermore, fuel ethanol from lignocelluloses may also open new employment opportunities in rural areas, and thus make a positive socio-economic impact 3. Developing ethanol as fuel, beyond its current role as fuel oxygenates will require developing lignocellulosic biomass as a feedstock because of its abundant availability and low cost. The world ethanol production in 2004 was estimated to be 40 giga litres (GL) (Berg, 2004; Kim and Dale, 2004) 4,5. Brazil and US are the world leaders, which together accounted for about 60% of the world ethanol production exploiting sugarcane and corn respectively. In India, 393
lignocellulosic biomass (crop residues, forestry and fruit and vegetable wastes and weeds) is available in plenty. Renewable fuels, particularly ethanol, should get more and more attention all over the world. The important issue that we wish to address affirmatively here is that the bioethanol production, without doubt, needs an economical approach to address the global fuel needs. Research efforts are needed to design and improve the process, which would produce sustainable and economically feasible transportation fuel. Improvement in process economics using new designed cellulases-enzyme cocktail are important factors in establishing a cost effective technology, besides the low cost of feedstoc 6,7. For the long haul, it is very important to understand bioethanol production technologies in terms of their economic viability, environmental feasibility and empowering employment opportunities before implementing a fuel ethanol policy. The choice of the best technology for lignocellulose to bioethanol conversion should be decided on the basis of overall economics (lowest cost), environmental (lowest pollutants) and energy (higher efficiencies) that is, comprehensive process development and optimization are still required to make the process economically viable. In reality, environmental considerations, energy and tax policies will determine the extent of fuel ethanol utilization in the future and therefore the role of one and all is very crucial to identify the gravity of the situation associated with bioethanol production and use of it as an alternative fuel 3. Ethanol production currently relies on the of sucrose from cane sugar and molasses or glucose derived from starch-based crops such as corn, wheat and cassava. There is a growing need for the industry to improve technology and expand production due to high oil prices, environmental pressures and 394 national security concerns. Zymomonas mobilis was found to be an alternative organism for starch based ethanol production. As they have been shown to ferment under fully anaerobic conditions with faster specific rates of glucose uptake and ethanol production, as well as, ethanol yields close to theoretical 8. OBJECTIVES The objectives of this proposed research work was to produce ethanol from corncob and utilizing corncob as substrate. To optimize the fungal pretreatment technique for the conversion of lignocelluloses to reducing sugars using yeasts and then subject to. To use agricultural waste to produce clean fuel. The present investigation carried out involved hydrolyszation of the corn cob by the Aspergillus flavus and using Saccharomyces cerviceae for the production of ethanol. MATERIAL AND METHODS The sun dried Corncob Substrate was grinded into small pieces of 5mm size and dried in an oven at 60 o C. The ten grams grinded corn cob was taken for further process 9,10,13. The dried substrate was chemically treated with 5% sodium hydroxide solution and then washed using distilled water. 12,13 After washing, substrate was dried again in oven at 60 o C, then sterilized in autoclave at 15 psi for 20 minutes 11,12. Corncob was pre-treated with Fungus like Aspergillus flavus in aseptic condition at 30 o C for 7 days in Richard s Media. In this Richards media we are taken instead of sucrose as a carbon source as biomass taken for the degradation (saccharifiaction) of the substrate (corncob). The resultant simplified sugar was analyzed by DNS method 14. Further, for the production of ethanol, the sterilized substrate was dissolved in 200 ml of distilled
water 9. The ph range taken for the study was 4.0 to 7.5. The micro-organisms were cultured in respective media of 100ml for 48 hours incubation at 30 o C in a 250 ml flask 3. The micro-organisms used for this work are listed in the Table 1. Table 1 : Microorganism used for ethanol production No Micro-organism Type Media Concentration in 100 ml (mg) 1. Saccharomyces cerviceae 180 &181 Yeast extract Dextrose Peptone 0.3 2.0 1.0 2. Aspergillus flavus Richard s media Potassium nitrate (KNO 3 ) Potassium dihydrogen ortho phosphate (KH 2 PO 4 ) Magnesium sulphate (MgSO 4 ) Ferric chloride (FeCl 3 ) Sucrose 1.0 0.05 0.25 0.002 5.0 The cultured media was inoculated to the substrate and incubated at 30 o C and kept for 7 days under anaerobic conditions. After 7 days samples were analyzed for quantification of ethanol. Analytical method Ethanol concentration was measured using UV- visible-double beam spectrophotometer (Double Beam, Elico Company, model no. Elico-SL 164). Ethanol was analyzed by the dichromate oxidation method which is based on the complete oxidation of ethanol by dichromate in the presence of sulfuric method (Mjean Telli Okur et al 2008) 15,16. Sugars are determined calorimetrically using dintrosalicyclic acid reagent (Systronics Colorimeter: model No 115) 14. RESULTS AND DISCUSSION Shide EG, et al (2004) 17 has reported the degradation of wood saw dust by Lentinus squarrosulussiger a Basidiomycetes produced ethanol of 0.66%. Gurav and Geetha (2007) 18-19 also have reported the degradation of rice straw and baggase following micorbial pretreatment using Phnerocheate chrysosporium, further the was done using yeast and bacteria and obtained 0.820%. The fungal (Aspergillus awamori and Pleurotus sajorcaju) pretreatment studies on rice husk and bagasse for the (yeast) ethanol production and obtained the yield of 0.85% and 0.98% (Seema J Patel et al., 2007) 20. (Humpry.C.Nzeble et al., 2007). 395
Table 2 : Ethanol production of corncob using micro-organisms in different ph & treated in chemical, Saccharomyces cereviceae 180 Reducing sugar by Aspergillus flavus produced % Ethanol Saccharomyces cereviceae 180 % ph 4.0 2.086 ph 4.0 1.565 ph 4.5 2.638 ph 4.5 1.971 ph 5.0 3.312 ph 5.0 2.821 ph 5.5 3.153 ph 5.5 2.962 ph 6.0 3.041 ph 6.0 2.923 ph 6.5 3.302 ph 6.5 2.708 ph 7.0 2.948 ph 7.0 2.046 ph 7.5 2.569 ph 7.5 1.968 Fig. 1 : Reducing sugars obtained by chemically treated corn cob using Aspergillus flavus 396
Fig. 2 : Ethanol produced by Saccharomyces cereviceae 180 using chemical treated corn cob Table 3 : Ethanol production of corncob using micro-organisms in different ph & not treated in chemical, Saccharomyces cereviceae 180 Reducing sugar by Aspergillus flavus produced % Ethanol Saccharomyces cereviceae 180 % ph 4.0 1.301 ph 4.0 0.579 ph 4.5 1.559 ph 4.5 0.995 ph 5.0 1.855 ph 5.0 1.223 ph 5.5 1.807 ph 5.5 1.622 ph 6.0 1.776 ph 6.0 1.367 ph 6.5 1.559 ph 6.5 1.186 ph 7.0 1.357 ph 7.0 1.041 ph 7.5 1.441 ph 7.5 0.992 397
Fig. 3 : Reducing sugars obtained by chemically untreated corn cob using Aspergillus flavus Fig. 4 : Ethanol produced by Saccharomyces cereviceae 180 using chemical untreated corn cob 398
Table 4 : Ethanol production of corncob using micro-organisms in different ph & treated in chemical, Saccharomyces cereviceae Reducing sugar by Aspergillus flavus produced % Ethanol Saccharomyces cereviceae 180 % ph 4.0 2.086 ph 4.0 1.765 ph 4..5 2.638 ph 4..5 1.996 ph 5..0 3.312 ph 5..0 2.788 ph 5..5 3.153 ph 5..5 2.977 ph 6..0 3.041 ph 6..0 2.891 ph 6..5 3.302 ph 6..5 2.741 ph 7..0 2.948 ph 7..0 2.179 ph 7..5 2.567 ph 7..5 1.901 Fig. 5 : Reducing sugars obtained by chemically treated corn cob using Aspergillus flavus 399
Fig. 6 : Ethanol produced by Saccharomyces cereviceae 181 using chemical treated corn cob Table 5: Ethanol production of corncob using micro-organisms in different ph & not treated in chemicals, Saccharomyces cereviceae 181. Reducing sugar by Aspergillus flavus produced % Ethanol Saccharomyces cereviceae 180 % ph 4.0 1.301 ph 4.0 0.536 ph 4.5 1.559 ph 4.5 1.028 ph 5.0 1.855 ph 5.0 1.213 ph 5.5 1.807 ph 5.5 1.531 ph 6.0 1.776 ph 6.0 1.339 ph 6.5 1.559 ph 6.5 1.151 ph 7.0 1.357 ph 7.0 1.077 ph 7.5 1.441 ph 7.5 1.015 400
Fig. 7 : Reducing sugars obtained by chemically untreated Corn cob using Aspergillus flavus Fig. 8: Ethanol produced by Saccharomyces cereviceae 181 using chemically untreated corn cob. 401
The production of ethanol from bitter kola (Garcinia kola) pulp an agro waste, using Aspergillua niger and yeasts results are 1.29%. 21 The Aspergillus are grown in Richard s media and (saccharifiacation) instead of sucrose we are using corn cob as carbon sources for the Aspergillus. The fugues degrade the lignin, hemicellulose, cellulose, in the form of reducing sugars. These sugars are analyzed by DNS method. In this experiment, the reducing sugars were found less in the chemically untreated substrate (Fig. 4 and Fig. 8) and the chemical treated substrate produced more reducing sugars (Fig. 2 and Fig. 6). The produced reducing sugars are further subjected to by using yeast (Saccharomyces cerveceae 181,180.) for the production of ethanol. The ethanol was analyzed by Potassium dichromate method. It was possible to produce more ethanol from chemically treated substrate (Fig.3 and Fig.7) than chemical untreated (Fig.5 and Fig. 9). In this experiment, it was observed that chemically treated substrate gets degraded by the fungus, which leads to production of more percentage of ethanol. CONCLUSION In the present study, the agriculture waste, corn cob can be utilized to produce bioethanol. In the experiment, fungal and chemical treated substrate produces more yield of ethanol compared to untreated substrate. REFERENCES 1. Bull S. R., Riley C. J., Tyson K. S. and Costella R.,Total fuel cycle and emission analysis of biomass to ethanol. Energy Biomass wastes XVI (Klass DL ed.) Institute of gas technology, Chicago, I.L. 1-14, (1992). 2. Kheshgi H. S., Prince R. C., Marland G., The potential of biomass fuels in the 402 context of global climate change; Focus on transportation fuels, Annual Rev. Ene. Environ., 25(2), 199-244, (2000). 3. Anju kumar Chandel, Chan Es, Ravindervaram, M Lakshmi Narasu, L.Venkateswar Rao and Pogaku Ravindra., Economics and Enviromental impact of bioethanol production technologies: an appraisal, Biotechnol. Mol. Boil. Rev., 2(1) 014-032, (2007). 4. Berg C. World Ethanol Production. The Distillery and Bioethanol Network. Available at www.distill.com/ world ethanol production.htm, (2004). 5. Kim S., Dale E.B., Global potential bioethanol production from wasted crops and crop residues. Biomass Bioene. 26(1), 361 375. (2004). 6. Mojovic L., Nikoli S., Rakin M., Vukasinovi M., Production of bioethanol from corn meal hydrolyzates, Fuel, 85(1), 1750-1755, (2006). 7. Gray K.A., Zhao L. and Emptage M., Bioethanol. Curr Opin Chem Biol, 10(2), 141-146, (2006). 8. Linda D., Peter R., John P. and Paul P., Evaluation of Zymomonas based ethanol production from a hydrolysed waste starch stream, Biom. En., 30(2), 809-814, (2006). 9. Mujgan T. O., Nurdan E. S., Fermentation of sunflower Seed Hull hydrolysate Ethanol by Pichia stipitis., Biores. Tech., 99(1), 2162 2169, (2008). 10. Sanjeev K. S., Krishna L.K. and Gurvinder S. K., Fermentation of enzymatic Hydrolysate of sunflower hulls for ethanol production and its scale up, Biom. Bioene., 27(2), 399 402, (2004). 11. Koutinas A. A., Vianoulis P., Gravalos K. and Koliopoulos K., A processing
scheme for industrial ethanol production from straw, Ene. Conc. Mgmt., 12(2), 131-135, (1981). 12. Ojjumu T. V., Solomon B., Obge B., Eriola L., Stephen K., and Amigun B., Cellulase production by Aspergillus Flavus linn isolate NSPR 101 fermented in saw dust, bagasse and corncob., Afr. J. biotech., 2(6), 150-152, (2003). 13. Sharma S. K., Krishna L. K. and Harmeet S. G., Fermentation of Enzymatically saccharified sunflower stalks for ethanol production and its scale up, Biores. tech., 85(1), 31-33, (2002). 14. Miller G. L., Use of DNS reagent for the measurement of reducing sugar, Anal. Chem., 31(2), 426-428, (1959). 15. Caputi A. Jr., Ueda M. and Brown T., Spectrophotometric determination of ethanol in wine., Am. J. Enol. Vitic. 19(1), 160-165, (1968). 16. Mujgan T. O. and Nurdan E. S., Fermentation of sunflower Seed Hull hydrolysate Ethanol by Pichia stipitis., Biores. tech., 99(2), 2162 2169, (2008). 17. Shide E. G., Wuyep P. A. and Nok A. J., studies on the degradation of wood sawdust by Lentinus squarrosulus (Mont.) Singer., Afri J. Biotech., 3 (8), 395-398, (2004). 18. Aguilar F. N. and Smith K., An ecofriendly approach to remove arsenic from agricultural waste, J. Environ. Res. Develop., 1(4), 331-341, (2007). 19. Manjunath S. Gurav and G.S.Geeta., Effectiveness of fungal treatment of agro residues on ethanol production by yeasts and Zymomonas mobilis, Karnataka J. Agr. sc., 20(2), 301-304, (2007). 20. Seema J. P., Onkarappa R. and Shobha K. S., Fungal treatment Studies on Rice husk and Bagasse for ethanol production., Elec. J. Environ. Agri. Food Chem., 6(4)., 1921-1926.,(2007). 21. Humprey. C. Nzelibe and Caritas U. O., Optimization of ethanol production from Garcinia kola (bitter kola) pulp agro waste., Afr. J. Biotech., 6(17), 2033-2037, (2007). Re USE Re DUCE Re CYCLE 403