Short Communication

Received: 13 April 2013, Accepted: 23 April 2013

ISSN: 2321-2969

Int. J. Pharm. Biosci. Technol.

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International Journal of Pharma Bioscience and Technology, Volume 1, Issue 1, May 2013, Pg 50-53

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FERMENTATIVE PRODUCTION OF ETHANOL FUEL FROM DOMESTIC WASTE BY PICHIA STIPITIS

Padmavathi Modugu*
Department of Biotechnology, DVR & Dr. HS MIC College of Technology, Kanchikacherla, A.P. INDIA. Corresponding Author* E-mail address- padmavathi_bt@yahoo.com

ABSTRACT: Production of Ethanol fuel from the garbage/kitchen waste was carried out with the main purpose of converting the domestic waste into a useful material. The conversion of food waste or garbage by acid hydrolysis was carried out to obtain fermentable sugars, which was converted into ethanol by fermentation process using Pichia stipitis. The present study indicated that at 36 h of incubation resulted in utilization of 29 g/L of glucose with yield of 9.2 g/L ethanol. Compared to various sugars the glucose resulted in the production of ethanol. Key words: Ethanol fuel, Garbage, Pichia stipitis, Biomass, Fermentation; Conversion INTRODUCTION Ethanol or ethyl alcohol is a volatile, flammable, colorless liquid. A psychoactive drug and one of the oldest recreational drugs known, ethanol produces a state known as alcohol intoxication when consumed in excess. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a solvent, and as a fuel. In common usage, it is often referred to simply as alcohol or spirits. The production of ethanol has two routes synthetic and biological. The synthetic ethanol production is commonly carried out by a catalytic hydration of ethylene in vapor phase and often as a by product of certain industrial operations [1]. Ethanol is produced from fermentation of sugars extracted mostly from crops. Saccharomyces cerevisiae is the most popular organism used for ethanol production due to its high ethanol yield and high tolerance. Now a days crops are the main source used for ethanol production [2,3]. Current industrial processes for bio ethanol production uses sugarcane or cereal grain as feed stocks, but they have to compete directly with the food sector [4]. Although these are the predominant feed stocks that are used today, projected fuel demands indicate that new, alternative, low priced feed stocks are needed to reduce ethanol production costs . Since the cost of feedstock Modugu P contributes more than 58% to the production cost, inexpensive feed stocks such as lignocelluloses biomass and agricultural food wastes are being considered to make bio ethanol competitive in the open market [5]. Lignocellulose is the major structural component of woody plants and non woody plants such as grass and represents a major source of renewable organic matter in the soil [6]. Large amounts of lignocelluloses waste are generated through forestry and agricultural practices, paper-pulp industries, timber industries and many agro industries and they pose an environmental pollution problem. Unfortunately much of the lignocelluloses waste is often disposed of by biomass burning, which is not restricted to developing countries alone, but is considered a global phenomenon [7]. Thus enzymes have significant potential applications in various industries including chemicals, fuel, food, brewery and wine, textile, animal feed etc. Basically lignocelluloses biomass comprises of cellulose, hemicelluloses and lignin [8]. The structure of the cellulose is rigid and harsh treatment is required to break it down [9]. Hemi cellulose consists of short, linear and highly branched chains of sugars [10]. Ethanol production has been taken into estimation depending upon the ratio of hexoses and Pg. 50

Int. J. Pharm. Biosci. Technol. pentoses. Pichia stipitis is predominantly haploid, heterothallic yeast related to pentose metabolizing ascomycetes (Fig. 1). Strains of this species are one of the best xylose fermenting yeasts in type culture collections. Increasing the capacity of these species for rapid xylose fermentation could enhance the productivity and sustainability of agriculture and forestry by providing new outlets for agricultural and wool harvest residues. The solid substrate in the form of garbage/kitchen-domestic waste was washed with water for 2-3 times and then allowed for sun drying for one day and then kept in hot air oven at 50 C until it was completely dried and used for acid hydrolysis. Cellulose can be converted to fermentable sugars by concentrated sugars. Concentrated acid processes generally give higher sugar yield and consequently higher ethanol yield compared to dilute acid processes. Estimation of reducing sugars 5 g of NaOH were added to 400 ml of distilled water and by continuous stirring 100 g of sodium potassium tartarate was added. After that 250 mg of sodium sulphite, 5 g of DNS and 1 ml of phenol were added to the solution. Then it was filtered and kept under constant stirring in dark room to avoid the exposure of DNS to light. The reagent was kept in a dark and cold place as DNS is light sensitive. Reducing sugars standard preparation The stock solution of 1000 g/ml was prepared in distilled water. From this stock solution, dilutions were prepared ranging from 100 g/ml to 1000 g/ml. 1 ml of DNS reagent was added to 1 ml of each dilution in separate test tubes. The mixture was vortexed well and kept in boiling water bath for 10 min. After 10 min the mixture were cooled down and absorbance was read at 540 nm against the blank containing 1 ml of distilled water instead of glucose dilution. Estimation of Ethanol The amount of Ethanol was determined from the supernatant according to the method described by Caputi et al. [11] Potassium Dichromate solution 3.4 g of potassium dichromate was added to 6.6 N of sulfuric acid. 5 ml of potassium dichromate solution was added to 1 ml of cell free culture filtrate and mixed well. The reaction mixture was kept at 60 C for 20 min in the water bath. Then the O.D was read at 600 nm after cooling it and concentration of ethanol was determined by computing the O.D. against the standard curve which was prepared using concentration of 0.2% to 2.5%. RESULTS AND DISCUSSION Acid hydrolysis of fermentable sugars garbage to produce Pichia stipitis increases the ethanol production at 5% concentrated sulphuric acid hydrolysate at temperature of 20 C to 30 C, pH 5.5. The effect of Pg. 51

Fig. 1. Haploid cells of Pichia stipitis Agricultural productivity would be greatly improved and incomes for famers would increase if there were larger markets for agricultural residues. Unlike yeast which regulates fermentation by sensing the presence of fermentable sugars such as glucose, it induces fermentative activity in response to oxygen limitation. MATERIALS AND METHODS A loop full of cells were added to each 250 ml of flasks containing 150 ml of sterile culture media containing - glucose 50 g/L, yeast extract 4 g/L, peptone 5 g/L, KH2PO4 1.5 g/L and MgSO4 0.5 g/L, were incubated in a rotary shaker at 30 C and at 300 rpm for 24 h. Substrates used are garbage i.e. vegetable waste, fruit waste collected from the local markets. The vegetables and fruits used are potatoes, Pineapples, Brinjal, Grapes, Apples, Oranges and Bananas. The substrate collected was initially separated into solid and liquid by squeezing the substrate and resultant filtrate was subjected to auto hydrolysis and the solid waste were subjected to acid hydrolysis. Auto hydrolysis Liquid faction which contains sugars are placed in an amber bottle and left for 7-10 days and filtered. The filtrate was added to the production media for ethanol along with Pichia stipitis.

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Int. J. Pharm. Biosci. Technol. various carbon sources on the ethanol production using this Pichia stipitis was estimated by adding various carbon sources (Fig 2). The effect of various carbon sources on the ethanol production using this Pichia stipitis was estimated by adding various carbon sources such as cellulose, glucose, xylose, galactose, mannose and trihalose in the concentration of 20 g/L. After adding various carbon sources to the production all the flasks were kept on rotary shaker for fixed time interval after which ethanol yield was calculated. Amongst tested sugars, glucose and xylose produced maximum amount of ethanol yield followed by cellobiose, galactose, mannose and trehalose. There was almost no ethanol yield when trehalose was used as carbon source. concluded that glucose liberated by hydrolysis of garbage/domestic waste, produced maximum of alcohol after 36 h of incubation. REFERENCES 1. Lodgsdon JE, Ethanol. In: Howe-Grant MK, Raymond E, Othmer DF, Kroschwitz J, editors. Encyclopedia of chemical technology 9. 4th ed. New York: Wiley; 1991. p. 812-860 2. Reijnders L. Ethanol production from crop residues and soil organic carbon. Resour. Conserv. Recycl. 2008; 52(4): 653658. 3. Pakarinen A, Maijala P, Stoddard FL, Santanen A, Tuomainen P, Kymlinen M, et al. Evaluation of annual bioenergy crops in the boreal zone for biogas and ethanol production. Biomass Bioenergy. 2011; 35(7): 30713078. 4. Rosillo-Calle F, Hall DO. Brazilian alcohol: Food versus fuel? Biomass. 1987; 12(2):97128. 5. Sarkar N, Ghosh SK, Bannerjee S, Aikat K. Bioethanol production from agricultural wastes: An overview. Renew. Energy. 2012; 37(1): 1927. 6. Wang D, Xu YH, Zhaog, Fermentation kinetics of difference sugars by apple wine yeast Sacharomyce cere vistae, J. Institute of Brewing, 2004; 110: 340-346. 7. Bernthsen A, Bansal RK. A textbook of organic chemistry; 2003. 8. Kunkee RE, Amerine MA. Sugar and Alcohol Stabilization of Yeast in Sweet Wine. Appl Microbiol. 1998; 16 (7): 1067-1075. 9. Christian, Gary D. Analytical chemistry, Vol. 1; 2003.p. 81-89 10. Grio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-ukasik R. Hemicelluloses for fuel ethanol: A review. Spec. Issue Lignocellul. Bioethanol Curr. Status Perspect. 2010;101(13):4775800 11. Caputi JA, Ueda M, Brown T. Spectrophotometric determination of ethanol in wine. Am. J. Enol. Vitic. 1968; 19(3): 160165.

Fig. 2. Screening of various sugars (20 g/L) for production of ethanol CONCLUSION Pichia stipitis was the best microorganism which can produce ethanol in a larger amount compared to normal yeast. After 24 h incubation glucose concentration was found 68 mg/ml where as Ethanol production concentration was found to be 6.9 g/L. But after 36 h the sugar concentration was found to be 29 g/L where as Ethanol production was found to be 9.2 g/L. It was concluded that Ethanol production was more by utilizing more amount sugar with a specific time period. In the present study, bioconversion of vegetable and fruits waste into commercially useful products was investigated. The substrate garbage i.e. potatoes, Pineapples, Brinjal, Grapes, Apples, Oranges and Bananas was taken. There extracts were prepared and used for ethanol and biomass production. The various analytical tests were performed to estimate total sugar, fermentable sugar, residual sugars, protein, and content before and after fermentation. The consumption of glucose on alcohol production was studied. From this it was

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Int. J. Pharm. Biosci. Technol. ___________________________________________ How to cite this article APA style Modugu, P. (2013). Fermentative Production of Ethanol fuel from Domestic Waste by Pichia stipitis. International Journal of Pharma Bioscience and Technology, 1(1), 5053. Elsevier Harvard style Modugu, P., 2013. Fermentative Production of Ethanol fuel from Domestic Waste by Pichia stipitis. Int. J. Pharm. Biosci. Technol. 1, 5053. Vancouver Style Modugu P. Fermentative Production of Ethanol fuel from Domestic Waste by Pichia stipitis. Int. J. Pharm. Biosci. Technol. 2013;1(1):5053. To receive bibliographic information in RIS format (For Ref Manager, ProCite, EndNote, Zotero) Send request to: ijpbst@yahoo.com ___________________________________________

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