Rice Straws and Husks Biofuel: Emphasizing on Selection of Pre-Treatment Method Elza Firdiani Shofia, Kharisma Bangsa Senior High School, Indonesia

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1 Rice Straws and Husks Biofuel: Emphasizing on Selection of Pre-Treatment Method Elza Firdiani Shofia, Kharisma Bangsa Senior High School, Indonesia Picture: Indonesian farmers are harvesting rice. There are many rice straw left. Encarta Encyclopedia.

2 Introduction Indonesia is a great agrarian country which has planted so many plants, especially grains, for food. Rice, or Oryza sativa, is commonly planted. In harvest months, the grains are harvested for food, but the straws are left. The grains are hulled but the husks left. Straws and husks are burned in open air and caused air pollution, as what happened in my hometown. It is better to use these straws and husks as raw materials for second-generation biofuel. Second-generation biofuel is derived from lignocellulosic crops, especially the inedible one, in example wastes from crops. Lignocelluloses are composed mainly of cellulose, hemicellulose, and lignin. It means, the raw material of second-generation biofuel should contain high level of these. Straws and husks, as parts of lignocellulosic crops waste, are rich in celluloses, hemicelluloses, and lignin, with total of 69.0% mass (33.4%, 28.2%, and 7.4%, respectively (He, Pang, Liu, Li, & Wang, 2008)). This project plan focuses on second-generation of advanced biofuel that made from rice straws and husks, emphasizing on the selection of pre-treatment method, because pre-treatment method seems affecting the effectiveness of the process of making biofuel, such as: the obtained amount of bioethanol (in which this project is focusing), time needed, cost, and simpler steps. Main Basis Biofuel is produced by fermenting plant-derived sugars, commonly celluloses, to ethanol (later mentioned bioethanol, due to its ingredient). Celluloses, hemicelluloses, and lignin are plant-derived sugars that combine into lignocelluloses. It is known that straws and husks are rich in it. Basically, there are two stages of producing bioethanol. 1. A pre-treatment phase (including hydrolysis), hydrolyzing lignocellulosic material into sugars. The sugar solution later separated from its residual materials, including excess lignin. 2. Microbial fermentation of the sugar solution using yeast to make bioethanol. Bioethanol later distilled to obtain 95% pure ethanol. Further dehydration will bring the ethanol concentration into almost absolute concentration (up to 99.5%) Cellulose and hemicellulose are different in structure, where cellulose only consists of anhydrous 1 4 glucose and hemicellulose consists of various sugars but both of them contain anhydrous glucose. Cellulose is stronger than hemicellulose but breaking intermolecular hydrogen bonds in cellulose is easier. Lignin has special structure, it has aromatic hydrocarbon chain and stronger than cellulose and hemicellulose. Lignocelluloses are derived from lignin, cellulose, and hemicellulose, but due to its complex bond, it is not easy for yeast to ferment it. They are not simply deposited in the cell wall because they are bonded by certain chemical bonds. The bonds form complex structure called lignin-carbohydrate complexes (LCC), which prevents effective fermentation. Ineffective fermentation decreases amount of produced bioethanol. Pre-treatment helps to hydrolyze linkages between sugars, which resulted in higher amount of exposed sugar to ferment. Higher amount of bioethanol will be produced later. That is why effective pre-treatment is needed. The more effective pre-treatment the more bioethanol will be produced. There are two methods of pre-treatment that are going to be compared in this project about its effectiveness in hydrolyzing LCC in rice straws and husks: adding either NaOH (alkali pre-treatment) or enzymes. They are capable of degrading cellulose, hemicellulose, and lignin, but hypothetically they are different in effectiveness. NaOH is capable of breaking some intermolecular hydrogen bonds through the breakage of 1 4-glycosidic linkages and intramolecular hydrogen bonds. This causes the degradation of cellulose and hemicellulose and makes them easier to be fermented by yeast (Saccharomyces cerevisiae). NaOH is also capable of degrading lignin. It breaks special bonds in lignin, such as deformation of aromatic ring and its linkage. Lignin is also degraded by ligninase. Cellulose and hemicellulose are degraded by cellulases that are able to hydrolyze the cellulose polymer to its monomer, the sugar glucose, which is directly fermented to ethanol by yeast. After pre-treatment, there will be a main treatment: fermentation of sugar solution by yeast, Saccharomyces cerevisiae. Yeast uses sugar as its energy source, producing ethanol as waste from its anaerobic metabolism. Ethanol later obtained by distillation, maximal concentration of 95% will be obtained. Further dehydration will bring the concentration up to 99.5% (almost absolute concentration).

3 Objective Making rice straws and husks biofuel effectively and comparing the effectiveness of two methods of pretreatment. Research Questions Method 1. Does the effectiveness of pre-treatment affect the effectiveness of making biofuel? 2. Which pre-treatment method is more effective, adding NaOH (alkali pre-treatment) or enzyme? 3. Can higher amount of ethanol be obtained if the more effective pre-treatment method is used? Section 1: Pretreatment For all chemical pre-treatment, first treat the biomass physically: cut the rice straw and husks into small pieces, the smaller the better. a. NaOH Place physically-treated straws and husks, and then add 6 g of NaOH per 100 g of straws and husks then 80 g of distilled water per 100 g of straws and husks to make the moisture content at 80% on a dry basis. This moisture content was determined by previous tests conducted in laboratory, (He, Pang, Liu, Li, & Wang, 2008) which was able to maintain them in the saturated state. Close the beaker and let it at 20 (± 2 C) for 3 weeks. Dry the pretreated rice straws and husks in oven. b. Enzymes Cellulases are produced by fungi Aspergillus niger and Trichoderma viride (isolated from soil) (Patel, Onkarappa, & Shoba, 2007). Ligninases are produced by bacteria Enterococcus casseliflavus and Enterococcus gallinarum (isolated from ruminant s colon) (Wahyudi, Cahyanto, Soejono, & Bachruddin, 2010). Incubate cellulases-producing fungi in separated potato dextrose broth at room temperature for seven days; they will produce cellulases to digest nutrition in broth during incubation. Grow ligninase-producing bacteria in solid media by Hungate method : Mix these material in an Erlenmeyer: 0.03g of KH 2 PO 4, 0.01g of MgSO 4, 0.01g of CaCl 2, 0.10g of NaCl, 0,10g of (NH 4 ) 2 SO 2, 0.10ml of Resazurin 0,1% solution, 0.02g of Cystein-HCl H2O, 0.40g of Na 2 CO 3, ml of rumen liquid, and 70.00ml aquadest. 1.00g substrate (Lignin, xylan or cellulose) added later after substrate sterilization by mixing it with 5 ml aquadest in a tube. Keep at ph 6,8 and heat until all ingredients dissolved. Transfer the flask aseptically in oxygen-free condition (CO 2 gas displacing all air) until red color faded, close the flask with rubber stopper, seal, then sterilize with its content in 12 psi for 20 minutes. (Wahyudi, Cahyanto, Soejono, & Bachruddin, 2010) Inoculate the bacteria there after sterilization. They will produce ligninase during incubation. After incubation, filter and centrifuge each fermented media. Enzymes are in the supernatant so take that out. Boil physically-treated straws and husks in distilled water with 10% biomass per gram of water and keep for sterilization. After sterilization, add 5% enzymes per gram of water for hydrolysis. Keep for 3 hours at 37 C. Section 2: Fermentation, Distillation, and Feasibility Ferment each pre-treated straws and husks by using Saccharomyces sp. for seven days of incubation at room temperature. Collect ethanol by distilling the fermented rice straws and husks at 80 C (boiling point of ethanol). Ethanol feasibility is checked by using specific gravity method. Pycnometer can be used for determining bioethanol specific gravity. Ethanol has a specific gravity of Ethanol concentration will be determined using spectrophotometer, so it will be known which pre-treated biofuel will result in higher concentration of ethanol. About this Plan I am confident about this plan feasibility: this project will satisfy the needed result and answer the research questions. However, this project plan is a short form of a longer idea that requires more space to write. Further studies need to be conducted to prove the feasibility of this plan and developing another factor of making biofuel effectiveness.

4 Bibliography Cellulase. (2011, February 17). Retrieved February 20, 2011, from Wikipedia: Cellulose. (2011, February 17). Retrieved February 20, 2011, from Wikipedia: Cellulosic ethanol. (2011, February 17). Retrieved February 20, 2011, from Wikipedia: Ethanol fuel. (2011, February 12). Retrieved February 20, 2011, from Wikipedia: He, Y., Pang, Y., Liu, Y., Li, X., & Wang, K. (2008). Physicochemical Characterization of Rice Straw Pretreated with Sodium Hydroxide in the Solid State for Enhancing Biogas Production. Energy and Fuels, unknown pages. Hemicellulose. (2011, January 12). Retrieved February 20, 2011, from Wikipedia: Hynes, E. (2008). Rice. Microsoft Encarta 2009 [DVD]. Redmond, WA, United States of America. Lignin. (2011, February 21). Retrieved February 21, 2011, from Wikipedia: Ligninase. (2010, December 19). Retrieved February 21, 2011, from Wikipedia: Patel, S. J., Onkarappa, R., & Shoba, K. S. (2007). Fungal Pretreatment Studies on Rice Husks and Bagasse. Electronic Journal of Environmental, Agricultural, and Food Chemistry, 6 (4), Prasad, M. P., Tamilarasan, R. S., & Subha, K. S. (2009, November). Production Of Bioethanol Using Various Agricultural Raw Materials By Two Step Enzymatic Process. Advanced Biotech, Relative density. (2011, February 12). Retrieved February 20, 2011, from Wikipedia: Saccharomyces cerevisiae. (2011, February 16). Retrieved February 20, 2011, from Wikipedia: Van Loon, L. R., & Glaus, M. A. (1998). Experimental and Theoretical Studies on Alkaline Degradation of Cellulose and its Impact on the Sorption of Radionuclides. Wettingen: National Cooperative for the Disposal of Radioactive Waste. Vasudevan, P., Sharma, S., & Kumar, A. (2005). Liquid fuel from biomass : An Overview. Journal of Scientific and Industrial Research, 64, Wahyudi, A., Cahyanto, M. N., Soejono, M., & Bachruddin, Z. (2010). Potency of Lignocellulose Degrading Bacteria Isolated from Buffalo and Horse Gastrointestinal Tract and Elephant Dung. Journal of Indonesian Tropical Animal Agricultural, 35 (1), Elza Firdiani Shofia Kharisma Bangsa Senior High School Jakarta, INDONESIA cha_xiaojie[at]mail.ru

5 Appendix: Pictures Picture 2: Chemical structure of Cellulose (Van Loon & Glaus, 1998) Picture 1: Harvesting Rice Encarta Encyclopedia Picture 3: Chemical structure of xylan, a hemicellulose (Van Loon & Glaus, 1998) Picture 4: Chemical structure of lignin Big picture: (Lignin: Wikipedia, 2011) Linkages: (He, Pang, Liu, Li, & Wang, 2008)

6 Picture5: Molecular hydrolysis of cellulose using NaOH. Similar process for hemicellulose. (Van Loon & Glaus, 1998) Picture 6: Molecular hydrolysis of cellulose using cellulase (Wikipedia: Cellulase, 2011) Picture 8: Fermentation of glucose ( obk/alcferm.gif) Picture 7: Molecular hydrolysis of lignin using NaOH (He, Pang, Liu, Li, & Wang, 2008)

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