DIRECT BIOCONVERSION OF AGRICULTURAL WASTE- RICE STRAW IN TO BIOETHANOL

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1 DIRECT BIOCONVERSION OF AGRICULTURAL WASTE- RICE STRAW IN TO BIOETHANOL Md. Zahangir Alam*, Nassereldeen A. Kabbashi and M. Hafiz Ismail Bioenvironmental Engineering Research Unit (BERU), Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia, (IIUM) Jalan Gombak, Kuala Lumpur (MALAYSIA) Received September 6, 2007 Accepted December 15, 2007 ABSTRACT Bioethanol production was carried out by solid state bioconversion method utilizing lignocellulosic material, rice straw (RS) as substrate with the aid of mixed cultures of Saccharomyces cerevisiae, T. harzianum and P. chrysosporium. The mixed culture of the microorganisms was established to investigate their compatibility for direct bioconversion of rice straw into bioethanol. The results indicated that the yeast, Saccharomyces cerevisiae and fungal strains T. harzianum, and P. chrysosporium were found to be the most suitable strains for the ethanol production using rice straw as major substrate. From the observation and estimation of ethanol yield, T. harzianum and S. cerevisiae are mutually grows together in liquid and solid media and proved to be excellent in producing ethanol compared to other combinations. The highest production of bioethanol found as 10.1% on the sixth day where the highest reading was recorded for the combination of P. chrysosporium, T. harzianum and S. cerevisiae. The other parameters glucosamine content as growth indicator, reducing sugar released and the ph were measured to evaluate the bioconversion process. Key Words : Bioethanol, Direct bioconversion, Compatible mixed culture, T. harzianum, S. cerevisiae, Agricultural waste During the past decades, global warming from the increased amount of greenhouse gases, mainly carbon dioxide, has become a major political and scientific issue. The main cause of global warming is believed to be the carbon dioxide formed by burning fossil fuels 17. By using biofuels, the net emission of carbon dioxide to the atmosphere can be reduced. Ethanol, a biofuel, which can be produced from various cellulosic materials, has been proposed as an alternative fuel. It can be manufactured from numerous natural materials containing *Author for correspondence INTRODUCTION 118 cellulose or starch. Ethanol can be obtained from a variety of renewable sources, such as biomass (i.e. sugarcane and corn), waste paper and domestic refuse 4. The major producers of ethanol are Brazil and US, which account for about 62% of world production 9. The major feedstock for ethanol in Brazil is sugar cane, while corn grain is the main feedstock for ethanol in the US 9. Another potential resource for ethanol is lignocellulosic biomass, which includes materials such as agricultural residues (e.g., corn stover, crop straw, sugar cane

2 bagasse), herbaceous crops (e.g., alfalfa, switchgrass), forestry wastes, waste paper, and other wastes 21. The utilization of lignocellulosic biomass for fuel ethanol is still under development 9. In recent years, much attention has been given to the utilization of fruit and vegetable processing industry waste into ethanol, and other useful chemicals 12. A wide range of raw material has been recognized to be a potential substrate for bioethanol production. Bioethanol have been produced from a various lignocellulosic materials such as wood, agricultural and forest wastes 7. Rice straw, wheat straw and corn stover are the most favorable bioethanol feed stocks in Asia 9. Most industrial processes are pertaining to find various ways to reduce the cost of production. The efficiency of ethanol production from biomass has steadily increased 20 however, the raw material accounts for 40-70% of the total ethanol production cost 7. Thus, to reduce the cost of production, the supply of cheap, renewable source is a necessity. Moreover, a fermentation substrate must be readily available throughout most of the year. In this study, bioethanol was produced using new type of substrate, rice straw, with the aid of Saccharomyces cerevisiae, Phanerochaete chrysosporium and Trichoderma harzianum. The objectives of this research are to study the compatibility of mixed culture of the stated microorganisms and to develop the direct solid-state bioconversion process for production of ethanol using rice straw as substrate by applying mixed culture of S. cerevisae, P. chrysosporium and T. harzianum. MATERIAL AND MEHTODS Fungal Strain Culture of Phanerochaete chrysosporium, Saccharomyces cerevisiae, Trichoderma harzianum and other lignocellulosic fungi were obtained from the laboratory stocks. All fungi were separately maintained on Potato Dextrose Agar (PDA) plates for spore production and incubated at 37 o C (P. chrysosporium), 30 o C (Saccaharomyces cerevisiae) and 32 o C (Trichoderma harzianum) until the entire plate was discovered by fungus. Mineral solution Mineral solutions were made with the following quantities of nutrient salts (NS) per liter: 2.0g NH 4 H 2 PO 4 : 0.86g urea: 0.6g KH 2 PO 4 : 0.4g K 2 HPO 4 : 0.5g MgSO 4.7H 2 O: 74.0mg CaCl 2.2H 2 O: 12.0mg ferric acid citrate: 6.6mg ZnSO 4.7H 2 O: 5.0mg MnSO 4 : 1.0 mg CoCl 2.6H 2 O: 1.0 mg CuSO 4.5H 2 O: and 0.1 mg thiamine hydrochloride (Eriksson et. al, 1980). Co-substrate Wheat flour was used as the cosubstrate and additional carbon source. The previous study reported it enhances the enzyme production 10. Preparation of inoculum and culture media Most of the fungal strains used were prepared in similar way except for S. cerevisiae (yeast). All fungal strains were cultured on 3.9% potato dextrose agar (PDA) as inocula sources 1. The inoculum of all fungal strains except yeast were prepared by washing the growing culture in PDA plate with sterilized distilled water and their spores were rubbed before filtering using Whatman no.1 filter paper. Yeast inoculum was prepared by putting 2 loopful of cells in Yeast Extract-Malt Extract (YM) media (2% Malt Extract and 1% Yeast Extract) and incubated for 24 hours in 25 C. Compatibility test The compatibility test was done as to determine which combination of fungal strain will be cooperated in producing ethanol. The set of combination were designed based of their functionality of degrading lignocellulose and producing ethanol. The combinations were listed as i) P. chrysosporium (PC) and S. cerevisiae (SC), ii) T. harzianum (TH) and S. cerevisiae (SC), iii) M. hiemalis (MH) and S. cerevisiae (SC), iv) A. nigers 103 (AN) and S. cerevisiae (SC). The evaluation of fungal possible 119

3 interaction was studied in liquid media (2% Malt Extract) for growing the fungal and solid media (PDA) for evaluation and observation. 1% of fungal (PC, TH, MH or AN 103) and 1% of yeast were added into flask of 50 ml of media for 2 replicate each combination and incubate at 150 rpm for 2 days. Then, each combination was sub-cultured by striking the plate and incubated for 6 days at 30 ± 2 C. Treatment of substrate Treatment of the substrate (RS) was based on the method suggested by Ghosh and Deb, The RS was thoroughly washed to make them dust free and then dried. After that, these were grinded into smaller particles in the range of µm in size. Solid state fermentation Treatment was carried out with 100 ml Erlenmeyer flask. Total weight of fermentation medium was 20g. Meanwhile, the total amount of moisture to be maintained during the solid state fermentation was 70% (v/w) 13 including 59% water, 5% mineral solution and 6% inoculum.100 ml Erlenmeyer flask was filled with 28% w/w (5.6g) RS followed by 2% w/w (0.4g) wheat flour. 59% v/w (11.8 ml) distilled water and 5% v/w (1ml) mineral solution were added. The medium was mixed and the cottonplugged flasks were autoclaved at 121 o C for minutes. After the fermentation medium was cooled down to room temperature, 6% v/ w (1.2ml) inoculum of Phanerochaete chrysosporium and Saccharomyces cerevisiae and Trichoderma harzianum were separately and evenly poured on the medium. The cotton-plugged flasks were incubated at 30 ± 1 ºC (Srivinas, 1994) for 8 days. The control also was handled in the same way but without inoculum. Direct bioconversion of rice straw for ethanol production by compatible mixed culture An experiment was carried out in 250 ml Erlenmeyer flasks for the development of the direct bioconversion process with compatible mixed culture. Four runs were designed to evaluate the process with potential mixed culture of fungi and yeast. Three types of fungi were used; T. harzianum, P. chrysosporium and M. hiemalis while the yeast used was S. cerevisiae. Each sample (20g) for every runs consisted of 28% of substrate (rice straw), 2% of co-substrate (wheat flour), 59% of sterile distilled water, 5% of minerals solution and 6% of inoculum which equally distributed depending how many types of microbes used 1. The experim ental design is shown in Table 1. Table 1 : Development of direct bioconversion with different fungi and yeast and incubation time Run Combination Incubation time Sampling time 1 T. harzanium and S. Both fungal at the same cerevisiae (TH-SC) time 2, 4, 6, 7, 8 2 TH was inoculated at the T. harzanium and S. beginning and SC on cerevisiae (TH-SC) the 5 th day of run 2, 4, 6, 7, P. chrysosporium, T. harzanium and S. cerevisiae (PC-TH-SC) P. chrysosporium and S. cerevisiae (MH-SC) PC was inoculated at the beginning, TH on the 4 th day and SC on the 6 th day of run MH was inoculated at the beginning and SC on the 5 th day of run 2, 4, 6, 7, 8 2, 4, 6, 7, 8

4 Extraction method The samples were taken out from incubator after 2, 4, 6, 7 and 8 days. The fermented substrate was extracted with 30 ml distilled water by shaking the mixture at 150 rpm for 2 hours at room temperature and the liquid portion was filtered and collected. The liquid portion was used for the analysis such as bioethanol determination, reducing sugar estimation and glucosamine determination and ph. RESULTS AND DISCUSSION Compatibility Test of Fungal Mixed Culture and Yeast For the liquid mixed culture, each combination of mixed cultures has shown the growth by changing the color and turbid. Only the PC-SC combination showed that S. cerevisiae (yeast) grown in the media and inhibited the growth of P. chrysosporium which usually grows in pellet-shaped in the shake flask (data not shown). Fig. 1 : Compatible mixed cultures showed in PDA plate cultures After 6 days, each combination of mixed cultures was cultured in the PDA plates for six days to observe the compatibility effect among them. The visual observation of compatibility test is shown in Fig. 1. Out of the four combinations, three shown up to be Ethanol (% v/v) TH-SC MH-SC PC-SC AN-SC Fungal mixed culture combination Fig. 2 : Production of ethanol (% v/v) with different fungal mixed culture and yeast 121

5 compatible with each other; which were TH- SC, MH-SC and AN-SC combinations. For PC- SC combination, P. chrysosporium did not grow with yeast in liquid media as well as the test in PDA plate also had shown the similar result. The content of ethanol after 6 days of fermentation is shown in Fig. 2. The results indicated that the highest ethanol production (14.2%, v/v) was observed by TH-SC combination whereas the lowest was 6.4 % (v/v) in AN-SC combination. From the observation and estimation of ethanol yield, T. harzanium and S. cerevisiae were mutually grown together in liquid and solid media and proved to be excellent in producing ethanol compared to other combinations. Development of direct bioconversion of rice straw for ethanol production by compatible mixed culture Production of bioethanol The production of bioethanol within the period of 8 days fermentation time is shown in Fig. 3. In overall, the productions of bioethanol are different for different run due to different types of strain being used. The highest production of bioethanol (10.1%, v/v) occurred during the third day for run RUN 1 RUN 2 RUN 3 RUN 4 Bioe thanol (%, v/v) F ermentation time, day Fig. 3 : Production of bioethanol by developed direct bioconversion of rice straw While for Run 1 the second highest production of bioethanol occurred at the second day. It seems that the factor that contributes and differentiates the result for Run 2 from others is due to the ligninolytic fungus P. chrysosporium. P. chrysosporium has drawn considerable attention as an appropriate host for the production of lignin-degrading enzymes or direct application in lignocellulose bioconversion processes 14,21. And the bioethanol increased again at the sixth day when S. cerevisiae was added. S. cerevisiae or yeasts can be used for ethanol and food production 16,17. S. cerevisiae facilitate in the fermentation of the raw material to ethanol. Release of reducing sugar in bioethanol production It is shown in Figure 4 that the reducing sugar released in bioethanol production has a decreasing trend with the highest glucose concentration was recorded by Run 1 during the second day. However, this is exceptional for Run 2 where the higher results were recorded during the sixth day. In the production 122

6 Run 1 Run 2 Run 3 Run Glu co smaine (mg /ml) Fe r mentatio n Pe riod, day Fig. 4 : Reducing sugar released in bioethanol production of ethanol, the concentration of reducing sugar released decreased with fermentation time, indicating the degradation of substrates occurred and decreased at the end of fermentation. The highest concentration of reducing sugar was obtained at day 2 by Run 1 of 3.2 g/l. A high concentration of reducing sugar released at the beginning of fermentation for Run 1 was due to the presence of cosubstrate and also the degradation of substrate occurred due to the presence of fungi. For Run 3, the concentration of reducing sugar was lower compared to other runs due to most reducing sugar were metabolized to produce ethanol, which is proved in the optimum yield of ethanol production for Run 3 compared to other runs. Determination of glucosamine as growth indicator As shown in Fig. 5, Glucosamine Run 1 Run 2 Run 3 Run Gluco smain e (mg/ml) F er men tatio n Pe riod, d ay Fig. 5 : Glucosamine determination in bioethanol production 123

7 concentration in ethanol production was observed, it increased with fermentation and decreased at the end of it. The highest glucosamine concentration was 1.2 g/l obtained at day 4 of fermentation by Run 1. However, in term of values obtained, they might not be very accurate. This is because the assay used was quite tedious. Besides that, the definition of SSF by Durand 5 shows that microorganism is intimately bound to the solid matrix, which involves difficulties for biomass measurements. It is expected that not all the biomass produced in the fermentation were extracted for this assay. CONCLUSION The results indicated that the compatible mixed cultures of yeast and lignocellulolytic fungi were able to produce ethanol by utilizing rice straw as a major substrate in the direct bioconversion process. Based on the ability to produce higher content of ethanol, combination of S. cerevisae, P. chrysosporium, T. harzianum were selected as the potential strains. This method would be beneficial in providing a more effective way in managing rice straw waste, development of an environmental friendly process for the production of ethanol, with less cost and also contribute to the country s economy. REFERENCES 1. Alam M.Z., Fakhru l-razi A. and Molla A.H., Evaluation of fungal potentiality for bioconversion of domestic wastewater sludge. Journal of Environmental Sciences 16(1), (2004). 2. Bosco F, Ruggeri B, and Sassi G. Performances of a trickle bed reactor (TBR) for exoenzyme production by Phanerochaete chrysosporium: influence of a superficial liquid velocity. Chem. Eng. Sci. 54, (1999). 3. Chang H.M (Eds) Lignin Biodegradation: Microbiology, chemistry and Potential Application, vol II. CRC Press, Boca Raton Florida, Cheung S.M. and Anderson B.C., 124 Laboratotary investigation of ethanol production from municipal primary wastewater solids. Bioresource Technology 59, (1996). 5. Durand H. and Clanet M., Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb. Technol. 10(6), (1988). 6. Eriksson K.E. and Vallander L. Biochemical Pulping. In: Kirk TK. Higuchi T. (1980). 7. Galbe M. and Zacchi G. A review of the production of ethanol from softwood. Appl. Microbial Bioethanol 59, (2002). 8. Ghosh, V.K. and Deb, J.K. Production and Characterization of Xylanase form Thielaviopsis basicola. Applied Microbiology and Biotechnology (1988). 9. Kim S. and Dale B.E. Global potential bioethanol production from wasted crops and crop residues. Biomass and Bioenergy 26 (2004). 10. Krishna C., Microbial enzyme production utilizing banana wastes. Ph.D Thesis, Cochin University of Science and Technology, India (1996). 11. Nguyen QA, Tucker MP, Keller FA, Beaty DA, Connors KM and Eddy FP. Dilute acids hydrolysis of softwoods. Appl. Biochem. Bioethanol (1999). 12. Nigam, J.N. Continuous ethanol production from pineapple cannery waste. Journal of Biotechnology, (1999). 13. Park Y.S., Kang S.W., Lee J.S., Hong S.I. and Kim S.W. Xylanase Production in Solid State Fermentation by Aspergillus niger mutant using Statistical Experiment Designs. Appl Microbiol Biotechol, (2002). 14. Ruggeri B. and Sassi G. Experimental sensitivity analysis of a trickle bed bioreactor for lignin peroxidases

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