BIOETHANOL: THE LIQUID GOLD OF TOMORROW FROM LIGNOCELLULOSIC WASTES

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1 Review Paper (NS-2) BIOETHANOL: THE LIQUID GOLD OF TOMORROW FROM LIGNOCELLULOSIC WASTES Rajni Kumari*, Navneet Kumar Dubey and K. Pramanik Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Orissa (INDIA) Received October 30, 2008 Accepted March 06, 2009 ABSTRACT Due to rapid depletion of fossil fuel reserves, alternative energy sources need to be renewable, sustainable, efficient, cheaper and eco-friendly. A worldwide interest in the utilization of bio-ethanol as the future transportation fuel has stimulated studies on the cost and efficiency of industrial process for ethanol production. Keeping this in view, improvements in the ethanol fermentation have been focused on the use of modified strains obtained by various methods including protoplast fusion technology to enhance the yield of ethanol production from various cheaper substrates like lignocelluloses. Ethanol is clean fuel. Its oxygen content decreases emissions when combusted with gasoline and because ethanol is derived ultimately from plant matter, its use as a fuel does not contribute to the net accumulation of carbon dioxide in the atmosphere. So, biomass and bio-energy markets are developing rapidly and pressure on available biomass resources is increasing day by day. Key Words : Ethanol, Fermentation, Lignocelluloses, Gasoline, Biomass. INTRODUCTION To meet the growing energy demand for transportation sector worldwide including India, there is a need for environmentally sustainable energy sources. In this context, bioethanol (ethanol from biomass) has been considered as an attractive transportation fuel. Ethanol as a fuel provides reduction in green house gas emission, carbon monoxide, sulfur, and helps to eliminate smog from the environment. Lignocellulosic wastes have emerged as cheaper and promising substrate * Author for correspondence for bioethanol production. Bioethanol can be produced from various lignocellulosic materials such as wood, agricultural and forest residues. However, the major technological hurdle is the conversion of these abundant and inexpensive wastes into ethanol in cost effective way. Hence, the technology development focuses on improved and engineered microorganisms for the production of ethanol from lignocellulosic materials. Therefore, this paper reviews the current status of bioethanol production, the microorganism involved, ecologic and economic impact and provides visions for the future prospects. 922

2 Lignocellulose Recent efforts have been concentrated on utilizing lignocelluloses to produce ethanol. Lignocellulosic biomass in nature is by far the most abundant raw material from hardwood, softwood, grasses, municipal solid waste and agricultural residues such as sugarcane baggase, wheat straw and corn cob. Lignocellulose consists of three main components: cellulose, hemicellulose and lignin, the first two being composed of chains of sugar molecules. These chains can be hydrolyzed to produce hexoses (Dglucose, D-galactose and D-mannose) and pentoses (D-xylose and L-arabinose) 1. Among these D- glucose is the most abundant sugar while D-xylose is next to it. Conversion of lignocelluloses to Ethanol Bioconversion of lignocellulosics to ethanol consists of three major steps such as pretreatment, hydrolysis and fermentation. Pretreatment Pretreatment is required to alter the biomass macroscopic and microscopic size and structure as well as its submicroscopic chemical composition so that hydrolysis of carbohydrate fraction to monomeric sugars can be achieved more rapidly and with greater yields 2,3.Pretreatment can be carried out in different ways such as, steam explosion 4, ammonia fiber explosion 5, acid or alkaline pretreatment 6,7 and biological treatment 8. However, biological pretreatment is favorable than other methods as it is ecofriendly. hydrolytic enzymes like cellulases, xylanases, hemicellulases and mannanases 9. Bacteria and fungi are the good sources of cellulases, hemicellulases that could be used for the hydrolysis of pretreated lignocellulosics 10. Some of them are Clostridium, Cellulomonas, Trichoderma, Penicillium, Neurospora, Fusarium, Aspergillus etc. Hydrolysis organisms 15. Saccharomyces cerevisiae, After pretreatment there are two types of yeast commonly used for ethanol production processes to hydrolyze the feed stocks into is capable of converting only hexose sugars monomeric sugar: acid and enzymatic to ethanol. Yeast such as Pichia stipitis, hydrolysis. Hydrolysis is employed for the Candida shehatae and Pachysolen conversion of hemicellulose and cellulose to tannophilus are characterized by best ability the sugars. There are two types of acid to produce ethanol from D-xylose. However, hydrolysis process commonly used - dilute their use for fermentation of complex and concentrated acid hydrolysis. Enzymatic lignocellulose hydrolysate is not very process utilizes mixtures of several economic, especially due a long time of 923 Fermentation Since the 1970's, several bioprocess configurations, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification cofermentation (SSCF), and direct microbial conversion (DMC) 11 were have been advanced for cellulosic ethanol fermentation 12,13. After final purification (by distillation and molecular sieves or other separation techniques), the ethanol is ready to be used as a fuel, either neat or blended with petrol. SSF involves breakdown of both carbon polymer, cellulose to glucose; and hemicellulose to xylose and arabinose; and simultaneous fermentation by recombinant yeast or the organism which has the ability to utilize both C5 and C6 sugars 14. Microorganisms involved The lack of a microorganism able to ferment efficiently all sugars released by hydrolysis from lignocellulosic materials has been one of the main factors preventing utilization of lignocellulose. The ability of microorganism to ferment pentoses and hexoses is not widespread among all

3 fermentation, low ethanol yield, difficult optimization of physicochemical parameters of the process and weaker tolerance for numerous inhibitors generated during pretreatment and hydrolysis of the lignocellulose material 16. For this reason it is desirable to develop hybrid strains from the above microbial strains to have the better characteristics of both these parental strains. Genetically engineered microorganisms that can convert pentose or hexose to ethanol can greatly improve ethanol production efficiency 17,18. The various techniques employed are recombinant DNA technology, mutagenesis and protoplast fusion. Recombinant pentose-fermenting Escherichia coli 19 and Klebsiella oxytoca 20 have been generated by introducing ethanologenic genes from Zymomonas mobilis. Protoplast fusion may be used to produce interspecific or even intergeneric hybrids. Protoplast fusion becomes an important tool of gene manipulation because it breakdown the barriers to genetic exchange imposed by conventional mating systems 21. Increasing the capacity of the strains for rapid xylose fermentation could enhance the productivity and sustainability of agriculture and forestry by providing new outlets for agricultural and wood harvest residues. Fermentation parameters The important parameters for the cell growth and product formation are aeration, ph, and temperature. Temperature : The effects of temperature on growth and ethanol concentration have been well studied in the xylose-fermenting yeasts. In P. stipitis, xylitol and residual xylose concentrations increase with temperature. The optimum ph range for growth and fermentation on xylose was 4-7 at 25 C. The limit for ethanol production with P. stipitis increased from 33 g/liter at 30 C to 43 g/liter at 25 C 22,23. Lucas and van Uden 24 found that ethanol enhances 924 thermal death of C. shehatae. Ethanol depressed the maximum temperature for growth from of C. shehatae 31 to 17.5 C and increased the minimum temperature for growth from 2.5 to 10 C of xylose fermenting yeasts. Some strains of xylosefermenting yeasts produce up to 47 g ethanol / liter at 30 C. ph : The ph and temperature optima for biomass accumulation by P. tannophilus on xylose were 3.7 and 31.5, respectively, at an initial xylose concentration of 50 g/liter 25. The best initial ph for ethanol production from D-xylose by C. shehatae and P.stipitis in batch fermentation was With a detoxified hemicellulose hydrolysate at ph between 6.0 and 7.5, P. tannophilus converted 90% of the available xylose into xylitol. Aeration : The dissolved oxygen tension (DOT) is particularly critical in attaining maximal ethanol production with xylosefermenting yeasts. P. stipitis and C. shehatae require aeration for maximal ethanol production. Native S. cerevisiae also requires oxygen to metabolize xylulose 27, and recombinant S. cerevisiae requires oxygen to produce ethanol from xylose 28. Kastner et. al. 29 showed that oxygen starvation induces cell death in C. shehatae when it is grown on D-xylose, but not when it is cultivated on D- glucose. Nutrient uptake : Membrane transport is mediated by two different systems in yeasts: facilitated diffusion and active proton symport. In yeasts that utilize both xylose and glucose, these sugars share the same transporter systems 30. Glucose can inhibit xylose uptake by competing with the xylose transporters. In S. cerevisiae genetically engineered for xylose uptake, glucose, mannose, and fructose inhibited xylose conversion by 99, 77 and 78%, respectively.

4 RESULTS AND DISCUSSION Ecological impact of ethanol As energy demand increases the global supply of fossil fuels cause harm to human health and contributes to the green house gas (GHG) emission 31. Bioethanol is the clean transportation fuel that releases less sulfur, carbon monoxide, particulates, and greenhouse gases. Process of ethanol production utilizes energy only from renewable sources: no net carbon dioxide is added to the atmosphere, making ethanol an environmentally beneficial energy source. Lignocellulosic biomass materials require fewer inputs, such as fertilizer, herbicides, and other chemicals that can pose risks to wildlife. Their extensive roots improve soil quality, reduce erosion, and increase nutrient capture. Economic impact of Ethanol production The cost for bioconversion of biomass to liquid fuel must be lower than the current gasoline prices so as to make it competitive and find economic acceptance 32. The cost of feedstock and cellulolytic enzymes are two important parameters for ethanol production. However, there is still huge scope to bring down the cost of biomass-to-ethanol conversion. The economics of the ethanol process is determined by the cost of substrate. Since, lignocellulosics cannot be digested by humans; the production of cellulose does not compete with the production of food. The price per ton of the lignocellulosic raw material is much cheaper than grains or fruits. Moreover, since cellulose is the main component of plants, the whole plant can be harvested. This results in much better yields per acre - up to 10 tons, instead of 4 or 5 tons for the best crops of grain. It is estimated that 323 million tons of cellulose containing raw materials that could be used to create ethanol are thrown away each year. Further, reduction of the disposal of solid waste through cellulosic ethanol conversion would reduce solid waste disposal costs. Future Prospects Apart from success stories of bioethanol, the ethanol production at plant scale still remains a challenging issue. Significant advances have been made in developing microorganism with improved fermentation performance; however, higher ethanol yields and increased ethanol concentrations and tolerances are the key targets that are still need to be achieved from lignocellulosic waste 34. Much improvement has been achieved during the last 10 years regarding pretreatment, enzymatic hydrolysis, fermentation and process integration. The next step is to implement all these improvements in a pilot scale process where all steps are integrated into a continuous pilot plant to verify the technology and to obtain data for scale-up to a demonstration or fullscale process. 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) so comprehensive process development and optimization are still required to make the process economically viable. CONCLUSION This review covers some of the important challenges faced and progress made in developing cellulosic ethanol biotechnology. The key now is to focus on critical needs: successful commercialization of current technologies, and the development of next-generation pretreatment and enzymatic hydrolysis technologies that can substantially reduce the cost of biomass processing to ethanol. With application of novel, tailored enzymes for the cellulose breakdown coupled with the recent development of genetically engineered microorganism those ferment all possible sugars in biomass to ethanol are the pivotal 925

5 factors for high productivity. The new generation of energy crops with enhanced sustainability, yield, and composition will pave the way for a biotechnology revolution with major benefits for the environment, the economy, and energy security. REFERENCES 1. Saha B.C., Iten L.B., Cotta M.A. and Wu Y.V., Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Proc. Biochem. 40, , (2003). 2. Sun Y. and Cheng J., Hydrolysis of lignocellulosic materials for ethanol production: a review. Biores Technol. 83, 1-11, (2005). 3. Moiser N., Wyman C., Dale B., Elander R., Lee Y.Y., Holtzapple M. and Ladisch M., Features of promising technologies for pretreatment of lignocellulosic biomass. Biores. Technol. 96, , (2005). 4. Gregg D.J. and Saddler J.N., Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnol Bioeng. 51, , (1996). 5. Kim H.T., Kim J.S., Sunwoo C. and Lee Y.Y. Pretreatment of corn stover by aqueous ammonia. Biores Technol. 90, 39-47, (2003). 6. Damaso M.C.T., Castro Mde, Castro R.M., Andrade M.C. and Pereira N., Application of xylanase from Thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse. Appl. Biochem. Biotechnol. 115, , (2004). 7. Kuhad R.C., Singh A. and Ericksson K.E.. Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Adv Biochem Eng Biotechnol. 57, , (1997). 8. Keller F.A., Hamilton J.E. and Nguyen Q.A., Microbial pretreatment of biomass: 926 potential for reducing severity of thermochemical biomass pretreatment. Appl. Biochem. Biotechnol. 27(41), , (2003). 9. Aristidou A. and Penttilä M., Metabolic engineering applications to renewable resource utilization. Curr. Opin. Biotechnol. 11, , (2000). 10. Lynd L.R., Weimer P.J., van Zyl W.H. and Pretorius IS., Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol Rev. 66, , (2002). 11. Singh A. and Kumar P.K., Fusarium oxysporum: status in bioethanol production. Crit. Rev. Biotechnol. 11(2), , (1991). 12. Wright J.D., Wyman C.E. and Grohmann K., Simultaneous Ssaccharification and F fermentation of Llignocellulose: Process Evaluation. Applied Biochemistry Biotechnology. 18, 75-90, (1987). 13. Spindler D.D., Wyman C.E. and Grohmann K., Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol. Biotechnology and Bioengineering. 34(2), , (1989). 14. Wingren A., Galbe M. and Zacchi G., Techno-Economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks. Biotechnol. Prog. 19, ,(2003). 15. Toivola A., Yarrow D., Van-den-bosch E., Van-dijken J.P. and Sheffers W.A., Alcoholic fermentation of D-xylose by yeasts. Appl. Microb. Biotechnol. 47, , (1984). 16. Hahn-Ha gerdal B. et al., An interlaboratory comparison of the performance of ethanol-producing microorganisms in a xylose-rich acid hydrolysate. Appl. Microbiol. Biotechnol. 41, 62 72, (1994).

6 17. Jeffries T.W. and Jin Y.S., Ethanol and thermotolerance in the bioconversion of xylose by yeasts. Adv Appl Microbio.l 47, , (2000). 18. Dien B.S., Cotta M.A. and Jeffries T.W., Bacteria engineered for fuel ethanol production current status. Appl. Microbiol. Biotechnol. 63, , (2003). 19. Ingram L.O. et al., Genetic engineering of ethanol production in Escherichia coli. Appl. Environ. Microbiol. 53, , (1987). 20. Burchhardt G. and Ingram L.O., Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca Appl. Environ. Microbiol. 58, , (1992). 21. Muralidhar R.V. and Panda T., Fungal Protoplast fusion: A revisit, Bioprocess Biosyst Engg. 22, , (2000). 22. Du Preez J.C., Bosch M. and Prior B. AXylose fermentation by Candida shehatae and Pichia stipitis: Effects of ph, temperature and substrate concentration. Enzyme Microb. Techno., 8, , (1986a). 23. du Preez J.C., Bosch M. and Prior B.A. The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis. Appl. Microbiol. Biotechno., 23, , (1986b). 24. Lucas C. and van Uden N. The temperature profiles of growth, thermal deathnand ethanol tolerance of the xylose-fermenting yeast Candida shehatae. J. Basic Microbiol,. 8, (1985). 25. Roebuck K., Brundin A. and Johns M., Response surface optimization of temperature and ph for the growth of Pachysolen tannophilus. Enzyme Microb. Technol., 17, 75-78, (1995). 26. Sanchez S., Bravo V., Castro E., Moya A.J. and Camacho F., The influence of ph and aeration rate on the fermentation of n-xylose by Candida shehatae. Enzyme Microb. Technol., 21, , (1997). 27. Maleszka R, and Schneider H., Involvement of oxygen and mitochondrial function in the metabolism of D-xylulose by Saccharomyces cerevisiae. Arch. Biochem. Biophys., 228, 22-30, (1984). 28. Kötter P., Amore R., Hollenberg C.P. and Ciriacy M., Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, Xyl2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr. Genet., 18: (1990). 29. Kastner J.R., Jones W.J. and Roberts R.S., Oxygen starvation induces cell death in Candida shehatae fermentations of D-xylose, but not D-glucose. Appl. Microbiol. Biotechnol. 51, , (1999). 30. Boles E. and Hollenberg C.P., The molecular genetics of hexose transport in yeasts. FEMS Microbiol. Rev. 21, , (1997). 31. Foody B., Ethanol from Biomass : The Factors Affecting It's Commercial Feasibility. Iogen Corporation, Ottawa, Ontario, Canada, (1988). 32. Wayman M. and Parekh S.R., Biotechnology of biomass conversion; Fuels and chemicals from renewable resources. Open University Press Milton Keynes, (1990). 33. Subramanian K.A., Singal S.K., Saxena M. and Singhal S., Utilization of liquid biofuels in automotive diesel engines: An Indian perspective. Biomass Bioenergy 29, 65-72, (2005). 34. Hohmann N. and Rendleman C.M., In Emerging Technologies in Ethanol Production, USDA Economic Research Service, Agriculture Information Bulletin No.663, (1993). 927