An overview of enzymatic production of biodiesel

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1 Available online at Bioresource Technology 99 (2008) Review An overview of enzymatic production of biodiesel Srivathsan Vembanur Ranganathan, Srinivasan Lakshmi Narasimhan, Karuppan Muthukumar * Department of Chemical Engineering, A.C. College of Technology, Anna University, Chennai , India Received 5 March 2007; received in revised form 29 April 2007; accepted 29 April 2007 Available online 25 June 2007 Abstract Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Lipase; Biodiesel; Transesterification; Immobilization; Whole cell 1. Introduction Biodiesel has gained importance in the recent past for its ability to replace fossil fuels which are likely to run out within a century. The environmental issues concerned with the exhaust gases emission by the usage of fossil fuels also encourage the usage of biodiesel which has proved to be eco-friendly far more than fossil fuels. Biodiesel is known as a carbon neutral fuel because the carbon present in the exhaust was originally fixed from the atmosphere. Biodiesel is a mixture of mono-alkyl esters obtained from vegetable oils like soyabean oil, jatropha oil, rapeseed oil, palm oil, sunflower oil, corn oil, peanut oil, canola oil and cottonseed oil (Peterson, 1986). Apart from vegetable oils, biodiesel can also be produced from other sources like animal fat * Corresponding author. Tel.: address: m_kumar77@yahoo.co.in (K. Muthukumar). (beef tallow, lard), waste cooking oil, greases (trap grease, float grease) and algae (Pearl, 2002). A method utilizing all the above mentioned sources was patented by Foglia et al. (1998) claiming the process to be a cost effective one as it uses inexpensive feedstocks. The direct usage of vegetable oils as biodiesel is possible by blending it with conventional diesel fuels in a suitable ratio and these ester blends are stable for short term usages. The blending process is simple which involves mixing alone and hence the equipment cost is low. But direct usage of these triglyceric esters (oils) is unsatisfactory and impractical for long term usages in the available diesel engines due to high viscosity, acid contamination, free fatty acid formation resulting in gum formation by oxidation and polymerization and carbon deposition. Hence vegetables oils are processed so as to acquire properties (viscosity and volatility) similar to that of fossil fuels and the processed fuel can be directly used in the diesel engines available. Three processing techniques are mainly used to convert vegetable oils /$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi: /j.biortech

2 3976 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) VEGETABLE OILS The transesterification process can be done in a number of ways such as using an alkali catalyst, acid catalyst, biocatalyst, heterogeneous catalyst or using alcohols in their supercritical state. The general reaction is shown below Vegetable oil þ Methanol Catalyst! Biodiesel þ Glycerol BIODIESEL TRANSESTERIFICATION to fuel form (Ma and Hanna, 1999) and they are pyrolysis, microemulsification and transesterification. Pyrolysis refers to chemical change caused by application of heat to get simpler compounds from a complex compound. The process is also known as cracking. Vegetable oils can be cracked to reduce viscosity and improve cetane number. The products of cracking include alkanes, alkenes, and carboxylic acids. Soyabean oil, cottonseed oil, rapeseed oil and other oils are successfully cracked with appropriate catalysts to get biodiesel (Ma and Hanna, 1999). By using this technique good flow characteristics were achieved due to reduction in viscosity. Disadvantages of this process include high equipment cost and need for separate distillation equipment for separation of various fractions. Also the product obtained was similar to gasoline containing sulfur which makes it less eco-friendly (Ma and Hanna, 1999). Microemulsification is another technique that has been reported to produce biodiesel and the components of a biodiesel microemulsion include diesel fuel, vegetable oil, alcohol, surfactant and cetane improver in suitable proportions (Ma and Hanna, 1999). Alcohols such as methanol, ethanol and propanol are used as viscosity lowering additives, higher alcohols are used as surfactants and alkyl nitrates are used as cetane improvers. Viscosity reduction, increase in cetane number and good spray characters encourage the usage of microemulsions but prolong usage causes problems like injector needle sticking, carbon deposit formation and incomplete combustion (Ma and Hanna, 1999). The most popular method of producing biodiesel is the transesterification of vegetable oils. Biodiesel obtained by transesterification process is a mixture of mono-alkyl esters of higher fatty acids. Transesterification is the alcoholysis of triglyceric esters resulting in a mixture of mono-alkyl esters and glycerol and the sequence of processes is shown in Fig. 1. The high viscosity component, glycerol, is removed and hence the product has low viscosity like the fossil fuels. The mixture of these mono-alkyl esters can hence be used as a substitute for fossil fuels. 2. Transesterification process GLYCEROL METHANOL Fig. 1. Biodiesel production sequence by transesterification. In the alkali process sodium hydroxide (NaOH) or potassium hydroxide (KOH) is used as a catalyst along with methanol or ethanol. Initially, during the process, alcoxy is formed by reaction of the catalyst with alcohol and the alcoxy is then reacted with any vegetable oil to form biodiesel and glycerol. Glycerol being denser settles at the bottom and biodiesel can be decanted. This process is the most efficient and least corrosive of all the processes and the reaction rate is reasonably high even at a low temperature of 60 C. There may be risk of free acid or water contamination and soap formation is likely to take place which makes the separation process difficult (Ma and Hanna, 1999; Fukuda et al., 2001; Barnwal and Sharma, 2005). The second conventional way of producing biodiesel is using an acid catalyst instead of a base. Any mineral acid can be used to catalyze the process; the most commonly used acids are sulfuric acid and sulfonic acid. Although yield is high, the acids, being corrosive, may cause damage to the equipment and the reaction rate was also observed to be low (Freedman et al., 1984). It has been recently found that enzymes such as lipase can be used to catalyze transesterification process by immobilizing them in a suitable support. The advantage of immobilization is that the enzyme can be reused without separation. Also, the operating temperature of the process is low (50 C) compared to other techniques. Disadvantages include inhibition effects which was observed when methanol was used and the fact that enzymes are expensive (Nelson et al., 1996; Shimada et al., 2002). Heterogeneous catalysts such as amorphous zirconia, titanium-, aluminium-, and potassium-doped zirconias have also become popular for catalyzing the transesterification of vegetable oils. Research is still in progress in order to solve the problems encountered in this process such as exhaustion of catalyst and to achieve higher conversions (Furuta et al., 2006). The transesterification process can be carried out even without catalyst but with considerable increase in temperature. Yield is very low at temperatures below 350 C and therefore higher temperatures were required. However at temperatures greater than 400 C thermal degradation of esters occurred (Demirbas, 2006). Recently it has been found that alcohols in their supercritical state produce better yield and researchers have experimented this process with methanol in its supercritical state. The process efficiency can be increased by using calcium oxide as a catalyst (Demirbas, 2006). Of all the methods mentioned above for production of biodiesel, only the alkali process is carried out in an industrial scale. It is cost effective and highly efficient. But problems arise in the downstream operations including separation of catalyst and unreacted methanol from

3 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) OIL TRANSESTERIFICATION SEPARATION ALKALI + MeOH EVAPORATION OF MeOH WASTE WATER- ALKALINE BIODIESEL EVAPORATION OF MeOH UPPER WASHING LOWER this review presents critical analysis of the methods reported which can contribute to the global effort of industrial implementation of the enzymatic production of biodiesel in the near future. The enzyme that was found to be capable of catalyzing methanolysis is lipase and lipase is obtained from micro-organisms like Mucor miehei, Rhizopus oryzae, Candida antarctica, Pseudomonas cepacia. Various alcohols are tried for the transesterification process including methanol, ethanol, iso-propanol and butanol but methanol is considered for industrial production because of its low cost and availability. 3. Transesterification by enzymatic technique SAPONIFIED PRODUCTS GLYCEROL PURIFICATION Enzymatic production is possible using both extracellular and intracellular lipases. In both the cases the enzyme is immobilized and used which eliminates downstream operations like separation and recycling. Hence in all the works reported in the literature either immobilized (extracellular) enzymes or immobilized whole cells (Intracellular enzymes) are used for catalysis. Both the processes are reported to be highly efficient compared to using free enzymes. OILS MeOH ENZYME Fig. 2. Production of biodiesel by alkali process. TRANSESTERIFICATION UPPER BIODIESEL SEPARATION Fig. 3. Enzymatic production of biodiesel. LOWER GLYCEROL biodiesel. The removal of the catalyst involves many complications and biodiesel requires repeated washing for attaining the necessary purity. Figs. 2 and 3 compare the difference in downstream operation required for alkali and enzymatic production. The production of biodiesel using a biocatalyst eliminates the disadvantages of the alkali process by producing product of very high purity with less or no downstream operations (Fukuda et al., 2001). This method of production of biodiesel using a biocatalyst was also patented by Haas (1997). But the process has not yet been implemented in an industrial scale due to certain constraints like enzyme inhibition by methanol, exhaustion of enzyme activity and high cost of enzymes. Research works have been reported in the literature in order to overcome these problems and 4. Extracellular lipase Mittelbach (1990) reported transesterification of sunflower oil with primary alcohols like methanol, ethanol and butanol using M. miehei and C. antarctica (Novozym 435) in the presence and absence of the solvent, petroleum ether. Yields obtained for ethanol and butanol were high even without the solvent but methanol was found to produce only traces of methyl esters without the solvent. Nelson et al. (1996) conducted batch experiments and found that C. antarctica was suitable for secondary alcohols (80% conversion) like iso-propanol and 2-butanol and M. miehei was efficient for primary short chain alcohols (95% conversion) like methanol, ethanol, propanol and butanol in the presence of hexane as a solvent. However in the absence of the solvent, methanol was the least efficient with a methyl ester yield of 19.4%. The low yield was attributed to the inhibitory effects caused by methanol on the immobilized enzyme. This was again confirmed by Abigor et al. (2000) who reported the conversion of palm kernel oil using methanol and ethanol as 15% and 72%, respectively. Noureddini et al. (2001) used methanol and ethanol for the tranesterification of soyabean oil in the presence of immobilized enzyme obtained from Pseudomonas flourescens and reported conversions 67% and 65% for methanol and ethanol respectively. But conversions as high as 97% is possible which was demonstrated by Linko et al. (1998) using 2-ethyl-1-hexanol for the transesterification of rapeseed oil. Similarly, usage of other alcohols as alternate acyl acceptors instead of methanol have been experimented and conversions as high as 90% were constantly obtained. Iso et al. (2001) reported 90% conversion of vegetable oil using P. fluorescens enzyme with butanol as the acyl acceptor. The reaction was carried out in a solvent free medium

4 3978 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) under an optimum condition of 0.3% water and 60 C. Propane-2-ol was used as an acyl acceptor by Modi et al. (2006) for the transesterification of Jatropha, Karanj and sunflower oil achieving a maximum conversion of 92.8%, 91.7% and 93.4%, respectively. With propane-2-ol, the reusability of lipase was maintained over 12 cycles while it dropped to zero after 7 cycles when methanol was used. In order to achieve high conversions with methanol as the acyl acceptor several attempts have been made to minimize the inhibitory effects of methanol. Using a common solvent for methanol and the oil was found to be effective by many researchers since insoluble methanol is responsible for inhibition. Iso et al. (2001) reported high conversion with methanol using 1,4 dioxane as a solvent. Rather than using butanol as an acyl acceptor, it was suggested to use tert-butanol as a solvent for methanolysis of oil. Li et al. (2006) reported the usage of tert-butanol as a solvent for the transesterification of rapeseed oil. A conversion of 95% was obtained using Lipozyme TI LM and Novozyme 435 in a suitable ratio (3:1) under optimum conditions. tert- Butanol as a novel solvent for the enzymatic production was again demonstrated by Royon et al. (2007) who used Novozyme 435 for the transesterification of cotton seed oil and yield as high as 97% was reported within 24 h at 55 C. With a continuous fixed bed reactor 95% conversion was reported under optimum flow conditions. Usage of tert-butanol as a solvent could hence be recognized as one possible solution for reducing the inhibitory effects of methanol and for the industrial implementation of the process. Pretreatment of the immobilized enzyme was believed to minimize the deactivation of the enzyme and Samukawa et al. (2000) illustrated this by preincubation of the enzyme in methyl oleate for 0.5 h and subsequently in soyabean oil for 12 h. Inhibitory effects were considerably reduced and high conversions were obtained. Chen and Wu (2003) suggested the usage of tert-butanol and 2-butanol for regenerating the activity of deactivated enzymes. They conducted experiments by completely deactivating Novozyme 435 with methanol and regenerating by washing the enzyme with tert-butanol and 2-butanol. The activity of the enzyme increased by 10 times compared to the untreated enzyme and the completely deactivated enzyme was restored to 56% and 75% of its original value when washed with 2-butanol and tert-butanol, respectively. The same process was also patented by Chen and Wu (2002). Recently, acyl acceptors other than alcohols are experimented to improve the efficiency of transesterification process. A novel acyl acceptor was developed by Du et al. (2004) who used methyl acetate for transesterification of soyabean oil with Novozyme 435. While a 1:1 molal ratio of methanol to oil caused serious inactivation of enzyme, even a 12:1 molal ratio of methyl acetate to oil did not have any noticeable negative effect on the enzyme. The activity of the enzyme was found to be unaltered even after 100 batches. Conversion, as high as 92%, was reported for soyabean oil. Crude soyabean oil was found to have equal conversion incontrast to methanolysis which gave only traces of methyl ester with crude soyabean oil. Since the by product triacetylglycerol is a valuable compound, this process was recommended for industrial production. Ethyl acetate was tried as an acyl acceptor by Modi et al. (2007) with Novozyme 435 and they reported conversions above 90% for oils such as jatropha, karanj and sunflower oil. Comparison was made with ethanolysis in which activity could not be maintained for more than 6 cycles whereas with ethyl acetate, even after 12 cycles there was no appreciable decrease in activity. Ethyl acetate to oil ratio of 11:1 was recommended for the process and even in such high concentrations no deactivation occurred. 5. Effective methanolysis using extracellular lipase Methanolysis yield was found to be low in the absence of a suitable solvent and even in the presence of such a solvent high conversions attained by other alcohols are not possible. Since the deactivation of the enzyme was due to insolubility of methanol, any methanol to oil ratio above 1.5:1 caused serious inhibition. To overcome this difficulty, Shimada et al. (1999) suggested stepwise addition of methanol through which 95% conversion was attained even after 50 cycles of operation. Similar to their work Watanabe et al. (2000) conducted experiments on two step batch wise addition of methanol and three step continuous addition of methanol. They reported high conversions of above 90% even after 100 cycles of repeated operation. Stepwise addition was also demonstrated by Samukawa et al. (2000) who preincubated the enzyme before usage, were successful in obtaining a conversion of 97% with three stepwise addition of 0.33 molar equivalents of methanol at h interval. For stepwise addition of methanol Kaieda et al. (2000) studied the difference in the usage of regiospecific and non-regiospecific lipases. Non-regiospecific lipases like Candida rugosa, P. cepacia and P. flourescens are tolerant to the inhibition of methanol. P. cepacia gave especially high conversion even with two to three molar equivalents of methanol. With regiospecific lipases like R. oryzae, 80 90% conversion was possible with stepwise addition of methanol in the presence of 4 30% water. Shimada et al. (2002) obtained conversions above 90% with waste cooking oil using stepwise addition of methanol. They also conducted experiments on continuous systems and obtained equally high conversions. Thus it was satisfactorily proved that the inhibitory effects of methanol can be minimized by stepwise addition of methanol and high conversions are possible even in a solvent free medium. Bako et al. (2002) suggested that the inhibitory effects are partly due to glycerol that is formed during the reaction and to reduce the inhibitory effects, they recommended in situ glycerol removal by dialysis. Experiments were conducted with two and three stepwise addition of methanol and glycerol was removed continuously by dialysis. This method was found to increase the effectiveness of the process it was reported that a minimum flow rate of 85 ml of

5 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) glycerol/liter of reacting mixture was required for effective separation to produce a conversion of 97% at 50 C. Xu et al. (2004) suggested that glycerol removal was also possible using iso-propanol in a process employing stepwise addition of methanol. Thermomyces lanuginosus (Lipozyme TL IM) was used to catalyze the transesterification of soyabean oil and a maximum yield of 98% was reported at 40 C and a conversion of 94% was maintained even after 15 repeated cycles. Nie et al. (2006) conducted optimization experiments on batch and continuous transesterification process and reported a maximum conversion of 96% for a batch process under optimal conditions with three stepwise addition of methanol using immobilized Novozyme 435. The lipase was found to retain its activity for more than 20 days of continuous operations. In the continuous process conversions of 92% and 93% were obtained for vegetable oil and waste cooking oil respectively and the process was recommended for industrial production. 6. Intracellular lipase The efficiency of the process can be highly increased by using intracellular lipase (whole cell immobilization) in the place extracellular lipase which requires complex purification steps before immobilization. A comparison of the immobilization process of extracellular and intracellular lipases is shown in Fig. 4. It can be clearly seen that considerable reduction in cost can be achieved with intracellular lipase. Whole cell biocatalyst was developed by Matsumoto et al. (2001) by immobilizing R. oryzae cells and permeabilizing them by air drying. It was then used for the production of methyl esters by three stepwise methanolysis of plant oil in solvent free and water containing system. The methyl ester content in the reaction mixture was reported a CULTIVATION SEPARATION PURIFICATION IMMOBILIZATION METHANOLYSIS b CULTIVATION + IMMOBILIZATION SEPARATION METHANOLYSIS Fig. 4. Comparison of steps involved in the immobilization of extracellular and intracellular enzymes: (a) extarcellular lipase and (b) intracellular lipase. to be 71 wt% after a 165-h reaction at 37 C with stepwise addition of methanol. Ban et al. (2001) utilized immobilized whole cell R. oryzae for the transesterification of vegetable oils and investigated the culture conditions, cell pretreatment effects and effect of water content on the production process. To enhance the methanolysis activity of the immobilized cells, several substrate related compounds were added to the medium, of which olive oil and oleic acid were found to be effective. It was reported that, with stepwise addition of methanol and with 15% water content, a high conversion of 90% was obtained which was comparative with the extracellular process. Inorder to stabilize R. oryzae cells, cross-linking treatment with 0.1% glutaraldehyhde was examined by Ban et al. (2002). It was reported that without glutaraldehyde treatment, in the stepwise methanol addition process, conversion level dropped to 50% after 6th batch cycle whereas with glutaraldehyde treatment conversion can be maintained at 72 83% even after 6 batch cycles. Fukuda and Kondo (2003) patented the process of producing biodiesel utilizing whole cell biocatalyst by pretreating the cells with lower alcohols. They claimed a times increase in the reaction rate using cells treated with lower alcohols compared to untreated cells. Whole cell immobilization was then experimented by many researchers in order to increase the efficiency of enzymatic transesterification. Hama et al. (2004) studied the increase in enzyme stability with the variation of the fatty acid composition of the cell membrane. The fatty acid composition of the cell membrane can be controlled by the addition of various fatty acids to the culture medium. It was reported that oleic acid and linoleic acid enriched cells showed higher enzymatic activity than saturated fatty acid enriched cells and palmitic acid enriched cells showed higher stability than unsaturated fatty acid enriched cells. Therefore an optimum ratio of unsaturated to total fatty acids was determined as a compensation for both activity and stability and it was found to be It was reported that the methanolysis yield was consistently high and even after 10 repeated cycles it was above 55%. Hama et al. (2006) reported the existence of two types of lipases, one bound to the cell wall (ROL 34) and the other to the cell membrane (ROL 31). They reported that the increase in enzyme activity with addition of olive oil or oleic acid was due to the increase in the production of membrane bound lipase (ROL 31) suggesting that ROL 31 plays a crucial role in the methanolysis activity. Hama et al. (2007) studied the production of biodiesel using a packed bed reactor utilizing R. oryzae whole cell biocatalyst by plant oil methanolysis. Compared with methanolysis reaction in a shaken bottle, the packed bed reactor enhanced repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. Lipase-producing R. oryzae cells were immobilized within 6 mm 6mm 3 mm cuboidal polyurethane foam biomass support particles (BSPs)

6 3980 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) Table 1 Comparison of various works on enzymatic production of biodiesel S.No Authors/year Oil/enzyme Acyl acceptor 1 Watanabe et al. Vegetable oil, Novozyme (2000) a Samukawa et al. Soyabean oil, Novozyme (2000) a 435 Conversion (%) Technique employed Cost of production Methanol Stepwise addition of methanol Moderate Methanol 97 Stepwise addition methanol and preincubation of High enzyme in methyl oleate and soyabean oil 3 Ban et al. (2001) b Vegetable oil, R. oryzae Methanol 90 Stepwise addition of methanol and application of Low glutaraldehyde for stability of enzyme 4 Iso et al. (2001) a Triolein, P. flourescens Butanol 90 Butanol was used as an acyl acceptor and no Moderate solvent was used 5 Shimada et al. Waste cooking oil, Methanol 90 Stepwise addition of methanol Low (2002) a Novozyme Bako et al. Sunflower oil, Novozyme Methanol 97 Stepwise addition of methanol and removal of High (2002) a 435 glycerol by dialysis 7 Du et al. (2004) a Soyabean oil, Novozyme Methyl 92 A novel acyl acceptor, methyl acetate which had no High 435 acetate inhibitory effects was used 8 Xu et al. (2004) a Soyabean oil, Novozyme Methanol 98 Stepwise addition of methanol and removal of High 435 glycerol using the solvent, iso-propanol 9 Li et al. (2006) a Rapeseed oil, Novozyme Methanol 95 Combined use of Lipozyme TL IM and Novozyme High 435 & Lipozyme TL IM 435 along with tert-butanol as solvent 10 Royon et al. Cotton seed oil, Novozyme Methanol 97 tert-butanol was used as a solvent High (2007) a Modi et al. Jatropha oil, Novozyme Ethyl 91.3 Ethyl acetate having no inhibitory effects was used High (2007) a 435 acetate 12 Hama et al. (2007) b Soyabean oil, R. oryzae Methanol 90 Stepwise addition of methanol in a packed bed reactor Low a Extracellular lipase. b Intracellular lipase. during batch cultivation in a 20-l air-lift bioreactor. Hama et al. (2007) reported that the emulsification of the reaction mixture resulted in increased yield (75.5%) due the increase in interfacial surface area whereas without emulsification a reasonable high conversion of 63% was obtained. During the investigation the flow rate of reaction mixture was varied between 5 and 55 l/h and higher flow rates caused exfoliation of immobilized enzyme whereas low flow rates resulted in decreased enzyme activity due to inefficient mixing. An optimum of 25 l/h was suggested to give a maximum conversion of 90%. Fukuda and Noda (2006) patented the process of using whole cell biocatalyst with waste oil containing water claiming no decrease in the efficiency of the process. This encourages the process employing intracellular lipase for commercialization. Table 1 summarizes the significant works reported in the literature with respect to enzymatic production of biodiesel. 7. Conclusions and future prospects The biodiesel fuel production has gained importance for its ability to replace fossil fuels, its environmental benefits and the fact that it is a renewable source of energy. Since the direct usage of vegetable oils as biodiesel is impractical, many processes have been developed to convert them into a suitable form. Pyrolysis and microemulsification are not satisfactory and hence only the transesterification process is accepted for large scale production of biodiesel. The alkali transesterification process has already been implemented on an industrial scale and it gives high conversion. However it has several drawbacks including the difficulty in recycling catalyst and the need for removal of glycerol. To overcome these drawbacks, which may limit the availability of biodiesel fuel, enzymatic processes using lipases have recently been developed. The enzymatic process is much simpler since recovery of unreacted methanol and glycerol requires less downstream operations. As the cost of lipase production is the main hurdle for commercialization of the lipase-catalyzed process, several attempts have been made to develop cost effective systems. The cost effective system can be achieved by reusing the enzyme which is possible with immobilization of enzyme in suitable support particles. Both extracellular and intracellular lipase can be immobilized and used for catalyzing the transesterification reaction. The choice of method is based on a balance between simplified upstream operations as in whole cell immobilization and high conversions as in the usage of extracellular lipases. However when methanol was used as the acyl acceptor the immobilized enzyme suffered serious inactivation. This is due to the inhibitory effect of undissolved methanol and glycerol present in the reaction medium. Although other acyl acceptors, with no inhibitory effects have been developed, usage of methanol has been extensively researched for its low cost and availability. Several methods have been developed by researchers to minimize the inhibitory effects of methnol and glycerol thereby increasing the overall yield of the process. The

7 S.V. Ranganathan et al. / Bioresource Technology 99 (2008) use of t-butanol as a common solvent for oil and methanol eliminates the inhibitory effects of methanol. Continuous glycerol removal by dialysis or solvent extraction reduces the inhibitory effects of glycerol on the enzyme activity. Highest conversion was obtained for the stepwise addition of methanol to oil by which methanol concentration in the medium was always kept low and thus eliminating enzyme deactivation. The conversion of whole cell biocatalyst process was enhanced to match the extracellular process by adding certain promoters and maintaining suitable reaction conditions. Combining the whole cell biocatalyst process with stepwise addition of methanol, significant reduction in the cost the production of biodiesel could be expected. Such novel system is promising for the industrial scale enzymatic production of biodiesel. 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