An overview of enzymatic production of biodieselIntroductionTransesterification processTransesterification by enzymatic techniqueExtracellular lipaseEffective methanolysis using extracellular lipaseIntracellular lipaseConclusions and future prospectsReferencesReviewAn overview of enzymatic production of biodieselSrivathsan Vembanur Ranganathan, Srinivasan Lakshmi Narasimhan,Karuppan Muthukumar*Department of Chemical Engineering, A.C. College of Technology, Anna University, Chennai 600 025, IndiaReceived 5 March 2007; received in revised form 29 April 2007; accepted 29 April 2007Available online 25 June 2007AbstractBiodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The productionof biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reactionrates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity productand 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 immobilizingin a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzymeis inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal ofglycerol, 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 enzy-matic process. 2007 Elsevier Ltd. All rights reserved.Keywords: Lipase; Biodiesel; Transesterification; Immobilization; Whole cell1. IntroductionBiodiesel has gained importance in the recent past for itsability to replace fossil fuels which are likely to run outwithin a century. The environmental issues concerned withthe exhaust gases emission by the usage of fossil fuels alsoencourage the usage of biodiesel which has proved to beeco-friendly far more than fossil fuels. Biodiesel is knownas a carbon neutral fuel because the carbon present in theexhaust was originally fixed from the atmosphere. Biodieselis a mixture of mono-alkyl esters obtained from vegetableoils like soyabean oil, jatropha oil, rapeseed oil, palm oil,sunflower oil, corn oil, peanut oil, canola oil and cotton-seed oil (Peterson, 1986). Apart from vegetable oils, biodie-sel can also be produced from other sources like animal fat(beef tallow, lard), waste cooking oil, greases (trap grease,float grease) and algae (Pearl, 2002). A method utilizing allthe above mentioned sources was patented by Foglia et al.(1998) claiming the process to be a cost effective one as ituses inexpensive feedstocks.The direct usage of vegetable oils as biodiesel is possibleby blending it with conventional diesel fuels in a suitableratio and these ester blends are stable for short term usages.The blending process is simple which involves mixing aloneand hence the equipment cost is low. But direct usage ofthese triglyceric esters (oils) is unsatisfactory and impracti-cal for long term usages in the available diesel engines dueto high viscosity, acid contamination, free fatty acid forma-tion resulting in gum formation by oxidation and polymer-ization and carbon deposition. Hence vegetables oils areprocessed so as to acquire properties (viscosity and volatil-ity) similar to that of fossil fuels and the processed fuel canbe directly used in the diesel engines available. Three pro-cessing techniques are mainly used to convert vegetable oils0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2007.04.060*Corresponding author. Tel.: +91 44 22203500.E-mail address: [email protected] (K. Muthukumar).Available online at www.sciencedirect.comBioresource Technology 99 (2008) 3975–3981to fuel form (Ma and Hanna, 1999) and they are pyrolysis,microemulsification and transesterification. Pyrolysis refersto chemical change caused by application of heat to getsimpler compounds from a complex compound. The pro-cess is also known as cracking. Vegetable oils can becracked to reduce viscosity and improve cetane number.The products of cracking include alkanes, alkenes, and car-boxylic acids. Soyabean oil, cottonseed oil, rapeseed oiland other oils are successfully cracked with appropriatecatalysts to get biodiesel (Ma and Hanna, 1999). By usingthis technique good flow characteristics were achieved dueto reduction in viscosity. Disadvantages of this processinclude high equipment cost and need for separate distilla-tion equipment for separation of various fractions. Alsothe product obtained was similar to gasoline containingsulfur which makes it less eco-friendly (Ma and Hanna,1999). Microemulsification is another technique that hasbeen reported to produce biodiesel and the componentsof a biodiesel microemulsion include diesel fuel, vegetableoil, alcohol, surfactant an d cetane improver in suitable pro-portions (Ma and Hanna, 1999). Alcohols such as metha-nol, ethanol and propanol are used as viscosity loweringadditives, higher alcohols are used as surfactants and alkylnitrates are used as cetane improvers. Viscosity reduction,increase in cetane number and good spray charactersencourage the usage of microemulsions but prolong usagecauses problems like injector needle sticking, carbondeposit formation and incomplete combustion (Ma andHanna, 1999). The most popular method of producing bio-diesel is the transesterification of vegetable oils. Biodieselobtained by transesterification process is a mixture ofmono-alkyl esters of higher fatty acids. Transesterificationis the alcoholysis of triglyceric esters resulting in a mixtureof mono-alkyl esters and glycerol and the sequence of pro-cesses is shown in Fig. 1. The high viscosity component,glycerol, is removed and hence the product ha s low viscos-ity like the fossil fuels. The mixture of these mono-alkylesters can hence be used as a substitute for fossil fuels.2. Transesterifica tion processThe transesterification process can be done in a numberof ways such as using an alkali catalyst, acid catalyst, bio-catalyst, heterogeneous catalyst