REVIEWA Review of the Current State of BiodieselProduction Using Enzymatic TransesterificationLene Fjerbaek, Knud V. Christensen, Birgir NorddahlInstitute of Chemical Engineering, Biotechnology and Environmental Technology,University of Southern Denmark, Niels Bohrs Alle´1, DK-5230 Odense M, Denmark;telephone: þ45-6550-7443; fax: þ45-6550-7443; e-mail: [email protected] 29 October 2008; accepted 16 December 2008Published online 8 January 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22256ABSTRACT: Enzymatic biodiesel production has beeninvestigated intensively, but is presently employed indus-trially only in a 20,000 tons/year pilot plant in China (Duet al. [2008] Appl Microbiol Technol 79(3):331–337). Thisreview presents a critical analysis of the current status ofresearch in this area and accentuates the main obstacles tothe widespread use of enzymes for commercial biodieseltransesterification. Improved results for enzymatic catalysisare seen with respect to increased yield, reaction time andstability, but the performance and price of the enzymes needfurther advances for them to become attractive industriallyfor biodiesel production. Critical aspects such as masstransfer limitations, use of solvents and water activity arediscussed together with process considerations and evalua-tion of possible reactor configurations, if industrial produc-tion with enzymes is to be carried out. Results of publishedstudies on the productivity of enzymes are also presentedand compared to the use of chemical catalysts.Biotechnol. Bioeng. 2009;102: 1298–1315.ß 2009 Wiley Periodicals, Inc.KEYWORDS: biodiesel; enzyme; lipase; transesterification;productivityIntroductionThere are several reasons for the introduction of biodieselas an alternative to conventional fossil based diesel. Theseinclude decreasing dependency on foreign energy supplyfrom declining fossil fuel resources; helping to reduce globalwarming by using renewable biofuels for the transportsector; and lowering emissions of particles, sulfur, carbonmonoxide and hydrocarbons (Demirbas, 2007; Meher et al.,2006; Mittelbach et al., 1983; Sheehan et al., 1998).Biodiesel can be produced from fat, lard, tallow, andvegetable oils. These mixtures of fatty acids (FFA) andtriglycerides (TAG) need to be chemically altered to fattyacid alkyl esters (FAAE) to be useful as biodiesel fuel forcurrently used diesel engines (Ma and Hanna, 1999; Meheret al., 2006; Mittelbach et al., 1983; Pryde, 1983; Srivastavaand Prasad, 2000).Catalysts investigated for transesterification are eitheracids, bases, both liquid and heterogeneous, as well as free orimmobilized (imm.) enzymes (Haas et al., 2006; Kaiedaet al., 1999; Komers et al., 2001; Ma and Hanna, 1999; Meheret al., 2006; Suppes et al., 2001, 2004).Most often used industrially today is alkaline transester-ification (Kaieda et al., 1999; Meher et al., 2006; Srivastavaand Prasad, 2000; Zhang et al., 2003), where raw materialwith a high water or free fatty acid (FFA) content needspretreatment with an acidic catalyst in order to esterify FFA(Freedman et al., 1984; Kaieda et al., 1999; Zhang et al.,2003), illustrated in Figure 1. Pretreatment is necessary toreduce soap formation during the reaction and ease theextensive handling for separation of biodiesel and glyceroltogether with removal of catalyst and alkaline wastewater(Meher et al., 2006; Mittelbach, 1990). The amount ofwastewater from a traditional biodiesel plant is around0.2 ton per ton biodiesel produced (Suehara et al., 2005).Therefore the wastewater treatment and eventual need forwater reuse is a severe problem both from an energyconsuming and environmental point of view.Contrary to alkaline catalysts, enzymes do not form soapsand can esterify both FFA and TAG in one step without theneed of a subsequent washing step. Thus enzymes are aninteresting prospect for industrial-scale production forreduction of production costs. This is especially the casewhen using feeds high in FFA such as rice bran oil (Lai et al.,2005), inedible Madhuca indica oil (Kumari et al., 2007) orsecond-generation raw materials like spent oils, animal fatand similar waste fractions, with high FFA and water contentand large variation in raw material quality. Besides areduction in the cost of biodiesel as spent oils are lessexpensive than virgin oils (Hsu et al., 2001; Kulkarni andDalai, 2006; Srivastava and Prasad, 2000), the use of wasteCorrespondence to: L. FjerbaekContract grant sponsor: The Danish Council for Strategic Research1298 Biotechnology and Bioengineering, Vol. 102, No. 5, April 1, 2009 ß 2009 Wiley Periodicals, Inc.oils etc. is also commendable as waste is turned into aresource reducing the pressure on farm land otherwise usedfor food production. Unfortunately, waste oils are muchmore complicated and expensive to transform into biodieselwith chemical catalysts (Freedman et al., 1984; Zhang et al.,2003), though Daka Biodiesel A/S (Løsning, Denmark)produces 2nd generation biodiesel from animal fat wastewith a capacity of 55,000 m3biodiesel per year using thisprocess.Enzymes are potentially useful compared to alkaline oracid catalyst, because they are: more compatible with variations in the quality of the rawmaterial and reusable; able to produce biodiesel in fewer process steps usingless energy and with drastically reduced amount ofwastewater; able to improve product separation and to yield a higherquality of glycerol (Fukuda et al., 2001; Kaieda et al., 1999;Kumari et al., 2007; Meher et al., 2006).Drawbacks for the use of enzymes are: low reaction rate (Zhang et al., 2003); their cost (Fukuda et al., 2001; Jaeger and Eggert, 2002;Ma and Hanna, 1999; Meher et al., 2006; Shimada et al.,1999) for industrial-scale use 1,000 US$ per kg comparedto 0.62 US$ (Haas et al., 2006) for sodium hydroxide; loss of activity, typically within 100 days of operation.These are the key issues to be addressed for industrial useof lipases in biodiesel production to be viable.This article presents a detailed review of the use ofenzymes, free or imm., for biodiesel production. Reactionmechanisms and the reported productivity of lipases fortransesterification are discussed. Lipases used together withdifferent kinds of biomass (oils and fats) for biodieselproduction, reaction conditions, and reactor configurations,together with stability/inactivation of the lipases when usedfor multiple cycles, are also included in the