Renewable Energy 34 (2009) 11851194

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Review

Enzymatic biodiesel synthesis Key factors affecting efciency of the process

Miros1awa Szczesna Antczak*, Aneta Kubiak, Tadeusz Antczak, Stanis1aw Bielecki
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland

a r t i c l e i n f o
Article history: Received 13 May 2008 Accepted 16 November 2008 Available online 16 December 2008 Keywords: Biodiesel Lipases Enzymatic transesterication Parameters

a b s t r a c t
Chemical processes of biodiesel production are energy-consuming and generate undesirable by-products such as soaps and polymeric pigments that retard separation of pure methyl or ethyl esters of fatty acids from glycerol and di- and monoacylglycerols. Enzymatic, lipase-catalyzed biodiesel synthesis has no such drawbacks. Comprehension of the latter process and an appreciable progress in production of robust preparations of lipases may soon result in the replacement of chemical catalysts with enzymes in biodiesel synthesis. Engineering of enzymatic biodiesel synthesis processes requires optimization of such factors as: molar ratio of substrates (triacylglycerols: alcohol), temperature, type of organic solvent (if any) and water activity. All of them are correlated with properties of lipase preparation. This paper reports on the interplay between the crucial parameters of the lipase-catalyzed reactions carried out in non-aqueous systems and the yield of biodiesel synthesis. 2008 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. Theoretical background The serious depletion of fossil resources and an increasing societal ecological awareness have led to a search for fuels from renewable sources such as plant biomass. Also the other alternative energy sources such as solar energy, energy of water, wind and gravitation, and energy derived through cleavage of radioisotopes have been intensively studied. All these renewable energy sources do not contribute to increasing carbon dioxide emission and are environment friendly. Plant oils were proposed to be used as a fuel for automobile engines in the beginning of 19th century by Rudolph Diesel [1]. However, it was found that products of their transesterication with methanol or ethanol were much better fuels due to more acceptable physicochemical properties like density and viscosity. Mixtures of methyl and/or ethyl esters of fatty acids (up to several dozen different species) derived through transesterication of triacylglycerols (TAGs) contained in plant oils were named biodiesel. Currently it has been possible to adapt engines and fuels distribution network for using biodiesel instead of petroleum oil. Biodiesel is the only alternative fuel to diesel that meets EPA requirements [2]. 1.2. Advantages of enzymatic catalysis in biodiesel production Chemical catalysts used for industrial biodiesel production (usually NaOH, KOH or sodium methoxide) provide the yield of
* Corresponding author. Tel.: 48 42 6313441; fax: 48 42 6366618. E-mail address: mirszcz@p.lodz.pl (M. Szczesna Antczak). 0960-1481/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2008.11.013

transesterication reaction close to 99% [36]. Despite this excellent productivity the overall biodiesel production is relatively limited, mainly due to such drawbacks as the necessity of application of rened plant oils, problems related to the recovery of pure glycerol (the main by-product) and formation of soaps, mono- and diacylglycerols, and pigments. The residues of all these by-products worsen biodiesel quality. The multi-step purication of transesterication reaction products comprises steps of the neutralization of the catalyst, deodorization and removal of pigments formed at elevated temperature at which the process is conducted (usually 70  C or higher). Besides, the concentration of free fatty acids (FFA) in plant oils used for biodiesel production has to be below 0.5% (otherwise the yield of transesterication is below 99%) [7]. Because the chemical esterication of free fatty acids requires application of the acid catalyst, oils rich in FFA have to be subjected to a 2-step treatment. Other drawbacks are the necessity of application of large amounts of alcohols (their molar excess has to be even several dozen-fold greater than that of plant oil) and complete removal of water. Because the anhydrous methanol is relatively cheap so the industrial production of biodiesel is limited to synthesis of methyl fatty acid esters. Ethyl alcohol produced from plant biomass [8] is available as 95% rectied spirit (azeotropic mixture with water) and requires dehydration, e.g. by using molecular sieve [5], prior to biodiesel production and this step increases process costs. In enzymatic biodiesel production, catalyzed by various lipases, some of the aforesaid drawbacks can be eliminated and therefore enzymatic processes are a promising alternative to the chemical ones. Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are produced by microorganisms (fungi and bacteria), animals and plants [912]. Their commercial preparations are derived mainly from microbial

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sources because of low costs of production and easy modication of properties. Both extracellular and intracellular lipases were used for biodiesel synthesis but the majority of research have been conducted by using commercial preparations (also immobilized) of extracellular lipases [13,14]. Enzymatic synthesis of biodiesel has been usually conducted at temperature between 20 and 60  C. On completion of transesterication process, the lower phase (glycerol) is simply separated from the upper phase (biofuel) and neither deodorization nor neutralization of the product is necessary. This in turn decreases duration of the batch. A small excess of alcohol provides high yield of biodiesel synthesis and the biocatalyst can be used several times (particularly the immobilized lipase). Also continuous enzymatic biodiesel production has been proposed. Lipase-catalyzed transesterication is applicable to rened and raw plant oils, free fatty acids, waste fats from frying, tallow and other waste fats, and various alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. Low water concentrations in reaction medium can have a positive impact on productivity of biodiesel. Concentration of FFA in the oils can be much higher than in case of processes catalyzed by base catalysts. Fats containing triacylglycerols (TAG) and FFA can be enzymatically converted to biodiesel in a one-step process because lipases catalyze both transesterication and esterication reactions (Fig. 1). Enzymatic synthesis of biodiesel can be carried out either in organic solvents or in solvent-free (only the mixture of substrates) systems. Usually, in organic solvent systems, lipases can catalyze conversion of oils (e.g. rapeseed oil, soybean oil) to fatty acid alkyl esters when the overall aliquot of alcohol is added at the beginning of the process. In the solvent-free systems, alcohol has to be added in several small portions to maintain its concentration at a relatively low level [15]. Costs of chemical biodiesel production have still been lower than those of the enzymatic processes, however if the pollution of natural environment is also taken into consideration, these costs are comparable [16]. Production of cheaper, robust lipase preparations and development of systems providing the long-term, iterative use of these biocatalysts can give rise to the replacement of chemical processes with enzymatic ones [57,13,1720]. 1.3. Enzymatic biodiesel synthesis crucial factors Crucial factors affecting productivity of enzymatic biodiesel synthesis are shown in Fig. 2. To achieve the economic viability, the suitable raw materials and lipase preparation have to be chosen. The latter can be modied to improve stability and catalytic efciency. These steps are followed by selection of organic solvent, optimization of molar substrates ratio, temperature, water activity, pH of enzymes microenvironment and the highest permissible glycerol concentration in reaction products (the so named subparameters). 2. Selection of lipase preparation Only few lipases have been found to be capable of efcient biodiesel synthesis and only some of them can catalyze reaction in
CH2-O-CO-R1 I n CH-O-CO-R2 + m R4-COOH + (3n+m) alkyl-OH I CH2-O-CO-R3

both systems: in organic solvent and in solvent-free system (Table 1). The majority of them are commercial preparations of extracellular enzymes, which were immobilized on different carriers [2127]. The preparation of Rhizopus oryzae intracellular lipase is an example of whole-cell biocatalyst used for biodiesel synthesis [2830]. Properties of commercial extracellular lipases and lipases available as whole-cell biocatalysts have been reviewed elsewhere [13,18]. The whole-cell biocatalysts are thought to be cheaper and more robust and thus more appropriate for industrial biodiesel manufacturing. 2.1. Differences in catalytic specicity of lipases Specicity of lipases used for biodiesel synthesis refers to their regiospecicity and specicity with respect to the length of hydrocarbon chain of fatty acid [39]. In terms of regioselectivity, i.e. the position of scissile ester linkage, lipases have been divided into three types: - sn-1,3-specic (hydrolyze ester bonds in positions R1 or R3 of TAG); - sn-2-specic (hydrolyze ester bond in position R2 of TAG); - nonspecic (do not distinguish between positions of ester bonds to be cleaved). Lipases characterized by the narrow regiospecicity are believed not to be applicable to biodiesel production and therefore the majority of lipases used for this purpose display both wide substrate specicity and regiospecicity. Their examples are lipases from Pseudomonas uorescens and Pseudomonas cepacia [29], Candida rugosa, Candida antarctica and Candida cylindracea [12,29]. However, also some sn-1,3-specic lipases, like those from R. oryzae [29] and Mucor miehei [40] efciently catalyze transesterication reactions and their yield exceeds the maximum theoretical yield, i.e. 66% [24]. This surprisingly high productivity of enzymatic transesterication reactions (attaining 90%) results from migration of acyl residue from position sn-2 to terminal positions in glycerol (sn-1 and sn-3). Lipase of Geotrichum candidum cleaves ester linkages in position sn-2 in TAGs with 2-fold higher rate than those in positions 1(3). The regioselectivity of lipases can be affected by various factors and some of them have not been recognized yet. It is known to depend on the structure of substituents in TAG molecules (e.g. preferences of Chromobacterium viscosum lipase change from sn-3 to sn-1 when the acylester in position sn-2 is replaced by alkyl residue linked by the non-ester bond [12]). Under appropriate conditions the yield of transesterication catalyzed by sn-1,3specic lipases can exceed 90% [24,32]. Du et al. [24] found how to achieve high yields of transesterication reactions catalyzed by regioselective lipases. They eliminated the negative impact of 1,3specicity of Thermomyces lanuginosa lipase (preparation Lipozyme TL IM) through addition of silica gel. This increased the rate of migration of acyl residue from position 2 and gave rise to the ultimate biodiesel yield of 90% [24].
CH2-OH I (3n+m) alkyl-O-CORn + n CH-OH + m H2O I CH2-OH

Rn = R1, R2, R3, R4 alkyl = methyl, ethyl, propyl-1, propyl-2, butyl-1, isobutyl
Fig. 1. Enzymatic, one-step (trans)esterication of fats containing free fatty acids with short aliphatic alcohols.

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Stabilization and/or modification of lipase

Choice of lipase Choice of substrates (fats & alcohols)

In organic solvent system Basic parameters

In organic solvent free system

Molar ratio of substrates (alcohol:fat) Temperature Water activity Glycerol concentration pH of lipase microenvironment

Subparameters

Amount of water added

Specific additives
Fig. 2. Crucial parameters affecting the yield of enzymatic synthesis of biodiesel.

Substrate specicity of lipases consists in capability of distinguishing structural features of acyl chains such as the length, the number, position, or conguration of double bonds, or the presence of branched groups, as well as the nature of the acyl source: free acid, alkyl ester, glycerol ester, etc. In reactions of triacylglycerols and alcohols lipases distinguish the length and type of FA contained in TAG and the length of alcohol (in biodiesel production: methanol or ethanol). For instance, R. oryzae lipase distinguishes between FA with different length. Under optimum conditions (in systems with organic solvent and solvent-free) it prefers C18 fatty acids and C2 C4 alcohols [28]. Isoforms A and B of G. candidum lipase display different preferences for acyl residues (the rst shows no preferences and the second prefers monounsaturated cis-9 fatty acids). These examples provide evidence that selection of lipase preparation is of the utmost importance for biodiesel production from various renewable raw materials. 3. Substrates for biodiesel production 3.1. Lipids Raw materials used for biodiesel production comprise all available plant oils (rened and raw, also non-edible), microbial oils and waste fats like that remained after frying, tallow, lard and the greasy by-product from u-3 FA production from sh oils. Chemical composition of some of these raw materials was reviewed in [6,20,41]. In model studies conducted in laboratory scale biodiesel has been produced from free FA (mainly C18:1) and rened oils (rapeseed, soybean, sunower and other). These plant oils are rich in C18 FA. Raw materials used for biodiesel synthesis are derived from oleaginous plants grown in a given geographical region.

Table 1 Microbial producers of lipases used in studies on biodiesel production in systems either containing organic solvent or not. Reaction in organic solvent Pseudomonas uorescens Pseudomonas cepacia Candida antarctica Rhizopus delemar Rhizopus oryzae Mucor miehei Geotrichum candidum Candida rugosa Ref. [21,31,32] [22,32,33] [33] [33] [28,34] [33] [33] [34] Solvent-free system Pseudomonas uorescens Candida antarctica Candida rugosa Rhizopus oryzae Mucor miehei Rhizomucor miehei Thermomyces lanuginosa Aspergillus niger Pseudomonas cepacia Ref. [21,31] [23,33,35] [15,36] [28] [33] [32] [21,32,35,37] [38] [22]

For instance, in South America (Brazil) biodiesel is produced mainly from sunower oil. Its transesterication is efciently catalyzed in organic solvent systems (hexane), by Rhizomucor miehei lipase (Lipozyme RM IM, applicable to butanol and methanol) [42] and by lipases from Aspergillus niger, C. rugosa, P. cepacia, R. oryzae and P. uorescens [32]. In solvent-free systems reactions of sunower oil transesterication with methanol are efciently catalyzed by lipases from R. miehei, P. uorescens and T. lanuginosa [32]. In South America and in the USA biodiesel has been produced through transesterication reaction catalyzed by M. miehei or P. cepacia lipases from soybean oil and methanol (in organic solvent containing systems) or ethanol (in solvent-free systems) or through Rhizopus delemar lipase-catalyzed transesterication with methanol [33]. In solvent-free systems methyl esters of fatty acids were derived from soybean oil by using lipases from C. antarctica (Novozyme 450), T. lanuginosa (Lipozyme TL IM), and R. miehei (Lipozyme RM IM) [35]. In Europe the principal raw material for biodiesel synthesis is rapeseed oil. Its transesterication with methanol and ethanol (in systems with organic solvents) has been catalyzed by lipases from M. miehei [33] and Mucor circinelloides [43]. In solvent-free systems transesterication of rapeseed oil with methanol has been catalyzed by lipases from T. lanuginosa (Lipozyme TL IM) [37,44], M. circinelloides [43] and other. Tallow is a much worse raw material for biofuel production than oils since it is solid at room temperature and is mainly a waste. It was successfully transesteried in hexane with methanol and ethanol by using M. miehei lipase [33]. In a solvent-free system tallow was efciently transesteried with isopropanol and 2butanol by using C. antarctica lipase [33]. Because biodiesel producers try to cut costs of raw materials Watanabe et al. [23] compared effectiveness of enzymatic transesterication of three forms of soybean oil: raw, degummed and pure (rened). The high degree of conversion and unchanged activity of C. antarctica lipase preparation were observed when biodiesel was produced from degummed and rened oils. When the raw oil was subjected to transesterication reaction, the degree of conversion was 3-fold lesser as compared to that of rened oil. For the raw oil the yield of methanolysis was only 10.3% in the rst batch and in the 5th batch it dropped to 7.1%. Watanabe et al. found that phospholipids contained in raw oils negatively affected transesterication reaction catalyzed by C. antarctica lipase. When concentration of phospholipids in reaction medium exceeded 1% the synthesis of fatty acid esters was completely stopped [23]. The preliminary analysis of costs revealed that the use of edible (rened) soybean oil for biodiesel production was not protable due to its high price. Only production of biofuel from useless or waste fats was found to be economically viable. Chinese researchers tried to use oil derived from rice bran for biodiesel production. This oil is cheap, inedible and contains more than 80% free fatty acids. Due to the high FFA content it cannot be used for base-catalyzed biodiesel manufacturing (only the expensive acid catalysts can be used for this purpose) but it can be enzymatically transesteried [45]. Also biodiesel production from waste fats (e.g. from restaurants) has been intensively studied [41]. Another interesting substrate is oil contained in waste activated bleaching earth (it accounts for almost 40% dry mass) being a waste from edible oil rening. Only in Japan 50,000 t of this waste have been deposited annually [46]. Because many different fats can be used for enzymatic synthesis of biodiesel, lipases seem to be competitive catalysts in relation to acids and bases. Another advantage of enzymatic biodiesel production is omitting the steps of initial oil purication (e.g. FFA removal or esterication in a separate process) and time consuming biodiesel purication. Therefore effective and cheap lipase

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preparations ensuring the cost efcient, large-scale transesterication of a given fat have been still looked for. 3.2. Selection of alcohol Alcohols used for biodiesel production comprise methanol, ethanol, propanol, isopropanol, 2-propanol, n-butanol, and isobutanol. Alcohols with higher molecular mass have the higher density and boiling temperature. Among these alcohols methanol and ethanol are the cheapest and produced in the largest scale and therefore they have been mainly used for industrial biodiesel production. However, these two alcohols are the stronger denaturing agents than longer aliphatic alcohols and inactivate enzymes [33]. Besides, the rate of lipase-catalyzed transesterication reaction usually increases with the length of hydrocarbon chain of alcohol. The overall yield of enzyme-catalyzed reaction depends on the interplay between reaction velocity and the rate of enzyme denaturation. The largest permissible aliquots of alcohol added to transesterication reaction mixtures depend on resistance of enzyme to alcohol and water concentration. The simultaneous presence of methanol and water can speed up enzyme denaturation while in systems containing ethanol, propanol, isopropanol, butanol and isobutanol the presence of small amounts of water is necessary [38]. Nelson et al. [33] compared the yields of transesterication of tallow with C1C4 aliphatic alcohols catalyzed by two commercial lipase preparations: Lipozyme IM 60 (M. miehei) and Novozyme SP435 (C. antarctica) which was conducted either in hexane or in a solvent-free system. In hexane, the yields of M. miehei-catalyzed transesterication with methanol, ethanol and isobutanol were higher than 95% (Table 2) while in solvent-free systems reaction yields grew with a length of hydrocarbon chain of alcohol and reached 19% for methanol, 65.5% for ethanol, and 97.4% for isobutanol. By contrast, for P. cepacia lipase preparation (lipase PS-30) used in alcoholysis of palm kernel oil [18] the highest yield of oil
Table 2 Lipase-catalyzed transesterication of fats with various alcohols. Alcohol Methanol Fat Tallow Lipase Mucor miehei IM60

transesterication in a solvent-free system was observed for ethanol (72%) and for C4 alcohols (1- and 2-butanol) the yield was lower. The yield of synthesis of esters of isobutanol and 2-butanol by C. antarctica lipase was higher in a solvent-free system than in hexane (Table 2). Table 2 presents yields of selected processes of fat transesterication with various alcohols catalyzed by different lipases in systems with or without organic solvent. Structure and concentration of alcohol affect operational stability of enzyme, i.e. decide how long the enzyme is capable of catalyzing transesterication. Selection of the reaction system (with or without organic solvent) is based on the operational stability of enzyme (usually organic solvents to some extent protect enzymes from denaturation caused by alcohols). It was emphasized above that one of the advantages of enzymatic transesterication consisted in using alcohols containing some water that were excluded in processes employing chemical catalysts. For instance, raw ethanol produced from lignocellulosic materials or its 95% recticate can be used as substrates in lipasecatalyzed transesterication reactions [8,13,15]. Besides, ethyl esters of fatty acids derived in enzyme-driven processes are superior to their methyl counterparts obtained by means of chemical catalysis because of the lower viscosity and higher cold lter plugging point (CFPP). Data collected in Table 2 provide evidence that selection of alcohol for enzymatic biodiesel synthesis is correlated with properties of enzymatic preparation. 3.3. Selection of molar ratio of the substrates used for transesterication reaction The molar excess of alcohol over fatty acids contained in TAGs always increases transesterication yield but it can also inactivate the enzyme, in particular when the alcohol is insoluble in reaction mixture (it forms emulsion and the size of droplets depends on intensity of stirring). Methanol and plant oils form a solution when

System Solvent-free hexane hexane Solvent-free Petroleum ether Solvent-free tert-butanol Solvent-free Solvent-free Solvent-free hexane Solvent-free Solvent-free Solvent-free Solvent-free hexane Solvent-free Solvent-free hexane Solvent-free Solvent-free hexane Solvent-free

Time [h] 5 5 8 24 24 7 10 48 8 8 5 5 5 24 8 8 16 16 8 8 5 5 8 8 16 16 8 8

Yield [%] 19.4 73.8 94.8 3.0 79.0 91.5 90.0 93.8 15.0 traces 65.5 98.0 83.0 82.0 72.0 35.0 90.3 51.7 24.0 16.0 97.4 98.5 42.0 40.0 96.4 83.8 42.0 40.0

Ref. [33]

Sunower oil Cotton oil Soybean oil Palm oil Coconut oil Ethanol Tallow

Pseudomonas uorescens Candida antarctica (Novozym-435) Pseudomonas cepacia Mucor miehei IM60

[47] [25] [48] [23] [49] [33] [26] [47] [49] [33] [49] [33] [49] [33] [49]

Sunower oil Palm oil Coconut oil Isopropanol Tallow Palm oil Coconut oil Isobutanol Tallow Palm oil Coconut oil 2-butanol 1-butanol Tallow Palm oil Coconut oil

Pseudomonas uorescens Pseudomonas cepacia Candida antarctica SP435 Pseudomonas cepacia Mucor miehei IM60 Pseudomonas cepacia Candida antarctica SP435 Pseudomonas cepacia

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their molar ratio is close to 1:1 (at 40  C). Addition of organic solvent to this mixture increases the solubility of alcohol (therefore it can be used in higher concentrations), protects enzymes from inactivation and ensures one-step enzymatic transesterication. High yields of biodiesel synthesis can be also achieved at stoichiometric concentrations of substrates. For instance, for the molar ratio methanol: sunower oil of 3:1 (in hexane) the degree of fatty acid conversion reached 72% in 24 h when the process was catalyzed by the immobilized P. uorescens lipase [32]. Under the same conditions, the yields of reactions catalyzed by immobilized lipases from Rhizopus miehei, T. lanuginosa and P. cepacia were close to 80% in 48 h reaction [32]. In an efcient one-step process (4 h, 40  C, a solvent-free system) catalyzed by T. lanuginosa IM lipase reaction yields were 75%, 92% and 80% for alcohol: TAGs molar ratio of 3:1, 4:1, 5:1, respectively [37]. However, the activity of lipase preparation dropped by approximately 90% and it could not be reused. Xu et al. [44] determined an effect of methanol: soybean oil molar ratio on the stability of T. lanuginosa IM lipase during transesterication and found that when it was 1.5:1 or higher the enzyme was inactivated. Much more resistant to denaturation by alcohol was lipase of P. uorescens capable of efcient transesterifying (yield above 90%) in a solvent-free system and at alcohol: fat molar ratio of 4.5:1 [21]. Shimada et al. observed inactivation of C. antarctica lipase by methanol and ethanol when alcohol: oil molar ratio was higher than 1:3 [15]. Since an excess of alcohol is a prerequisite of high transesterication yields this substrate is added in portions to reaction mixtures to maintain its concentration at a relatively low level. Soumanou and Bornscheuer [32] added alcohol in 3 successive portions (in a molar ratio to oil of 1:1) when reactions were catalyzed by R. miehei and T. lanuginosa lipases and after 24 h achieved 84% and 40% biodiesel synthesis yields, respectively [32]. The same method was used in processes catalyzed by P. cepacia lipase [22]. It was found that the solubility of alcohol considerably increased when fatty acid esters appeared in reaction mixtures [50]. The optimum molar ratio of substrates used for enzymatic biodiesel synthesis has to be determined separately for a given system: alcoholfatlipase. Usually, in organic solvent systems a slight excess of alcohol (over the stoichiometric alcohol: fat molar ratio of 45:1) is necessary to achieve a satisfactory yield of transesterication process. In solvent-free systems the alcohol should be poured to reaction mixture in successive small portions and the relatively poor and temperature-dependent solubility of alcohols in oils should be taken into consideration in the rst step of transesterication processes. 4. Role of organic solvent and its selection Laane et al. [3,29,51] found that the fundamental criterion describing the inuence of polarityhydrophobicity of organic solvents on enzymatic catalysis was the log P. The optimum organic solvent ensures good solubility of substrates and maintains enzymatic activity. In many cases, the optimum system for enzymatic reaction consists of a mixture of organic solvents [52]. Organic solvents are used in enzymatic biodiesel synthesis in order to increase transesterication rate through improved mutual solubility of hydrophobic TAGs and hydrophilic alcohols. It is to note that the solubility of propanol and butanol in oils is much higher than that of methanol and ethanol, and therefore these two rst alcohols do not need the additional organic solvent [32]. Besides, as it was mentioned above organic solvents protect enzymes from denaturation by high concentrations of alcohols such as methanol and ethanol. Among organic solvents used for enzymatic synthesis of biodiesel the most suitable were found to be hydrophobic ones, such

as isooctane (log P 4.7), n-heptane (log P 4.0), petroleum ether 60120 (log P 3.54.3), petroleum ether 4060 (log P 3.2), nhexane (log P 3.5) and cyclohexane (log P 3.1) [32] as well as tert-butanol with log P 0.83 [53]. Hydrophilic organic solvents strongly interact with the essential water layer coating enzyme molecules and therefore they are much less useful in enzymecatalyzed synthesis reactions [48]. For example, Iso et al. [21] noted that in acetone (log P 0.24) the yield of fatty acid ester synthesis catalyzed by lipases of P. uorescens (AK), T. lanuginosa (TL), and P. cepacia (PS) was low (520%) while in hydrophobic aliphatic hydrocarbons (log P > 3.0) it reached 80%. When the same authors produced biodiesel by using P. uorescens lipase, they found that also application of water miscible, hydrophilic 1,4-dioxane (log P 1.1) ensured the high transesterication yield [21,32]. When concentration of the latter solvent was 50 wt% and 90 wt% the degree of conversion of trioleate to methyl esters was 23 and 70%, respectively [21]. Also the use of the hydrophilic tert-butanol gave rise to efcient transesterication reactions [53]. Synthesis of biodiesel was carried out in numerous organic solvents and yields of this process usually depended on lipase preparation and composition of fat (see Table 3). Recently, processes of transesterication have been also conducted in less conventional solvents, e.g. in supercritical gases like butane [55] and carbon dioxide that is well known for its compatibility with lipases [56]. CO2 is also regarded as a green solvent owing to its low toxicity, non-ammability, and its environmentally benign character. Novozyme 435 efciently catalyzed oleic acid methanolysis in supercritical carbon dioxide (turnover number of 13 molecules/s) [57] but the yield of methanolysis of edible oils like palm oil and groundnut oil was only 6070% even after 8 h [58]. More sophisticated and rather expensive solvents are ionic liquids (ion pairs that are liquid at ambient temperature) that were also used in biodiesel synthesis [59]. Ionic liquids are non-volatile, non-toxic and thermally stable solvents and moreover, their hydrophobicity/hydrophilicity balance can be adjusted depending on the nature of the constitutive cation and anion. Besides, they dissolve many polar and non-polar molecules participating in transesterication processes and can stabilize some enzymes. Optimization of enzymatic biodiesel synthesis can be based on certain methods used to increase activity of lipases in organic solvents, like formation of homogeneous systems through lipase solubilization in the organic solvents (e.g. in reverse micelles [60]), obtaining surfactant coated lipase (e.g. by using dialkyl glucosyl glutamates [61] or PEG [62]) or by using the known lipase-activating substances, such as cyclodextrins, crown ether or other [6368]. Reassuming, the main reason of addition of organic solvents to oilalcohol mixtures consists in improving mutual solubility of these substrates and increasing operational stability of lipase preparations. Organic solvents eliminate the necessity of adding alcohols in portions (in particular methanol which rapidly inactivates enzymatic proteins). However, the removal of organic solvent on completion of the reaction may also be difcult. Therefore the method devised by Koijma et al. [69] who used diesel oil as a solvent in synthesis of fatty acid methyl esters from waste activated bleaching earth is interesting. Since volatility, ammability and toxicity of organic solvents can impair scale-up of biodiesel production, the enzymatic transesterication in solvent-free systems has been intensively investigated. 5. Temperature of enzymatic alcoholysis Optimum temperature for activity of various lipases used for transesterication of oils with methanol, ethanol, propanol,

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Table 3 Enzymatic alcoholysis of oils in various organic solvents. Organic solvent n-hexane Oil sunower sunower sunower sunower edible sunower sunower sunower edible sunower sunower sunower edible sunower sunower sunower waste sunower sunower sunower sunower sunower sunower waste waste triolein cottonseed rapeseed rapeseed Molar ratio alcohol: oil 5:1 3:1 3:1 3:1 3:1a 3:1 3:1 3:1 3:1a 3:1 3:1 3:1 3:1a 3:1 3:1 3:1 3:1a 3:1 3:1 3:1 3:1 3:1 3:1 3:1a 3:1 3:1 6:1 4:1 4:1
a

Lipase Rhizomucor miehei Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Candida sp. Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Candida sp. Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Candida sp. Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Candida sp. Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Pseudomonas uorescens Thermomyces lanuginosa Rhizomucor miehei Candida sp. Candida sp. Pseudomonas uorescens Candida antarctica Thermomyces lanuginosa Candida antarctica

Volume of organic solvent/amount of oil 10 ml/20 mM 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 5 ml/2 g 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 5 ml/2 g 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 5 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 5 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 2 ml/0.2 mmol 5 ml/2 g 5 ml/2 g 90 wt%/30 g 32.5 vol%/18 g 0.75:1 (v/v) tert-butanol/oil 1:1 (v/v) tert-butanol/oil

Temp. 40  C 40  C 40  C 40  C 40  C 40 C 40  C 40  C 40  C 40 C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40  C 40 C 50  C 50 C 35  C 35  C
   

Yield [%] 94% 80% 73% 72% 96% 73% 35% 65% 94% 70% 68% 68% 94% 80% 73% 60% 93% 80% 65% 60% 20% 8% 20% 40% 83% 60% 90% 75% 90%

Ref. [42] [32]

[54] [32]

cyclohexane

[54] [32]

n-heptane

[54] [32]

petroleum ether

[54] [32]

isooctane

acetone

[32]

[54] [54] [21] [48] [53]

Chloroform 1,4-dioxane tert-butanol

Stepwise addition of alcohol.

isopropanol, butanol and isobutanol in hexane ranges between 30  C and 55  C [38]. Studies of Iso et al. [21] who synthesized ethyl oleate by using P. uorescens lipase, showed that in the temperature range 30  C55  C the rate of transesterication reaction increased with temperature. Optimum temperatures for transesterication reactions catalyzed by various lipases in solvent-free systems are in the same range (30  C50  C). Xu et al. observed a rise in biodiesel synthesis rate catalyzed by T. lanuginosa IM lipase (at the molar substrate ratio of 1:1) when temperature gradually rose from 30  C to 40  C but further increment in temperature to 50  C caused no increase in reaction velocity [44]. When the molar ratio alcohol: oil was higher, a rise in temperature had an adverse effect since it caused faster inactivation of T. lanuginosa lipase [44]. Du et al. [37] observed the similar relationship between the temperature, transesterication rate and stability of T. lanuginosa IM lipase in repeated-batch processes. A rise in temperature from 30  C to 40  C speeded up the reaction but also decreased (by around 50%) the enzyme stability in continuous-batch reactions while at 30  C almost 90% of enzymatic activity was maintained after 10 successive batches. Also other studies, carried out at molar ratio alcohol: TAGs of 0.75:1 (solvent-free system), showed that 30  C was the optimum process temperature. The rate of reaction was not increased when the temperature was elevated to 50  C and a decrease in temperature to 25  C resulted in a lower reaction rate [70]. Thus the optimum temperature for enzymatic transesterication results from the interplay between the operational stability of the biocatalyst and the rate of transesterication. It also depends on molar ratio alcohol: oil, type of organic solvent and thermostability of enzymatic preparation.

6. Content of water in transesterication system Water content in reaction mixture can be expressed as water activity (aw) or percentage concentration (%). In non-aqueous media used for biodiesel synthesis the degree of medium hydration is frequently expressed as water activity (aw), dened as a ratio of vapor pressure over the given system versus that over pure water p=pH2 O . Results of numerous studies on enzymatic synthesis of biodiesel indicate that in practice it is more convenient to present the yield of transesterication as a function of percentage water content in reaction system (%) since water is usually added to this system to increase catalytic efciency of enzyme and its concentration is also optimized [15,22,71]. Determination of optimum percentage water content in transesterication system is necessary since waste fats are usually contaminated with water [41]. At the optimum water content, the hydrolysis of ester linkages is kept at the minimum level and this ensures the highest degree of transesterication and yield of biodiesel synthesis. 6.1. Water activity Water activity (aw) of reaction system is a function of water activities of individual components of this system. The value of aw denes the amount of water which is not bound in the system and can be evaporated. It was found that the optimum water activity, corresponding to the highest activity of enzyme was characteristic of both the enzyme and reaction medium composition (it depended on substrates and organic solvent) and the type of enzymecatalyzed reaction.

M. Szcz sna Antczak et al. / Renewable Energy 34 (2009) 11851194 e

1191

Ma et al. [34] used immobilized lipases of C. antarctica and R. oryzae for transesterication of ethyl caprylate with hexanol conducted in diisopropyl ether (at water activity varying between 0.05 and 0.95) and determined the rates of transesterication and hydrolysis of ethyl caprylate. The rst of them was the highest at aw 0.35 and the second at aw 0.750.95 when the catalyst was C. antarctica lipase. For R. oryzae lipase the fastest transesterication was noted at aw 0.05 and a slight increase in aw decreased the rate of this reaction and increased the rate of ethyl ester hydrolysis. The latter reaction was the fastest at aw 0.35. Thus the fastest increase in hexanol caprylate concentration was achieved at aw 0.56 and 0.33 for C. antarctica and R. oryzae lipases, respectively. The similar results obtained by Chowdary et al. who determined the effect of water activity on transesterication of butanol and ethyl butyrate in n-hexane catalyzed by C. antarctica lipase [36]. At high water activity (aw 0.96) synthesis of butyl butyrate was very slow while the hydrolysis of ester was very fast. When aw was decreased to 0.33 the rate of transesterication was considerably increased. Also the rate of butanoic acid formation through hydrolysis was much lower [36]. Application of an in situ immobilized M. circinelloides lipase [72,73] for transesterication of rapeseed oil with ethanol and methanol (in petroleum ether) showed that water activities of all reaction mixture components affected to higher or lesser extent the yield of product synthesis [43,40]. Effects of aw of two of these components (lipid and enzymatic preparation) were determined by means of mathematic modeling (see Figs. 3 and 4) [43,74]. Presented examples provide evidence that water activity has to be optimized separately for each transesterication reaction system because it is affected by values of aw of all constituents of this system. It also relates to the enzyme preparation, particularly when it is hydrophilic. 6.2. Addition of water Water concentration in reaction mixture (usually assayed by Karl-Fischer method and expressed as percentage content, %) is a characteristic and one of the most important factors deciding about lipase-catalyzed transesterication reaction rate and yield of biodiesel synthesis. Numerous studies revealed that addition of a small aliquot of water to enzyme-catalyzed reaction mixture (this aliquot cannot shift the reaction equilibrium in favour of hydrolysis) increased the rate of fatty acid ester synthesis [21,54,71,75]. For example, Noureddini et al. [22] who used P. cepacia lipase (in a solvent-free system) for transesterication of soybean oil with methanol and ethanol found that an increment in the initial water
90 80 70 60 50 40 30 20 10 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Ethanolysis Methanolysis

80 70 60

Yield [%]

50 40 30 20 10 0 0.2 0.3 0.4 0.5 0.6 0.7 Ethanolysis Methanolysis 0.8 0.9

aw of whole-cell lipase preparation

Fig. 4. The yield of processes of methanolysis and ethanolysis of rapeseed oil (o aw 0.530) in organic solvent system [43] as a function of water acivity of Mucor circinelloides whole-cell lipase preparation.

content from 0.01 to 2 g (in the reaction mixture containing 10 g of soybean oil and 3 g of methanol or 5 g of ethanol) increased concentration of free fatty acids. Furthermore, in reaction of ethanolysis they observed a decreased synthesis of esters when water content was above the optimum (0.20.5 g) while for the methanolysis reaction the addition of 0.012 g of water resulted in an increased FFA concentration but did not decrease transesterication yield [22]. Shah and Gupta [71]determined the effect of water content on transesterication of Jatropha oil with ethanol catalyzed by P. cepacia lipase and found that a rise in amount of water added in the range 110% (w/w enzyme) increased transesterication yield. After 5 h it reached 98% for water content of 5% and only 70% when water was not added [71]. The interplay between water added and transesterication and hydrolysis yields is shown in Fig. 5. Shimada et al. investigated methanolysis of waste plant oils catalyzed by C. antarctica lipase in a solvent-free system and found that water generated through ester synthesis from FFA contained in these oils was bound by glycerol and did not impair the transesterication process. Moreover, the concentration of methyl esters gradually increased during the process [15]. Reassuming, water content in reaction mixture is a crucial factor determining the yield of enzymatic biodiesel synthesis. Water activity is a parameter, which more accurately describes the degree of hydration of non-aqueous systems but its assay is more difcult than determination of the percentage of water content because of the necessity of application of specic device. To adjust aw of the overall system to a given value, the substrates, organic solvent and a biocatalyst have to be kept over the saturated, aqueous solutions

acids

Yield [%]

Yield..

alkyl esters

(Optimal amount of water*) increase in amount of water

Fig. 5. The effect of water concentration in reaction mixture on the yield of products of enzymatic transesterication (alkyl esters) and hydrolysis (acids). *) The optimum water concentration depends on the type of lipase preparation and reaction mixture composition.

aw of rapeseed oil
Fig. 3. The yield of processes of ethanolysis and methanolysis of rapeseed oil catalyzed by whole-cell Mucor circinelloides lipase preparation (with aw 0.530) in organic solvent system [43] as a function of water activity of this oil.

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of appropriate salts or dehydrated by using molecular sieves. Low water activity (low content of free water) in reaction mixtures positively affects synthesis of fatty acid esters but the effect of this parameter on the productivity of transesterication is not so unambiguous. Determination of optimum water added to the reaction system (usually at the start of the process) is much easier. This optimum corresponds to such an aliquot of water added, which increases the rate of ester synthesis and concomitantly maintains concentration of FFA in the reaction mixture at the minimal level. 7. Lipase microenvironment Electrostatic charges of polar groups contained in enzyme molecules depend on pH of their microenvironment and affect the rate of reactions taking place in non-aqueous media. Polar groups on amino acid residues in proteins dissolved in aqueous solutions bear positive or negative charges dependently on pH of these solutions. They bear the same charges when the proteins have been transferred to the non-aqueous medium like organic solvent or oil. This phenomenon is termed the pH memory of proteins [76,77]. For that reason, optimization of enzymatic activity in non-aqueous systems relates also to adjustment of pH of their microenvironment [27,78]. Application of enzymes bearing appropriate charges on polar groups (given through preincubation in buffer solutions with suitable pH) in reactions of esterication/transesterication carried out in organic solvents should contribute to the higher catalytic activity of these enzymes. However, there are no reports on such an approach in case of enzymes used for biodiesel synthesis. Regulation of pH memory of the biocatalyst can be necessary when standardization of enzymatic preparation is difcult and it displays too low activity. The adjustment of pH of enzymes microenvironment can also contribute to its increased stability. Therefore enzymatic preparations used for large-scale biodiesel production may need this pre-treatment. 8. The effect of glycerol on enzymatic transesterication Results of studies on the effect of glycerol on efciency of transesterication indicate that it inactivated enzymes, particularly in continuous and repeated-batch processes (repeated batches catalyzed by the same preparation of enzyme). Dossat et al. [31] and Du et al. [37] observed the considerable drop in activity of lipases immobilized in hydrophilic matrices when exposed to glycerol. They found that glycerol molecules were adsorbed on the surface of these carriers thereby forming the hydrophilic coating which made enzyme molecules inaccessible to hydrophobic substrates. Addition of another hydrophilic substance like acetone or silica gel to the reaction system partially removed glycerol from the lipase environment (through absorption on silica gel) and this led to efcient transesterication [31]. Du et al. noticed that also washing of the immobilized lipase with isopropyl alcohol restored its activity since glycerol was removed from the carrier [37]. Another approach neutralizing the adverse inuence of glycerol was proposed by Li et al. [53] who produced biodiesel by using the immobilized preparations: Lipozyme TL IM (T. lanuginosa lipase) and Novozyme 435 (C. antarctica lipase). Reaction of rapeseed oil transesterication with methanol was carried out in a relatively hydrophilic organic solvent (tert-butanol) in which glycerol was well soluble and therefore it did not form lms on the carriers. The tert-butanol is a good solvent not only for glycerol but also for methanol and therefore the latter did not denature the enzymes. This in turn increased the operational stability of both the lipases. Washing with tert-butanol or 2-propanol was proposed by Chen and Wu [79], as an efcient method of regeneration of immobilized C. antarctica lipase (Novozyme 435) used preparations.

9. Conclusions Owing to the necessity of reducing energy production from fossil resources and preventing pollution of natural environment, the newly developed biotechnologies attract attention of manufacturers and politicians. Environmentally safe production of energy and biofuels from renewable materials ranks among the most captivating solutions. Biodiesel production from plant oils by using base catalysts has been widely accepted. The replacement of chemical catalysts with enzymes can make this process even more acceptable due to the following advantages: - Ethanol (preferably 95% recticate) derived from renewable materials can be used for enzymatic biodiesel synthesis, - Certain additional steps that are of crucial importance for chemical synthesis, like saponication or acid-catalyzed esterication of free fatty acids abundant in fat substrates of worse quality, catalyst neutralization, removal of soaps and product purication, e.g. deodorization, are omitted, - Raw oils (no need of rening) and waste fats can be used as lipid substrates, - Enzymatic process is carried out at much lower temperature and consumes much less energy, - Enzymatic preparations can be reused and/or applied in continuous biodiesel synthesis processes if their operational stability is sufciently high, - Biocatalysts are biodegradable and non-toxic unlike some chemical catalysts. Robust and highly active biocatalysts and optimization of enzymatic transesterication conditions have currently been objectives of numerous collaborative research projects. Their results comprise construction (by means of genetic engineering methods) of lipases displayed on microbial cells (lipase-displaying cells) that are also used for transesterication reactions [8082] and lipases characterized by an increased activity and stability in organic solvents and alcohols [83]. Acknowledgements The authors gratefully acknowledge the nancial support of the Minister of Science and Higher Education of Poland for this work under grant No. N N205 1448 33. References
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