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Biotechnological production of biodiesel fuel using biocatalysedtransesterification: A reviewWilson Parawira a

a Institute of Food Science, Nutrition and Family Sciences, University of Zimbabwe, Harare, Zimbabwe

First Published:June2009

To cite this Article Parawira, Wilson(2009)'Biotechnological production of biodiesel fuel using biocatalysed transesterification: Areview',Critical Reviews in Biotechnology,29:2,82 — 93

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Critical Reviews in Biotechnology, 2009; 29(2): 82–93

R E V I E W A R T I C L E

Biotechnological production of biodiesel fuel using biocatalysed transesterification: A review

Wilson Parawira

Institute of Food Science, Nutrition and Family Sciences, University of Zimbabwe, Harare, Zimbabwe

Address for Correspondence: aparawira@yahoo.co.uk

( Accepted 20 January 2009)

Introduction

Biofuels are considered in part a solution to such issues as sustainable development, energy security and a reduction of greenhouse gas emissions. Biodiesel fuel for use in motors with internal combustion and compression ignition is an environmentally friendly fuel similar to petro-diesel in com-bustion properties. Biodiesel is biodegradable, non-toxic and has a low emission profile. The environmental issues concerned with the exhaust gases emission by using fossil fuels also encourages the use of biodiesel which has proved to be far more eco-friendly than fossil fuels (Ranganathan et al., 2008). The use of biodiesel fuel is becoming increas-ingly important due to diminishing petroleum reserves and environmental regulations. Biodiesel is a methyl or ethyl ester of a fatty acid made from renewable biological resources such as vegetable oils (both edible and non-edible), recycled waste vegetable oils and animal fats (Demirbas, 2000; Kinney and Clemente, 2005; Modi et al., 2007; Recep et al., 2000). Thus biodiesel is a renewable fuel derived from: vegetable oils such as sunflower oil, palm oil, castor oil, soybeans oil,

rape seed oil, canola oil, peanut oil, cottonseed oil, Jatropha oil and others; animal fats (beef tallow, lard) and fish oil; or from waste cooking oil and greases. The source for biodiesel production is chosen according to the availability in each of the producing countries. The prices of edible vegetable oils such as soybean oil, rapeseed oil and sunflower oil are higher than those of diesel fuel, therefore waste vegetable oils and non-edible crude vegetable oils should be preferred as potential low-priced biodiesel sources. Since biodiesel comes from renewable sources, it does not contribute to the emission of new carbon dioxide (one of the gases responsi-ble for the greenhouse effect), unlike fossil diesel.

Vegetable oils are a very promising alternative to diesel oil since they are renewable and have similar properties to fossil diesel. The use of vegetable oils as alternative fuels started around 1900, when the inventor of the diesel engine (Rudolph Diesel) first tested peanut oil in his compression-ignition engine (Shay, 1993). However, due to the avail-ability of inexpensive petroleum products, the use of such

ISSN 0738-8551 print/ISSN 1549-7801 online © 2009 Informa UK LtdDOI: 10.1080/07388550902823674

AbstractBiotechnological production of biodiesel has attracted considerable attention during the past decade com-pared to chemical-catalysed production since biocatalysis-mediated transesterification has many advantages. Currently, there are extensive reports on enzyme-catalysed transesterification for biodiesel production; the related research can be classified into immobilised-extracellular and immobilised-intracellular biocatalysis and this review focusses on these forms of biocatalyst for biodiesel production. The optimisation of the most impor-tant operating conditions affecting lipase-catalysed transesterification and the yield of alkyl esters, such as the type and form of lipase, the type of alcohol, the presence of organic solvents, the content of water in the oil, temperature and the presence of glycerol, are discussed. However, there is still a need to optimise lipase-cata-lysed transesterification and reduce the cost of lipase production before it is applied commercially. Optimisation research of lipase-catalysed transesterification could include development of new reactor systems with immobi-lised biocatalysts, the use of lipases tolerant to organic solvents, intracellular lipases (whole microbial cells) and genetically modified microorganisms (intelligent yeasts). Biodiesel fuel is expensive in comparison with petrole-um-based fuel and 60–70% of the cost is associated with feedstock oil and enzyme. Therefore ways of reducing the cost of biodiesel with respect to enzyme and substrate oils reported in literature are also presented.

Keywords: Biodiesel; transesterification; lipase; biocatalysis; vegetable oils; biotechnology; renewable energy

http://www.informapharmascience.com/bty

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Biotechnological production of biodiesel fuel using biocatalysed transesterification 83

non-conventional fuels has only recently become a practi-cal proposition, and has received considerable attention recently worldwide, because of its many advantages and the need to replace fossil fuels which are likely to run out within the present century. Biodiesel derived from surplus edible oils, such as soybean oil, sunflower oil and rapeseed oil, is already being used in the USA and Europe to reduce air pollution, reduce dependency on already-depleting fossil fuels localised in specific regions of the world and to counteract increases in crude oil prices (Agarwal, 2007; Berchmans and Hirata, 2008; Foidl et al., 1996; Ma and Hanna, 1999; Meher et al., 2006; Openshaw, 2000; Sarin et al., 2007). The use of edible oil to produce biodiesel in Africa and other developing continents is not feasible because of the huge gap between the demand and the sup-ply of such oils in the developing world. There is therefore a need to explore alternative non-edible oils for use in the production of biodiesel. To this end, several non-edible oils have already been found to be suitable for biodiesel production (Azam et al., 2005).

The direct use of vegetable oil (triglyceric esters) as a biodiesel is possible but is unsatisfactory for long-term usage in today’s direct and indirect diesel engines (Ma and Hanna, 1999). This is because they have high viscosity (about 10 times that of No. 2 diesel fuel), are contaminated by acid, phospholipids and water; and form gum due to oxidation and polymerisation of free fatty acids during storage and combustion; they deposit carbon on engines and thicken lubricating oil (Banapurmath et al., 2008; Crabbe et al., 2001; Foglia et al., 1996; Openshaw, 2000; Srivastava and Prasad, 2000). Consequently vegetable oils are processed so as to acquire viscosity and volatility characteristics similar to No. 2 diesel fuel (Agarwal, 2007; Demirbas, 2008). The main processing techniques used to convert vegetable oils into the fuel form are blending, pyrolysis, micro-emulsification and transesterification (Demirbas, 2000; Ma and Hanna, 1999). Among these, transesterification is by far the most important step in the production of a cleaner and environmentally safe fuel from vegetable oils and animal fats.

The transesterification (alcoholysis) process

The transesterification (an alternative term that is frequently used is alcoholysis) of vegetable oils is the most popular method of producing biodiesel and it yields the best products (Antolin et al., 2002). It is the reaction of a fat or oil (a trigyl-ceride) with an alcohol to form fatty acid alkyl esters (valuable intermediates in oleo chemistry), methyl and ethyl esters (which are excellent substitutes for biodiesel) and glycerol (Figure 1). To obtain the biodiesel, the esters (biodiesel) in the vegetable oil are separated from glycerine. Transesterification is the process of exchanging the alk-oxy group of an ester com-pound with another alcohol. The overall process is a sequence of three consecutive and reversible reactions, in which diglyc-erides and monoglycerides are formed as intermediate com-pounds (Ma and Hanna, 1999). The stoichiometric reaction requires 1 mol of triglycerides and 3 mol of alcohol (Figure 1).

The reaction is reversible and therefore excess alcohol is used to shift the equilibrium to the products’ side (Barnwal and Sharma, 2005; Meher et al., 2006).

The alcohols that can be used in the transesterification process are methanol, ethanol, propanol, butanol and amyl alcohol, with methanol and ethanol being frequently used. Transesterification as an industrial process is usually car-ried out by heating an excess of the alcohol with vegetable oils under different reaction conditions in the presence of an inorganic catalyst. The reactions are often catalysed by homogeneous catalysts such as an acid or a base, by het-erogeneous catalysts such as metal oxides or carbonates, or by lipase, so as to improve the reaction rate and yield.

The process of transesterification brings about drastic changes in viscosity of the vegetable oil. The high-viscosity component (glycerol) is removed and hence the product has a low viscosity, like the fossil fuels. The biodiesel pro-duced is totally miscible with mineral diesel in any propor-tion. The flash point of the biodiesel is lowered after trans-esterification and the cetane number is improved. The yield of biodiesel in the process of transesterification is affected by several process parameters which include the presence of moisture and free fatty acids (FFAs), reaction time, reac-tion temperature, catalyst type, molar ratio of alcohol and oil, and organic co-solvent addition (Sharma et al., 2008).

Variables affecting the transesterification reaction

In the conventional transesterification of vegetable oils and fats for biodiesel production, the gylceride should have an acid value of < 1 and all materials should be substantially anhydrous. An acid value > 1 requires that the process uses more sodium hydroxide to neutralise the free fatty acids. Transesterification yields are significantly reduced if the reactants do not meet this requirement (Dorado et al., 2002; Freedman et al., 1986; Goodrum, 2002; Ma et al., 1998). The presence of water causes the transesterification reac-tion to partially change to saponification, which produces soap, thus lowering the yield of esters. Saponification also renders the separation of ester and glycerol difficult since it increases the viscosity and forms gels (Berchmans and Hirata, 2008). Free fatty acids and water always produce negative effects, since their presence causes soap formation, consumes catalyst and reduces catalyst effectiveness, all of which result in a low conversion rate (Kasudiana and Saka, 2004). Most of the biodiesel is currently made from edible

CH2–OOC–R1 R1–COO–R′ CH2–OH

CH–OOC–R2 + 3R′OH Catalyst R2–COO–R′ + CH–OH

CH2–OOC–R3 R3–COO–R′ CH2–OH

Triglyceride(vegetable oil)

Alcohol Mixture of alkylesters

Glycerol

Figure 1. Transesterification reaction of triglycerides.

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84 Wilson Parawira

oils by using methanol and alkaline catalyst. However, there are large amounts of low-cost oils and fats that cannot be converted to biodiesel using methanol and alkaline cata-lyst because they contain high amounts of free fatty acids and water. In some instances, crude vegetable oil quality gradually deteriorates due to improper handling and inap-propriate storage conditions, which cause various chemical reactions such as hydrolysis, polymerisation and oxidation to occur. Two-step processes, such as acid-catalysed esteri-fication followed by base-catalysed transesterification, have been developed for these oils in which initially the free fatty acids are converted to fatty acid methyl esters by an acid-catalysed pretreatment and then transesterified using an alkaline catalyst in the second step (Ghadge and Raheman, 2005; Velkovic et al., 2006). A two-stage transesterification process for crude Jatropha curcas L. seed oil with a high content of free fatty acids was studied by Berchmans and Hirata (2008). The first stage was an acid pretreatment proc-ess which reduced the free fatty level to < 1%. The second stage was an alkali-base catalysed transesterification proc-ess which gave a 90% methyl ester yield.

The conversion rate increases with reaction time and temperature, and therefore these factors are important in the transesterification process, which can occur at different tem-peratures depending on the oil used. Generally the reaction is carried out close to the boiling point of methanol (60–70°C) at atmospheric pressure and at a molar ratio (alcohol to oil) of 6:1 (Huaping et al., 2006; Pramanik, 2003; Ranganathan et al., 2008; Srivastava and Prasad, 2000). Freedman et al. (1986) observed that temperature clearly influenced the reaction rate and yield of esters when they investigated transesterification of soybean oil with methanol (6:1) at 32°C, 45°C and 60°C.

The stoichiometric ratio for transesterification requires 3 mol of alcohol per mole of triglyceride to yield 3 mol of fatty esters and 1 mol of glycerol. The transesterification reaction is shifted to the right by using excess alcohol or continuously removing one of the products from the reac-tion mixture. A molar ratio of 6:1 (with alkali as the catalysts) is normally used in industrial processes to obtain yields of methyl esters higher than 98% by weight. Ratios greater than 6:1 do not increase the yield, but tend to interfere with the separation of glycerol because there is an increase in glyc-erol solubility. When glycerine remains in solution, it helps drive the equilibrium back to the left, lowering the yield of esters (Tomasevic and Marinkovic, 2003). When using acid catalysts the desirable product is obtained with 1 mol% of sulphuric acid with a molar ratio of 30:1 at 65°C, and a con-version of 99% is achieved in 50 h.

The effects of catalysts

To make the transesterification process possible, a catalyst in the form of an alkali or acid, or a heterogeneous catalyst such as an enzyme, etc is required (Du et al., 2004; Hama et al., 2004; Noureddini et al., 2005; Oda et al., 2005; Shieh et al., 2003; Zhang et al., 2003a). Bases catalyse the reaction by removing a proton from the alcohol, thus making it more

reactive, while acids can catalyse the reaction by donating a proton to the carbonyl group, thus also making it more reactive.

Alkali catalystThe commercial production methodology frequently uses alkaline media for the transesterification of the oil or fats, in the presence of an alcohol, producing methyl esters of fatty acids and glycerol. The detailed reaction mechanism of alkali-catalysed transesterification has been described as a three-step process (Ma and Hanna, 1999; Meher et al., 2006). Sodium hydroxide or potassium hydroxide is used as the basic catalyst, with either methanol or ethanol as well as the vegetable oil. Sodium hydroxide is less expensive and produces a high product yield and is therefore the one that is widely used in large-scale processing (Demirbas, 2003). The alkaline catalyst concentrations in the range of 0.5–1% by weight yield a 94–99% conversion rate of most vegetable oils into esters (Barnwal and Sharma, 2005; Srivastava and Prasad, 2000).

Alkali-catalysed transesterification is much faster than acid-catalysed transesterification and is also less corrosive to industrial equipment. Consequently it is the most often used process commercially (Agarwal, 2007; Ma and Hanna, 1999; Marchetti et al. 2007). There are several disadvantages in using an alkaline catalysis process, although it does offer high conversion levels of triglycerides to their corresponding methyl esters with short reaction times. The process is energy intensive, recovery of glycerol is difficult, the alkaline cata-lyst has to be removed from the product, alkaline wastewater generated requires treatment and the level of free fatty acids and water greatly interfere with the reaction. The content of free fatty acids present in oils should be lower than 0.5% wt% (Ma and Hanna, 1999) and the water concentration should be limited to 0.1 wt% or less (Hass, 2004). The risk of free acid or water contamination results in soap formation, mak-ing downstream recovery and purification very difficult and expensive (Barnwal and Sharma, 2005; Fukuda et al. 2001). For substrates such as waste cooking oil with a 2% (w/w) normal concentration of free fatty acid, pretreatment of the oil by esterification with alcohol using sulphuric acid is rec-ommended, continuing afterwards with the normal alkali-catalysed transesterification process (Canakci, 2007). For producing diesel, Tiwari et al. (2007) found that the optimum combinations for reducing the free fatty acids of Jatropha curcas oil from 14% to < 1% were 1.43% (v/v) sulphuric acid catalyst, 0.28 (v/v) methanol-to-oil ratio and 88 min reaction time at 60°C compared to 0.16 (v/v) methanol-to-pretreated oil ratio and a 24 min reaction time at 60°C. This process gave an average yield of biodiesel of > 99%.

Acid catalystBiodiesel can also be produced conventionally by using an acid to catalyse the transesterification process. Sulphuric acid, sulphonic acids and hydrochloric acids in methanol are the usual acid catalysts but the most commonly used is sulphuric acid (Demirbas, 2005; Freedman et al., 1986; Ma

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Biotechnological production of biodiesel fuel using biocatalysed transesterification 85

and Hanna, 1999). The mechanism of acid-catalysed trans-esterification of vegetable oil proceeds via protonation of the carbonyl group of the ester. This leads to the carbonation which, after a nucleophilic attack of the alcohol, produces a tetrahedral intermediate. This intermediate eliminates glycerol to form a new ester and to regenerate the catalyst (Meher et al., 2006). Acid catalysts are used if the trigyl-ceride has a higher free fatty acid content and more water. The amount of acid catalyst that is required to be added to the reactor varies from 0.5 mol% to 1 mol%, although some authors have used 3.5% (Zhang et al., 2003b). In general, a 1 mol% of sulphuric acid is a reasonable amount for a final conversion rate of 99% in a time of around 50 h. Although the yields can be high, the corrosiveness of the acids may cause damage to the equipment and the reaction rate can also be low, sometimes taking more than a day to finish (Freedman et al., 1986). According to some authors, the reactions are also slow, requiring typically temperatures above 100°C and more than 3 h to complete the conversion (Meher et al., 2006). For example, Freedman et al. (1986) studied the transesterification of soybean oil in the pres-ence of 1% sulphuric acid with an alcohol/oil molar ratio of 30:1 at 65°C, and the conversion was completed in 20 h. As in the alkali-catalysed transesterification process, if excess alcohol is used in the experiment then a better conversion of the triglycerides is obtained, but recovering glycerol becomes more difficult and that is why the optimal rela-tionship between the alcohol and the raw material should be determined experimentally, taking into account each process as an individual problem. Recently, Guan et al. (2009) investigated the transesterification of corn oil with methanol using ptoluene sulphonic acid in the presence of dimethyl ether. They obtained a 100% oil conversion using a methanol/oil ratio of 6:1 at 80ºC for 2 h in the presence of dimethyl ether.

Heterogeneous catalystsHeterogeneous catalysts, such as enzymes, amorphous zirconia, titanium and potassium zirconias, heterogenised on organic polymers have also been used for catalysing the transesterification of vegetable oils. Huaping et al. (2006) demonstrated the potential of preparing biodiesel from Jatropha curcas oil catalysed by a solid super base of cal-cium oxide with its excellent refining process. When treated with ammonium carbonate solution and calcinated at high temperature, calcium oxide becomes a solid super base, which shows high catalytic activity in transesterification. Under optimum conditions, the conversion of Jatropha cur-cas oil can reach a level of 93%. The heterogeneous catalyst eliminates the additional costs associated with the homoge-neous sodium hydroxide used to remove the catalyst after transesterification.

Lipase catalystRecently, it has been found that enzymatic catalysis spe-cifically synthesise alkyl esters resulting in a high purity product, allows simple recovery of the glycerol, the total

transesterification of the free fatty acids and the use of mild conditions in the process, with yields of at least 90%. The enzymatic process is therefore a sustainable biotechnologi-cal alternative to chemical catalysis (Devanesan et al., 2007; Mamoru et al., 2001; Oznur and Melek, 2002). The advan-tages and disadvantages of the methods of catalysing the transesterification for biodiesel production are summarised in Table 1.

The enzymatic catalysed transesterification can be car-ried out by extracellular and intracellular lipases. Lipase-catalysed transesterification is more appropriate for the production of biodiesel from feedstocks containing high free fatty acids and water, such as waste or recycled oils and greases, because the free fatty acids are directly esterified into biodiesel (Canakci, 2007; Hsu et al., 2003; Nelson et al., 1996; Rivera et al., 2007). Biocompatibility, biodegradability and environmental acceptability of the biotechnological procedures when using lipase as a catalyst are the desired properties in this alternative biodiesel production method (Devanesan et al., 2007; Marchetti et al., 2007). Two major problems in using lipases to catalyse the transesterification process for biodiesel production are that the lipase activity is lower than that of chemical catalysts and the enzyme is liable to deactivation by lower alcohols. Harding et al. (2007) recently made a life-cycle comparative study between the alkali and the enzyme catalysis for the production of biodiesel using five flow-sheet options from rapeseed oil. The life-cycle assessment showed that the enzymatic-catalysed biodiesel production route was environmen-tally more favourable with improvements seen in all impact categories such as global warming, acidification and photochemical oxidation. The lower pressures and temperatures obtained in biological catalysis help give it the more favourable life-cycle assessment results. The advantages and disadvantages of using lipase as catalysts for the transesterification for biodiesel production are given in Table 2.

Table 1. Comparison of the different transesterification methods for the production of biodiesel (Crabbe et al., 2001; Chen and Wu, 2003; Watanabe et al., 2000).

Variable Alkali catalysis Acid catalysis Lipase catalysis

Reaction temperature (°C)

60–70 55–80 30–40

Free fatty acid in raw materials

Saponified products

Esters Methyl esters

Water in raw materials

Interference with reaction

Interference with reaction

No influence

Yields of methyl esters

Normal Normal Higher

Recovery of glycerol Difficult Difficult Easy

Purification of methyl esters

Repeated washing Repeated washing

None

Production cost of catalyst

Inexpensive Inexpensive Relatively inexpensive

Reaction time Short Short (9 h) Long (36 h)

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86 Wilson Parawira

Biocatalysed transesterification for biodiesel production

The enzyme that was found to be capable of catalysing transesterification is lipase (glycerol ester hydrolase EC 3.1.1.3). This enzyme is produced intracellularly and extra-cellularly in several microorganisms, such as Mucor miehei, Rhizopus oryzae, Candida antarctica, Thermomyces lanugi-nous, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas mendocina and Pseudomonas cepacia (Du et al., 2004; Hama et al., 2004; Noureddini et al., 2005; Oda et al., 2005; Shieh et al., 2003). Enzymatic biodiesel produc-tion is possible using both intracellular and extracellular lipases.

In all of the work in the literature on lipases, the enzymes or whole cells are immobilised and used for catalysis since they are usually more stable than free lipases and whole cells. The advantage of immobilisation is that the enzyme can be reused without separation and the immobilised process is highly efficient compared to the use of free enzymes and whole cells (Mateo et al., 2007). Immobilising the enzyme (both intracellular and extracellular) in a suitable biomass support particle (BSP) increases the cost-effectiveness of the enzymatic process. The immobilisation of enzymes extends their utility since immobilisation promotes recovery and reuse, and the development of continuous processes which

reduce processing costs (Hsu et al., 2004a). Immobilisation is also used to increase enzyme stability. However, the cost of enzymes remains a barrier to their industrial implemen-tation (Nelson et al., 1996; Shimada et al., 2002).

Immobilised-extracellular-lipase catalysed transesterification for biodiesel production

Most of the researches on the transesterification of vegeta-ble oils for biodiesel production use the pure commercial enzymes in several reaction media (solvents, the presence of additives, polar ions solutions, supercritical fluids or using the enzyme immobilised on celite and polymers). Several reports using immobilised extracellular lipases have dem-onstrated the high transesterification activity of various fat and oil substrates including low-value feedstocks such as greases. These immobilised lipases catalyse the alcoholysis of animal fats and vegetable oils with primary and second-ary alcohols. For example, Nelson et al. (1996) demonstrated the lipase-catalysed production of biodiesel from soybean oil, rapeseed oil, and tallow and recycled restaurant grease. They established conditions for converting tallow and recy-cled grease to alkyl esters with more than a 95% yield using various commercial lipases. They also developed conditions effective for transesterifying feedstocks high in free fatty con-tent to their respective alkyl esters. Simple alkyl esters for use as a biodiesel were readily produced from tallow and greases using phyllosilicate-sol-gel-immobilised Pseudomonas cepa-cia lipase (Hsu et al., 2001, 2002, 2004a). The final conversion of grease or tallow to alkyl esters was aided by the addition of molecular sieves (0.4 wt% of substrates) to the reaction mix-ture. The phyllosilicate sol-gel-matrix-immobilised lipase effectively converted grease and tallow to ethyl esters with a > 95% yield using ethanol. The immobilised enzyme could be reused at least five times without losing its activity.

Wanatabe et al. (2000) demonstrated the conversion of oils to biodiesel using Candida antarctica lipase immobilised on a macroporous acrylic resin. Shimada et al. (2002) produced esters from vegetable oils using immobilised Candida antarc-tica lipase in a continuous reaction process, which is neces-sary for the economic production of these esters as biodiesel. Lipases from C. antarctica and Thermomyces lanuginosa supported on granulated silica also have high esterification activity for ester synthesis (Christensen et al., 2003). Hsu et al. (2004b) compared the production of alkyl esters from fats and oils using lipase from Pseudomonas cepacia and Thermomyces lanuginosa immobilised in a phyllosilicate sol-gel matrix. At 50°C and 48 h reaction time, immobilised Thermomyces lanuginose lipase gave higher alkyl ester yields (70–100%) from fats and oils regardless of chain length or the degree of unsaturation of the acyl groups in the triacylglycerols than did immobilised Pseudomonas cepacia lipase (20–90%), which preferred unsaturated oils. Both immobilised lipases catalysed ester formation (80–90%) from greases containing a range of free fatty acids (2.6–36%). However, the addition of molecu-lar sieves had no effect on ester yields in the Thermomyces lanuginosa lipase-catalysed transesterification of greases but

Table 2. The advantages and disadvantages of using lipases in transesterification (Chen and Wu, 2003; Fukuda et al., 2001, 2007; Marchetti et al., 2007).

Advantages Disadvantages

Biocompatible, biodegradable and environmental acceptability

Loss of some initial activity due to the volume of the oil molecule

Lipases catalyse more specific reactions than chemical catalysts, and hence produce purer products

The immobilised lipase is deactivated by lower alcohols such as methanol and ethanol

There is the possibility of regeneration and reuse of the immobilised residue, because it can be left in the reactor if the reactive flow is maintained

The number of support enzymes is not uniform

The use of enzymes in the reactors allows the use of high concentrations of them and that allows for longer activation of the lipases

The production of commercial enzymes is still prohibitively costly, although the potential costs are being reduced

Immobilisation of the lipase could protect it from the solvent that could be used in the reaction and that will prevent all enzyme particles getting together

The activity of the lipase is relatively lower than that of chemical catalysts

Separation of the product will be easier using this catalyst, producing a product of very high purity with less or no downstream operations

–

A greater thermal stability of the enzyme due to the native state in which they are used

–

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Biotechnological production of biodiesel fuel using biocatalysed transesterification 87

did improve yields (5–10%) in the immobilised Pseudomonas cepacia lipase-catalysed reactions.

Shah and Gupta (2007) reported transesterification of Jatropha oil with ethanol using Pseudomonas cepacia lipase immobilised on celite. They optimised the process with regard to pH tuning, immobilisation, varying water content in the reaction media, varying enzyme concentration and varying temperature of the reaction. The best yield (98% w/w) was obtained at 50°C in the presence of 4–5% (w/w) water at 8 h. The immobilised biocatalyst could be used four times without any loss of activity.

From the literature, it seems that different enzymes have different requirements for biodiesel synthesis. Hernandez-Martin and Otero (2008) carried out a comparative study of different immobilised lipase preparations for the synthesis of biodiesel from different vegetable oils (sunflower, borage, olive and soybean) in methanol and ethanol. They observed that loss of lipase activity induced by the nucleophile was greater with methanol than with ethanol, and was greater for the sn1(3)regio-specific lipase Lipozyme® (a registered trademark of Thermomyces. lanuginosus lipase from Novo Nordisk, Bargsvaerd, Denmark) TL IM than for the non-specific lipase Novozyme® 435 (a registered trademark of Candida antarctica type B lipase, from Novo Nordisk, Bargsvaerd, Denmark). Maximum conversion rates were obtained with Lipozyme® TL IM with a molar ratio of alco-hol to fatty acid residues of 0.33. In contrast, Novozyme® 435 required at least a 2:1 ratio. Alcoholysis of the vegetable oils was faster with Lipozyme® TL IM than with Novozyme® 435. The use of a high loading of Novozyme® 435 (50% w/w) and a large molar excess of ethanol were required to obtain an initial rate similar to that obtained with Lipozyme® TL IM at a lower enzyme loading (10% w/w) and an equimolar ratio of ethanol and free fatty acid residues. Novozyme® 435 pro-duced quantitative conversions in only 7 h at 25°C, but com-plete conversions were not obtained with Lipozyme® TL IM in a single step process. The three-stage stepwise addition of ethanol yielded 84% conversion to ethyl esters for Lipozyme® TL IM. Hence the choice of Novozyme® 435 to catalyse the transesterification process should always be made.

Immobilised whole-cell-catalysed transesterification for biodiesel production

The use of extracellular lipase as a catalyst requires compli-cated recovery, purification and immobilisation processes for industrial applications (Ban et al., 2001). Consequently, the direct use of a whole-cell biocatalyst of intracellular lipases has received considerable research attention (Devanesan et al., 2007; Kaieda et al., 1999; Li et al., 2007; Matsumoto et al., 2001; Tamalampundi et al., 2008; Fukuda et al., 2008). The cost of the transesterification process using lipase can be significantly decreased by using immobilised intracellular lipase (whole-cell immobilisation) instead of extracellular lipase which demands complex purification stages before immobilisation. Whole-cell biocatalysts are prepared simply by cultivation, and the enzymes trapped inside the cells are

regarded as immobilised and can be separated easily. This can clearly reduce the cost of the transesterification produc-tion as reported by Ban et al. (2001) and Hama et al. (2004; 2007) where they used R. oryzae for the transesterification process of vegetable oils. Yeast cells containing high lipase activity were developed, permeated by air drying and used as whole-cell biocatalysts for methanolysis in a solvent-free and water-containing system (Matsumoto et al., 2001). The methyl ester content in the reaction mixture reached 71% (wt) after 165 h reaction at 37°C with the stepwise addition of methanol. These results indicated that an efficient whole-cell biocatalyst can be prepared by intracellular overproduc-tion of lipase in yeast cells and then permeabilised.

Processes using lipase-producing microorganisms immo-bilised within BSPs as whole-cell biocatalysts are much more straightforward since no purification of lipase is necessary and cell immobilisation can be achieved naturally during batch cultivation. Ban et al. (2001; 2002) found that dried Rhizopus oryzae cells immobilised within BSPs efficiently catalysed methanolysis in the presence of 4–20% water and that the lipase activity of Rhizopus oryzae cells treated with 0.1% glutaraldehyde solution was maintained during the six repeated-batch reaction cycles, with the methyl ester con-tent in each cycle reaching 70–80% within 72 h.

Tamalampundi et al. (2008) recently reported that whole-cell R. oryzae immobilised on to BSPs catalysed the meth-anolysis of Jatropha oil more efficiently than Novozym 435 in their comparative study of immobilised whole-cell and commercial lipases as biocatalysts. The presence of water in Jatropha oil had a significant effect on the rate of methanol-ysis, and Rhizopus oryzae cells exhibited their highest activ-ity in the presence of 5% (v/v) of added water. In contrast to Rhizopus oryzae cells, Novozym 435 activity was inhib-ited by the presence of added water and it needed almost anhydrous media for efficient catalysis. Several researchers (Du et al., 2004; Shimada et al., 1999) have reported that the commercially available Novozym 435 (Candida antarctica lipase B immobilised on acrylic resin) was the most effec-tive catalyst among the lipases tested for the production of biodiesel fuel. However, the laborious and expensive puri-fication processes of this lipase from culture broth restrict its application in biodiesel fuel production on an industrial scale. The direct use of lipase-producing Rhizopus oryzae cells is thus an effective way to reduce the cost of lipase production, which is the main hurdle to the commercialisa-tion of the enzymatic process (Ban et al., 2002; Li et al., 2008; Zeng et al., 2006).

The facilitatory effect of immobilised lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel fuel pro-duction was reported by Oda et al. (2005). Rhizopus oryzae cells producing 1,3positional specificity were cultured with polyurethane-foam BSPs in a scaled-up 20 L air-lift bioreactor. The cells, immobilised within BSPs, were used as a whole-cell biocatalyst in a repeated batch-cycle methanolysis reaction of soybean oil. This repeated batch methanolysis reaction using the dried BSP-immobilised cells maintained a methyl ester content of 65–80% during 20 batch reaction cycles. The

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hydrolytic activity of the whole-cell biocatalyst, on other hand, was stable regardless of the number of reaction cycles. Fukuda et al. (2007) investigated the methanolysis reaction in a packed-bed reactor (PBR) using dried Rhizopus oryzae cells immobilised within BSPs in preparation for a large-scale prac-tical biodiesel fuel production. The reaction was carried out in a glass column (of 25 mm internal diameter and 400 mm in height). The reaction conditions were investigated in an attempt to increase the reaction rate and the lipase stability. Comparison with the methanolysis reaction in a shaken bot-tle suggested that the PBR enhances repeated-batch meth-anolysis by protecting immobilised cells from physical dam-age and excess amounts of methanol. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. When the flow rate was varied between 5 L/h and 55 L/h, they obtained the highest methyl ester content of over 90% at a flow rate of 25 L/h in the first cycle, and over 80% was still being maintained after the tenth cycle. They concluded that this was a promising process for industrial biodiesel fuel production.

Ban et al. (2002) also reported the stable repeated use of whole-cell biocatalysts immobilised within BSPs for biodiesel fuel production. The lipase activity of Rhizopus oryzae cells immobilised on BSPs without glutaraldehyde treatment decreased considerably (to 50% after six batch cycles) in the presence of methyl esters produced by meth-anolysis, but the activity of cells treated with 0.1% glutaral-dehyde solution showed no significant decrease during six batch cycles and the methyl ester in the reaction mixture reached 70–83% in each cycle. Also, good methanolysis of 90% methyl ester yield was reported by Ban et al. (2001) when using Rhizopus oryzae cells immobilised within BSPs as whole-cell biocatalysts for biodiesel production. To enhance the methanolysis activity of the immobilised cells under the culture conditions used, various substrate-related compounds were added to the culture medium, of which olive oil and oleic acid were significantly effective.

The cost of oil sources is another influential factor on biodiesel cost; producing biodiesel from refined vegeta-ble oils is obviously not competitive and therefore there is a need to explore the use of relatively low-cost crude and acidified oils for biodiesel production. Different feedstocks (refined, crude and acidified rapeseed oil) were adopted by Li et al. (2007) for biodiesel production in a tert-butanol system using whole-cell Rhizopus oryzae (IFO 4697) cells immobilised within BSPs as a catalyst. They found that when acidified rapeseed oil was used as a feedstock, the reaction rate and final methyl ester yield were significantly higher than that of refined and crude rapeseed oil. The reaction rate increased with the increase of free fatty acid content in oils, the water content had a varied influence on reaction rate and biodiesel yield, and using adsorbent to remove excess water increased biodiesel yield significantly.

For the industrial transesterification of fats and oils, Pseudomonas sp. immobilised with sodium alginate gel can be used directly as a whole-cell biocatalyst (Devanesan et al. 2007; Foidl et al., 1996; Mohamed and Uwe, 2003;

Yong and Shiyi, 2007). Devanesan et al. (2007) reported a maximum yield (72%) of biodiesel from the transesterifi-cation of Jatropha oil and a short-chain alcohol (methanol on hexane) using a commercial immobilised Pseudomonas fluorescens MTCC 103 in sodium alginate gel as a whole-cell biocatalyst. The optimum conditions for the transesterifi-cation were a temperature of 40°C, pH of 7.0, molar ratio of 1:4, amount of beads of 3 g and reaction time of 48 h.

Biodiesel is usually produced from food-grade vegeta-ble oils that are more expensive than diesel fuel (Canakci, 2007). Therefore, biodiesel produced from food-grade vege-table oil is currently economically infeasible. The feedstock in many studies for biodiesel production was focussed on edible vegetable oils such as sunflower oil, soybean oil or cottonseed oil, but the researches on animal fats are few. Waste cooking oils, restaurant grease and animal fats are potential low-cost feedstocks for biodiesel production and are available in large amounts in many countries. Edible and non-edible lard can be used as biodiesel feedstocks owing to their highly centralised generation in slaughter/process-ing facilities and their historically low prices. Immobilised Candida sp. 99–125 whole cells were successfully used in enzymatic esterification for biodiesel production from lard by Lu et al. (2007). There the optimal conditions for process-ing 1 g of lard were as follows: 0.2 g of immobilised whole cells, 8 mL of nhexane as solvent, 20% of water based on the fat weight, a temperature of 40°C and the three-step addi-tion of methanol. The fatty acid methyl esters yield was 87% and the immobilised Candida sp. 99–125 whole cell proved to be stable when used repeatedly for 180 h.

The display of novel enzymes on the yeast cell surface is a very powerful method for the development of efficient whole-cell biocatalysts, because the diffusion problem of substrate and product is circumvented (Kondo and Ueda, 2004). Kondo and Fukuda (2007) developed a new method for displaying novel enzymes and their applicability to the production of biofuels from biomass. Plasmids were constructed of various lengths for the cell surface display of lipases by fusion with the cell-wall anchoring proteins -agglutinin and flocculin flo 1p, and introduced into yeast cells. The biodiesel production from plant oil and methanol catalysed by yeast cells displaying Rhizopus oryzae lipase was investigated in a water-containing system without an organic solvent. It was found the methyl ester yield reached 80% by the stepwise addition of methanol to the reaction mixture (Matsumoto et al., 2001). The displayed enzymes are regarded as a type of self-immobilised enzyme on the cell surfaces. Cell-surface engineered Saccharomyces cerevi-siae exhibiting the expression of Rhizopus oryzae lipase was successfully used by Ueda et al. (2002) to produce biodie-sel. Matsumoto et al. (2002) constructed a yeast whole-cell biocatalyst overproducing lipase with a pro-sequence from Rhizopus oryzae IFO 4697 and successfully used it for biodie-sel production. Such whole-cell biocatalysts overproducing intracellular lipase can be used to reduce the cost of lipase preparation significantly and offer a promising prospect for industrial biodiesel production.

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Other attempts to improve lipase-catalysed transesterification

Various alcohols are being investigated for the transesteri-fication process using lipase including methanol, ethanol, isopropanol and butanol. However, immobilised lipase is frequently deactivated by lower alcohols such as methanol and ethanol. Pretreatment of immobilised Candida ant-arctica lipase enzyme (Novozyme® 435) for biodiesel fuel production from plant oil was demonstrated by Samukawa et al. (2000) to reduce the deactivation of the lipase. The methanolysis reaction was observed to be much faster when Novozyme® 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. When preincubated Novozyme® 435 was used, the methyl ester content reached over 97% within 3.5 h by the stepwise addition of 0.33 mol equivalent of methanol at 0.25–0.4 h intervals. Improvement in the lipase-catalysed synthesis of fatty acid methyl esters from sunflower oil was achieved by carrying out the alco-holysis in organic solvents (Soumanou and Bornscheuer, 2003). The highest conversion (80%) was found in nhexane and petroleum ether.

Jackson and King (1996) used immobilised lipases as biocatalysts for the transesterification of corn oil in flowing supercritical carbon dioxide and reported an ester conver-sion of more than 98%. But the activity of the immobilised enzyme is inhibited by the methanol and glycerol present in the mixture. The use of tert-butanol as a solvent, the con-tinuous removal of glycerol and the stepwise addition of methanol are found to reduce the inhibitory effects, thereby increasing the cost-effectiveness of the process (Li et al., 2006; Royon et al., 2007; Samukawa et al., 2000).

The effect of alcohol on the enzymatic production of biodiesel from vegetable oils was also investigated. In the absence of organic solvents, Samukawa et al. (2000) and Shimada et al. (2002) have both reported that biodiesel production increased when the methanol concentration increased up to an oil-to-methanol ratio of 3:1 equivalents, and decreased when methanol concentration was further increased. Effective methanolysis using extracellular lipase has been reported to improve with the stepwise addition of methanol through which a 90–95% conversion can be achieved even after 50 and 100 cycles of repeated operation (Samukawa et al., 2000; Shimada et al., 2002; Soumanou and Bornscheuer, 2003; Watanabe et al., 2000).

There have been several further attempts to overcome the problem of deactivation of immobilised enzyme by lower alcohols such as methanol and ethanol, and to improve the lipase activity. For example, Nelson et al. (1996) used hexane as a diluent to prevent the deactivation by lower alcohols. Samukawa et al. (2000) attempted to maintain a very low concentration of methanol during the reaction. However, these methods have some inherent problems. Using a diluent decreases the reaction rate and the precise control necessary to maintain the methanol concentration at a very low level is not appropriate for the production of biodiesel on a large scale. From an economic point of view, a

continuous reaction process without the use of any organic solvent is needed for the industrial production of biodiesel. A better solution is to regenerate immobilised lipase for transesterification when its activity becomes lower than a set value. Immobilised lipase is frequently deactivated by lower alcohols with the deactivation being caused by the immis-cibility between triglycerides and methanol and ethanol. When these lower alcohols are adsorbed to the immobilised enzyme, the entry of triglycerides is blocked, which causes the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-butanol, can regener-ate the deactivated immobilised enzyme. Chen reported that the activity of immobilised lipase could be significantly increased when such alcohols were used for an immersion pretreatment of the enzyme. The activity of Novozyme® 435 increased about ten-fold in comparison to the enzyme not being subjected to any pretreatment. Following the complete deactivation of lipase by methanol, washing with 2-butanol and tert-butanol successfully regenerated the enzyme and restored it to about 56% and 75% of its original activity level, respectively.

Apart from substrate concentrations, temperature has also been reported to affect the rate of the transesterification reaction considerably. The trend observed in all studies that investigated the effect of temperature on the production of biodiesel by lipase was that initially the reaction increased as the temperature increased. However, the reaction rate decreased sharply at the onset of denaturation of the enzyme in all the studies that investigated the effect of temperature on the production of biodiesel by lipase (Al-Zuhair et al., 2003). The optimum operating temperature for lipases from different sources is reported to be approximately 40°C.

Biodiesel production can also be improved by using a mixture of lipases. Kim et al. (2007) optimised biodiesel pro-duction by using a mixture of immobilised Rhizopus oryzae and Candida rugosa lipases. The ratio of Rhizopus oryzae and Candida rugosa lipases in the mixture was optimised to 3:1 (w/w). It was found that when 3 mmol of methanol was initially added to the reaction medium and 3 mmol of methanol was added every 1.5 h during biodiesel produc-tion, biodiesel conversion reached over 98% at 4 h. In addi-tion, when the mixture of immobilised lipases was reused, biodiesel conversion was maintained at levels exceeding 80% after five reuses.

Room-temperature ionic liquids (organic salts entirely composed of ions) are recently emerging as desirable sub-stituents for volatile, toxic and flammable organic solvents, which are major causes of environmental pollution. Ha et al. (2007) reported improved lipase-catalysed biodiesel pro-duction from soybean oil in ionic liquids. Among tested 23 ionic liquids, the highest fatty acid methyl esters production after 12 h at 50°C was achieved in [Emim] [TfO].” [Emim] [TfO] from C-TRI (Suwon, Korea) was one of the 23 ionic liquids used for production of fatty acid methyl esters and compared with tert-butanol, a commonly used solvent. The production yield of 80% was eight times higher compared to the conventional solvent-free system, and it was 15% higher

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than the fatty acid methyl esters production system using tert-butanol as an additive. The effect of substrate molar ratio (methanol: soybean oil) fatty acid methyl esters production was also investigated. The optimum substrate molar ratio of methanol to soybean oil for fatty acid methyl esters produc-tion in [Emim] [TfO] was found to be 4:1. Methanolysis in [Emim] [TfO] was significantly decreased when molar ratio of methanol to soybean oil was increased to 8:1 and greater. This might be explained by the inactivation of Novozym 435 caused by high methanol concentration. These high produc-tion yield results in ionic liquids show that they are potential reaction media for biodiesel production. However, ionic liquids are considered to be expensive solvents and their use would increase the cost of biodiesel production.

Optimisation of the reaction parameters involved in lipase-catalysed biodiesel production is commonly made by varying one factor at a time and keeping the others constant (Hsu et al., 2003; Iso et al., 2001; Salis et al., 2005, 2008). But this classical method is inefficient as it fails to explain the relationships between the variables and the responses when there are interactions between the variables. Response sur-face methodology (RSM) is an effective statistical technique for the investigation of complex processes like lipase-cata-lysed biodiesel production because it has many advantages (Rodrigues et al., 2008). The use of RSM for optimising enzy-matic biodiesel production has been reported by various authors. Chang et al. (2005) investigated the alcoholysis of canola oil by Novozym 435 using RSM and central compos-ite design (CCD), and obtained a methyl ester yield of 99% under optimum conditions. Shieh et al. (2003) studied the optimisation of lipase-catalysed synthesis for biodiesel using RSM in combination with CCD (soybean oil methyl ester) by Lipozyme IM77 and obtained a maximum molar conver-sion of 92%. Dermirkol et al. (2006) reported the optimisa-tion of enzymatic methanolysis of soybean oil by RSM using Lipozyme RM IM. Rodrigues et al. (2008) optimised lipase-catalysed ethanolysis of soybean oil in a solvent-free system using CCD and RSM and obtained a yield conversion of 96% under the optimum conditions with a relatively low enzyme content and in a short time. The improvement of biodiesel production by lipozyme TL IM-catalysed methanolysis using RSM and an acyl migration enhancer was reported by Wang et al. (2008). They optimised the process of biodiesel production from corn oil catalysed by lipozyme TL IM with regard to the effect of enzyme dosage, the ratio of tbutanol to oil (v/v) and the ratio of methanol to oil (mol/mol) on the methyl esters yield of the methanolysis. Waste oil was found to be a suitable feedstock and could yield 93.7% methyl esters under the optimum conditions that they created. Adding triethylamine, an acyl migration enhancer, could effectively improve the methyl esters yield of the methanolysis of corn oil from 85% to 92%.

Using immobilised whole cells such as Rhizopus oryzae as catalysts for biodiesel production has many advan-tages; however, the stability of whole-cell biocatalysts was reported to be poor in solvent-free systems. Li et al. (2008) investigated the factors influencing the stability of whole

cells during biodiesel production in a solvent-free and tert-butanol system. During repeated Rhizopus oryzae whole-cell catalysed biodiesel production, whole-cell stability was still poor even with the stepwise addition of methanol, while it improved considerably in a tert-butanol system compared to that in a solvent-free system. The difference in whole-cell stability was found to be due to the differences of product accumulation between the solvent-free and the tert-butanol systems. After a 144 h reaction, glycerol and methyl ester accumulated in the cell in the solvent-free sys-tem to an extent of about 1000 mg/g and 350 mg/g dry mass, respectively, while in the tert-butanol system, the glycerol and methyl ester were maintained at a relatively low level of approximately 100 mg/g and 2 mg/g dry mass, respectively. They concluded that the accumulated glycerol influenced the whole-cell stability through mass transfer only, while the accumulated methyl ester influenced whole-cell sta-bility through both mass transfer limitations and product inhibition.

Research gaps

The studies on biodiesel production from vegetable oil using lipase in the literature are purely experimental. Kinetic information on the rate of product formation and the effects of changes in operating conditions are essential for the designing of suitable reactors, control systems and process optimisation. There are only limited reports on the kinetics of transesterification reactions in the literature (Al-Zuhair et al., 2006, 2007; Jansen et al., 1999). Experimental deter-mination of the separate effects of palm oil and methanol concentrations on the rate of their enzymatic transesterifi-cation was used to propose the Ping Pong Bi Bi mechanism as the suitable kinetic model for the production of biodiesel (Al-Zuhair et al., 2007). The reactions were carried out in an n-hexane organic medium with lipase from Mucor miehei. Kinetic studies reported in the literature investigated the esterification of free fatty acids rather than the transesterifi-cation of triglycerides (oil) which is the substrate of industrial interest (Jansen et al., 1999; Watanabe et al., 2002). There is a need for more kinetic studies on the production of biodiesel from vegetable oils using lipase in single- and multi-step processes.

There is also a need to carry out experiments on lipase catalysed in pilot-scale or full-scale reactors to confirm the benefits of using lipase-catalysed transesterification. Most of the experimental data in the literature were obtained using reactors ranging from 2 mL (Eppendorf tubes) to 50 mL (Ban et al., 2001; Iso et al., 2001; Shah et al., 2004; Watanabe et al., 2002) with the exception of the excellent work in a 20l packed-bed reactor by Hama et al. (2007). The mixing was efficiently done using orbital shakers or magnetic stirrers and therefore cannot replicate the large-scale industrial reactors.

Studies in the literature have shown that different enzyme preparations have different requirements for the synthe-sis of biodiesel from different vegetable oils and therefore transesterification reactions need to be optimised for each

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application. Several studies have shown the possibility of recycling the biocatalysts when immobilised, but the reac-tion time is still unfavourable when compared to the alkali-catalysed transesterification process. Some critical enzyme properties always must be improved before their implemen-tation at an industrial scale, such as stability, activity, inhibi-tion by reaction products and selectivity.

Biodiesel is usually produced from food-grade vegetable oils that are more expensive than diesel fuel. With the cur-rent shortage of food and the high prices worldwide, there is a need to focus research on non-food-grade oils, restaurant waste oils and on rendered animal fat as low-cost feedstocks for biodiesel production. There are large amounts of restau-rant waste oils and rendered animal fats in many countries that are potentially available for biodiesel production. Waste restaurant oils and animal fats have relatively high levels of saturation, are highly contaminated with free fatty acids and moisture in varying amounts, and the technique for biodie-sel production from them needs to be improved. Levels of free fatty acids were reported to vary from 0.7% to 41%, and moisture from 0.01% to 55% in waste cooking oils, restaurant grease and animal fat by Canakci (2007). These wide ranges indicate that an efficient process for converting waste grease and animal fats which tolerate a wide range of feedstock properties needs to be developed.

Conclusions

Developing alternative renewable energy sources to replace traditional fossil fuel has recently become more and more attractive due to the high energy demand, the limited resources of fossil fuel and environmental concerns. Of the several methods available for producing biodiesel, trans-esterification of vegetable oils is currently the method of choice. Generally, biodiesel is produced by alkali-catalysed transesterification of the oils. There are several difficulties associated with the chemical methods currently used to produce biodiesel. Therefore the enzymatic method is pre-ferred over chemical methods because, among other advan-tages, it produces a high-purity product and enables easy separation from the by-product, glycerol. Lipases of various origins have been widely employed to catalyse the transes-terification of vegetable oils under different conditions. The present paper presents an overview of the biotechnological production of biodiesel that has been reported in the litera-ture. Since biocatalysts are more expensive than traditional chemical catalysts, it is essential to be able to recycle them so as to overcome economical constraints. This is possible through enzyme immobilisation on solid support, which allows continuous processes. Methanol, the most frequently used alcohol, can be enzyme denaturing and mixes poorly with the oil or the fat. In order to overcome these drawbacks, a multi-step addition of methanol to the reaction medium has succeeded in improving the lipase-catalysed transes-terification of vegetable oils to biodiesel.

Optimisation of the reaction parameters involved in lipase-catalysed biodiesel production is commonly made by

varying one factor at a time and keeping the others constant. RSM and CCD analyses were successfully used by many authors to further optimise enzyme-catalysed biodiesel pro-duction in solvent-free systems.

Both immobilised extracellular lipase and whole-cell intracellular lipase can be used to catalyse the transesteri-fication process. Several researches have demonstrated that immobilised Rhizopus oryzae whole cells could efficiently catalyse the methanolysis of refined vegetable oils for biodie-sel production in solvent-free systems. The stepwise addi-tion of methanol was recommended so as to minimise the negative effect of methanol on Rhizopus oryzae whole cells. However, the stability of the whole cell during repeated uses was poor. In many studies, tert-butanol was demonstrated as an ideal media for biodiesel production, in which the stability of the biocatalysts could be enhanced significantly. A large percentage of the biodiesel fuel cost is associated with feedstock oil and enzyme. The cost of enzyme remains a barrier to its industrial implementation and much effort is being made to improve the cost-effectiveness of the process. Several research programmes have been reported which are searching for a lipase which is readily available as an industrial enzyme and which works well for catalysing the transesterification. The high cost of the enzymes that are currently being investigated should not be allowed to thwart the attempts of carrying out more extensive research to identify the most promising ones and to determine the optimal conditions for their application. In fact, the results of such research should provide the incentive for the com-mercial development aimed at finally producing the lipase enzymes economically on a large scale. The costs of new lipase development are expected to decrease as technol-ogy and techniques advance, as fermentation becomes optimised and as less-expensive growth substrates are explored for the cultivation of the parent microorganisms and as improvements are made in downstream process-ing. Utilising immobilised whole-cell biocatalysts instead of immobilised lipase is a feasible way to reduce the costs of the enzymatic process since it can avoid the complex procedures of isolation, purification and immobilisation. Several attempts are being made to explore ways of reduc-ing the cost of raw material including using waste or used oil and the use of non-edible oils. In general, biocatalysts offer promising prospects for industrial biodiesel produc-tion, although much further work, such as scaling up and optimization, require further investigation.

Acknowledgements

Declaration of interest: The author reports no conflicts of interest.

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