Biotechnological Methods to Produce Biodiesel

C H A P T E R

13 Biotechnological Methods to Produce Biodiesel Denise Maria Guimara˜es Freire*, Joab Sampaio de Sousa, Elisa d’Avila Cavalcanti-Oliveira Universidade Federal do Rio de Janeiro, Instituto de Quı´mica, Av. Athos da Silveira Ramos, 149 - CT, Bloco A, lab. 549-1, CEP 21941-909 Rio de Janeiro, RJ, Brazil *Corresponding author: E-mail: [email protected]

1 INTRODUCTION The process currently being used for industrial-scale biodiesel production makes use of an alkali catalyst (usually NaOH, KOH, or sodium methoxide) in the transesterification of triacylglycerol (TAG) with methanol. Industry has favored this process because of the high conversion obtained in a short time and the low cost of the catalysts. However, there are some drawbacks in the process that have encouraged researchers and business people to look into different biodiesel production methods. The choice of catalyst is fundamental, as this determines the characteristics of the raw material, the reaction conditions, and the purification steps in the process, as shown in Table 1. The use of enzymes (lipases) as catalysts in biodiesel production overcomes the problems inherent to alkali catalysts. Lipases are a group of enzymes that are initially described by their capacity to catalyze the hydrolysis of ester bonds in long-chain TAGs, producing free fatty acids (FFAs) and glycerol. For this reason, they have been defined as glycerol ester hydrolases (E.C. 3.1.1.3). These enzymes not only catalyze the cleaving of carboxyl-ester bonds (hydrolysis), but can also catalyze the reverse reaction (esterification and transesterification) in water-restricted systems. However, despite the advantages of using enzymes (table 1), biodiesel production plants using lipases are not yet an industrial-scale reality. The reason for this is that there are some challenges that are yet to be overcome before biocatalysts can be made feasible for biodiesel production, such as their higher cost, biodiesel productivity, and inhibition by reactants and products.

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TABLE 1 General Considerations About the Different Types of Processes for Biodiesel Production (Al-Zuhair, 2007; Balat and Balat, 2010; Fijerbaek et al., 2009; Loreto et al., 2005; Marchetti et al., 2007; Nielsen et al., 2008; Zhang et al., 2003a,b) Type of Process

Composition of Raw Material

Reaction Conditions

Purification Step

Alkaline catalyst

• The raw material must be of high quality in order maintain the yield of the process: free of FFAs (<0.5%) and water (<0.1–0.3%); • FFAs react with the catalyst and form soap; • The water promotes hydrolysis of the alkyl esters to FFAs; • The raw material of high quality can represent 70–95% of the final cost of biodiesel

• High conversion (
99%); • Short reaction time (90 mim); • Temperature around 60 C; • Molar ratio methanol/oil 6:1; • 1% of catalyst based on the mass of oil; • Homogeneous and lowcost catalyst

• If there is soap formation, the separation step become unfeasible; • The catalyst is usually homogeneous and can not be reused; • The catalyst has to be removed from the product and a large volume of alkaline wastewater is generated and must be properly treated; • Glycerol (co-product) is contaminated with salts of catalyst neutralization, exhibiting low sale value

Acid catalyst

• Low-quality low-price raw materials can be employed; • FFAs of raw material are esterified to biodiesel, but the presence of water may diminish the reaction conversion

• High conversion (>90%); • The transesterification of TAGs is slow, only the esterification of FFAs is rapid; • Temperature of 60-120 C; • Homogeneous and lowcost catalyst; • The acid catalyst is corrosive to the equipment

• The catalyst is usually homogeneous and can not be reused; • Neutralization and removal of the catalyst;

Enzymatic catalyst

• Low-quality low-price raw materials can be employed; • FFAs are converted into biodiesel, without loss of raw material; • For some lipases, the water does not negatively interfere in reaction conversion;

• High conversion (>95%); • Mild reaction conditions (30–40  C and atmospheric pressure); • Long reaction time (8-72h); • Low energy consumption; • High costs of the biocatalyst

• Easy separation of biodiesel and biocatalyst by filtration; • Easy separation of biodiesel and glycerol by decanting; • Fewer steps process; • The immobilized enzyme can be reused; • Glycerol is of high quality and has a high sale value; • Enzymes are biodegradable

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TABLE 1 General Considerations About the Different Types of Processes for Biodiesel Production (Al-Zuhair, 2007; Balat and Balat, 2010; Fijerbaek et al., 2009; Loreto et al., 2005; Marchetti et al., 2007; Nielsen et al., 2008; Zhang et al., 2003a,b)—Cont’d Type of Process Non-catalytic (supercritical)

Composition of Raw Material • Low-quality low-price raw materials can be employed; • FFAs are converted into biodiesel, without loss of raw material

Reaction Conditions

Purification Step

• High conversion (98%); • Short reaction time (7–15 min); • High temperature (250-300 C) and pressure (10-25 MPa); • High energy consumption; • Possible generation of thermal degradation products; • No catalyst cost

• No catalyst separation step; • Easy separation of biodiesel and glycerol by decanting

2 ENZYMATIC TRANSESTERIFICATION Transesterification is a term that is widely used to describe an important class of organic reactions, where one ester is converted into another. This transfer of an acyl group can happen between an ester and an acid (acidolysis), one ester and another ester (interesterification) or between an ester and an alcohol (alcoholysis; Gunstone and Herslo¨f, 2004). In broad terms, the transesterification reaction between TAGs and alcohol to produce biodiesel is a sequence of three consecutive and reversible reactions, by which DAG and MAG are formed as intermediates. There are some factors that influence conversion by enzymatic transesterification, such as the substrate used (TAG and alcohol), the molar ratio between the substrates, the water content in the reaction medium, whether a solvent is used, the temperature, whether the enzyme is free or immobilized, the lipase concentration, and others. Despite the many reports in the literature describing biodiesel synthesis using different lipases, it is hard to make any generalizations about the optimal reaction conditions because lipases from different sources tend to respond differently to changes in the reaction medium (Bajaj et al., 2010; Jothiramalingam and Wang, 2009).

2.1 Alcohol Biodiesel can be produced using primary short-chain alcohols like methanol, ethanol, propanol, and butanol, as well as secondary alcohols like isopropanol and 2-butanol (Fijerbaek et al., 2009). The prerequisites for selecting the alcohol for industrial-scale biodiesel production are that it must be cheap and in plentiful supply. Currently, only methanol and ethanol meet these two requirements, and of these two substances, methanol is the more widely used as it is cheaper and more readily available in most countries although ethanol has the dual

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13. BIOTECHNOLOGICAL METHODS TO PRODUCE BIODIESEL

advantages of being renewable and less toxic (Antczak et al., 2009; Deng et al., 2005; Demirbas, 2007). Meanwhile, in enzyme catalysis, it is generally the case that the shorter the alcohol chain, the more likely it is to deactivate the lipase. It is believed that this is because it strips off the microlayer of water surrounding the lipase, which is essential for the optimal conformation of the enzyme (Fijerbaek et al., 2009; Zheng et al., 2009). Shimada et al. (1999) studied the transesterification of vegetable oil with methanol catalyzed by an immobilized lipase from Candida antarctica (Novozym 435) in a solvent-free system. What they found was that when the methanol concentration exceeded its solubility level, it deactivated the lipase irreversibly. To prevent lipase deactivation and keep the stoichiometric ratio required for the total conversion of the TAG, the reaction involved adding methanol in three steps. This three-step process converted 98.4% of the oil to its corresponding methyl esters in 48 h, and the immobilized lipase was able to be reused 50 times. In another study, Watanabe et al. (2000) used a two-step strategy for adding the alcohol. They started by adding one-third of the alcohol at the beginning of the reaction, but then found that after it had been converted into biodiesel (10 h reaction time), they could add the rest of the alcohol in a single step, since its solubility was raised by the presence of the biodiesel. As such, the reaction time was reduced to 36 h and the enzyme was reused 70 times, achieving >95% conversion. This stepwise addition of a short-chain alcohol was adopted by researchers investigating other lipases, such as Candida sp. (Lu et al., 2007), Pseudomonas fluorescens (Soumanou and Bornscheuer, 2003), Rhizopus oryzae (Chen et al., 2006). Shimada et al. (2002) explained the lower enzyme deactivation by longer-chain alcohols (>3 carbons) by the fact that they are more apolar and more soluble in oil at the stoichiometric ratio. However, as already mentioned, each lipase has different properties. With P. fluorescens, high conversion (>90%) was possible with 4.5 molar equivalent of methanol added at the beginning of the reaction (Soumanou and Bornscheuer, 2003). In another study (Salis et al., 2008) the use of two lipases, from P. fluorescens and Pseudomonas cepacia (now Burkholderia cepacia), resulted in 58% and 37% conversion, respectively, in the presence of 1:8 oil/methanol molar ratio in a solvent-free system, while another six lipases tested were completely inactive under these conditions. The excess alcohol above and beyond the stoichiometric ratio increases the reaction rate, but may also deactivate the enzyme, compromising the number of times the enzyme can be reused or even the conversion of the reaction when enzyme deactivation is more severe (Antczak et al., 2009). There are also some arguments against using excess alcohol in industrial-scale processes, such as higher energy consumption, larger equipment requirements, and the need to treat the unreacted alcohol (Fijerbaek et al., 2009). To prevent the alcohol deactivating the enzyme, many researchers have used organic solvents in the reaction medium in a bid to increase the solubility of the alcohol and reduce its concentration. (Iso et al., 2001; Mittelbach, 1990; Nelson et al., 1996; Ranganathan et al., 2008; Royon et al., 2007).

2.2 Water Content It is known that the water content in nonaqueous media affects the activity of enzymes, reducing their rigidity and consequently enhancing their activity. When biodiesel production is catalyzed by lipases, if the water content exceeds the optimal concentration, biodiesel

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conversion is affected because a competing inhibition reaction takes place that enables the hydrolysis of the TAGs, DAGs, MAGs, and alkyl esters (Shah et al., 2004). The ideal water content in the reaction medium varies greatly depending on the enzyme and the reaction medium, and so must be studied on a case-by-case basis. For example, Kaieda et al. (2001) found that the water concentrations that resulted in the best conversions were 8-20% for Candida rugosa lipase, 4-20% for P. fluorescens lipase, and 1-2% for P. cepacia lipase. Deng et al. (2005) tested several immobilized commercially available lipases and found that with the exception of C. antarctica, the conversion obtained from the transesterification reaction with all the others (Thermomyces lanuginosus, Rhizomucor miehei, P. cepacia, and P. fluorescens) was higher when anhydrous ethanol was replaced with hydrous ethanol (4% water). Kaieda et al. (1999), who used a R. oryzae lipase and the stepwise addition of methanol, observed that the addition of 4-30% water in proportion to the substrate mass resulted in higher conversions. It is also very important to take account of the water present in the reagents and even in the enzyme in order to design appropriate reaction medium. Studies of lipase reutilization at different water concentrations have to be carried out since water can influence enzyme stability, making it crucially important for designing an economically feasible process (Deng et al., 2005; Triantafyllou et al., 1995). Some authors have noted that adding water to the reaction medium can protect lipases against deactivation in the presence of short-chain alcohols (Kaieda et al., 1999; Kaieda et al., 2001; Noureddini et al., 2005; Pizarro and Park, 2003). Those lipases that respond well to reaction media with a higher water content are of interest for use with raw materials containing water, as this would rule out the need for a dehydration pretreatment stage. For example, exchanging anhydrous ethanol for ethanol containing 5% water, which is cheaper, had no impact on the transesterification reaction catalyzed by a P. cepacia lipase (Shah and Gupta, 2007). However, the water content in the biodiesel must be kept within the specifications required by law. Thus, unless the raw material already contains water, it is preferable to maintain a low water concentration in the reaction medium (Deng et al., 2005).

2.3 Organic Solvent Use Organic solvents are used in the enzymatic production of biodiesel to obtain a homogeneous reaction medium by ensuring greater solubility of both the hydrophobic compounds (like TAG and biodiesel) and the hydrophilic compounds (e.g., alcohol and glycerol). Solvents also serve to reduce the viscosity of the reaction medium, enabling a higher diffusion rate to be achieved and reducing mass transfer problems (Fijerbaek et al., 2009). A suitable solvent must therefore be found, which both enhances the catalytic activity of the enzyme and keeps it stable. Soumanou and Bornscheuer (2003) studied methanolysis using six different solvents and found that for the apolar solvents (hexane, cyclohexane, n-heptane, isooctane, and petroleum ether) the three lipases being studied (P. fluorescens, T. lanuginosus, and R. miehei) achieved good conversions (60-80%); yet when acetone was the solvent, conversion into biodiesel was low for all the lipases (< 20%). Kojima et al. (2004) assessed the lipase activity of C. cylindracea (now C. rugosa) after incubation for 72 h in different solvents and found the same behavior: the polar solvents reduced enzyme activity, while the hydrophobic solvents kept it stable. Polar solvents may alter the native conformation of the enzymes by disrupting hydrogen bonding and hydrophobic interactions, leading to a very low alcoholysis rate

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(Soumanou and Bornscheuer, 2003). Also, polar solvents tend to strip the water molecules present on the surface of the enzyme, causing a reduction in its catalytic activity (Gorman and Dordick, 1991 cited in Lara and Park, 2004). One important organic solvent is tert-butanol, which is relatively hydrophilic and has been used successfully as a novel reaction medium for the lipase-mediated methanolysis of biodiesel production. Due to steric hindrance, this alcohol is not accepted by the lipases as a substrate, and as a solvent it has the ability to dissolve oil, methanol, and glycerol, leading the authors to believe that the negative effects caused by methanol and glycerol on lipase performance could be entirely eliminated (Li et al., 2006; Royon et al., 2007). Using a combination of immobilized lipases from T. lanuginosus and C. antarctica in a system containing tertbutanol, 95% conversion into biodiesel was obtained and the enzymes were reused in over 200 cycles without any obvious loss in lipase activity (Li et al., 2006). Using lipase from C. antarctica lipase, 95% conversion was obtained in the reaction with t-butanol, while the lipase itself was used for 500 h without any loss of activity (Royon et al., 2007). One interesting option is to use fossil diesel as a solvent in enzymatic transesterification reactions, as this way the solvent does not have to be separated from the product at the end of the reaction. The group that investigated this solvent (Kojima et al., 2004; Park et al., 2008) studied biodiesel using waste activated bleaching earth as a substrate, and obtained 97-100% conversion. The use of solvents resolves several problems, but their use on an industrial scale is not desirable because of the cost of the solvent itself and the cost of recovering it at the end of the reaction. The use of solvents also makes it necessary to use larger reactors, since it occupies a large volume, and also raises operational risks because of solvents are toxic and flammable. Although similar conversions have been obtained with and without solvents (Kumari et al., 2007; Soumanou and Bornscheuer, 2003), solvent-free enzymatic biodiesel production is characterized by lower reaction rates than when solvents are used (Mittelbach, 1990), which is something that must be improved to make it feasible (Fijerbaek et al., 2009).

2.4 Types of Biocatalysts Free and immobilized lipases have been studied for biodiesel production, including, more recently, as whole-cell immobilized lipases. Each type of biocatalyst has its strengths and weaknesses when it comes to reducing the contribution of the biocatalyst in the final cost of the biodiesel. 2.4.1 Free Biocatalysts Free enzymes are far cheaper than immobilized lipases. They can be purchased in an aqueous solution composed of the enzyme solution plus nothing more than a stabilizer to prevent enzyme denaturation (e.g., glycerol or sorbitol) and a preservative to inhibit microbial growth (e.g., benzoate; Nielsen et al., 2008). Several studies have obtained high biodiesel conversions (>90%) using soluble lipases from C. rugosa, P. cepacia, and P. fluorescens (Kaieda et al., 2001), R. oryzae (Kaieda et al., 1999), and C. cylindracea (now C. rugosa; Park et al., 2008). To prevent the addition of water to the reaction medium, the solution containing the free lipase can be freeze-dried. However, this combined freezing and drying process sometimes reduces enzyme activity. It has been reported that pH tuning (when an enzyme solution is freeze-dried in a buffer whose pH is the same as the optimal pH of the enzyme in an

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321

aqueous medium) may protect enzymes from deactivation (Roy and Gupta, 2004). However, Nielsen et al. (2008) strongly recommend that this kind of nonformulated enzyme preparation be used with care and on a small scale, because the powder containing the enzyme is allergenic if inhaled. Even free lipases are considerably more expensive than the chemical catalysts currently used in biodiesel production. If they are to be economically feasible, lipases must be reusable. When free lipases are used in biodiesel production, they can be partially recovered in the aqueous phase. However, their indefinite reuse is restricted by the build-up of glycerol (Nielsen et al., 2008). Furthermore, most enzyme molecules are insoluble in anhydrous media, and tend to clump together, which reduces the surface area of the biocatalyst. One way of getting round both these problems is to immobilize the enzyme (Bisen et al., 2010; Shah et al., 2003). 2.4.2 Immobilized Biocatalysts Enzyme reutilization is an important key to making the production of a commodity like biodiesel possible by enzymatic means (Hsu et al., 2001). The longer an enzyme can be reused, the lower its contribution to the overall price of the product. Immobilized lipases have attracted most interest by researchers for their potential use in biodiesel production, since immobilization serves not only to enable recovery and reutilization, but also to enhance enzyme stability (Ranganathan et al., 2008). The reason immobilized enzymes are more stable is because their molecular mobility is lower. This helps prevent denaturation, which can be caused by chemicals or high temperatures, while they are also protected from mechanical damage inside the support (Ranganathan et al., 2008). The immobilized biocatalysts can be recovered at the end of the reaction by filtration alone, or can be packed in columns for use in a continuous-flow process (Nielsen et al., 2008). Different immobilization techniques have been tried for lipases for biodiesel production: adsorption, covalent attachment, entrapment, and cross-linkage (Ganesan et al., 2009). Adsorption, the simplest and most widely used technique for immobilizing lipases, consists of bonding the lipase to the support surface through weak forces such as van der Waals or hydrophobic interactions. However, given the low bond strength between the enzyme and the support, it may be desorbed throughout the reaction (Jegannathan et al., 2008 cited by Tan et al., 2010). The most widely studied lipase for biodiesel production is the C. antarctica lipase immobilized by adsorption on acrylic resin (Novozym 435—manufactured by Novozymes). This lipase has been reported to obtain over 95% conversion into biodiesel (Shimada et al., 1999; Watanabe et al., 2000) and has been reused 70 times without any reduction in the conversion (Watanabe et al., 2000). Some studies have used lipases immobilized by covalent bond onto a support matrix for biodiesel production. T. lanuginosus lipase immobilized by covalent attachment onto polyglutaraldehyde activated styrene-divinylbenzene copolymer catalyzed the conversion of 97% rapeseed oil into biodiesel in 24 h, and was reused for 10 reactions with no loss of activity (Dizge et al., 2009). A relatively new immobilization method involves cross-linked enzyme aggregates (CLEAs) and protein-coated microcrystals (PCMCs). These have been tested for production of biodiesel with a P. cepacia lipase in solvent-free conditions, obtaining 92% conversion with CLEAs and 99% with PCMCs, both after 2.5 h (Kumari et al., 2007).

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On the other hand, the use of immobilized lipases can incur some problems, since large molecules (TAG, FAME) have to diffuse through small pores to access the enzyme, while low-solubility reagents (methanol) have to penetrate the oil-filled pores in the support (internal diffusion). There are also external restrictions on mass transport, with the possibility of a film being produced around the support. The formation of a layer of reagents or products around the immobilized enzyme (external diffusion) can usually be minimized by increasing the agitation rate in the reactor, or increasing the flow rate if it is a fixed-bed reactor (Ranganathan et al., 2008). At the start of a transesterification reaction catalyzed by immobilized lipases, the reaction system is made up of three immiscible phases (TAG, alcohol, and immobilized enzyme), but as the alkyl esters are formed, they operate as a solvent for the substrate and the reaction becomes a two-phase (liquid and solid) reaction, ameliorating the diffusion-related problems in the system (Noureddini et al., 2005). 2.4.3 Whole-Cell Biocatalysts Whole-cell immobilized lipases have been studied for biodiesel production. This kind of biocatalyst should be cheaper to produce because it does not require many of the steps in the downstream process, such as the isolation and purification of the enzyme after fermentation (Ban et al., 2001; Li et al., 2007a; Zeng et al., 2006). Qin et al. (2008) used lyophilized free whole cells of R. chinensis for biodiesel production, obtaining yields of 86% FAME. Torres et al. (2003) used whole-cell lipase of Aspergillus flavus to catalyze methanolysis combined with oil extraction. The authors obtained 92% conversion after 96 h of reaction. However, the free cells in the reaction mixture are difficult to reuse, so cell immobilization could solve this problem. Ban et al. (2001) did just this, immobilizing a whole-cell biocatalyst of R. oryzae on reticulated polyurethane foam. The fungal biomass was immobilized on the support spontaneously during fermentation and a high conversion of 90% on biodiesel could be achieved. In a later work, Ban et al. (2002) showed that when this same biocatalyst was treated with a solution of glutaraldehyde (cross-linking), it enhanced the stability of the intracellular lipase from R. oryzae and yielded 80% conversion into methyl esters in a solvent-free system (Tamalampudi et al., 2008). Ying and Chen (2007) studied the cells of lipase-producing Bacillus subtilis encapsulated within a net of hydrophobic carrier with magnetic particles. This biocatalyst was recoverable by magnetic separation. When methanolysis was carried out using waste cooking oil, the proportion of methyl esters in the reaction mixture reached about 90% after 72 h in a solvent-free system. Matsumoto et al. (2001) constructed a strain of S. cerevisiae with high-level expression of intracellular R. oryzae lipase. They obtained 71% conversion into methyl esters after 165 h with permeabilized cells. The yeast S. cerevisiae cell surface display system for the lipase from R. oryzae was developed by Matsumoto et al. (2002), who obtained 78% methanolysis after 72 h in a solvent-free system. These differences in yield and conversion rate of methyl esters might be attributed to the easier access of molecules from the substrate to the cell surface displayed lipase, which did not need to be permeabilized for methanolysis to occur.

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3 ENZYMATIC ESTERIFICATION Some basic differences have been identified between the transesterification of TAGs and the esterification of FFAs. The former is a sequence of three reactions (DAG and MAG are formes as intermediates), while esterification involves parallel reactions of FFAs to make biodiesel, which is quicker (Marchetti et al., 2008). The greater polarity of FFAs than TAGs makes the short-chain alcohols more soluble in the reaction medium (Du et al., 2007). Also, water is one of the products of esterification reaction and shifts the equilibrium toward hydrolysis when the concentration exceeds optimal levels. A variety of waste oils and fats have been used for enzymatic biodiesel production (Fijerbaek et al., 2009; Table 2); indeed, the use of low-quality raw materials with a high FFA concentration (low aggregate value) is one way of reducing the overall cost of producing biodiesel, helping make the use of more expensive catalysts like lipases economically feasible. The effect of the presence of FFAs on the tolerance of lipases to alcohol (methanol) has been studied using a C. antarctica lipase as a model. In a previous work, Shimada et al. (1999) showed that when refined oil (100% TAG) was used as a substrate, the maximum methanol concentration that could be employed without deactivating the lipase was a 1.5:1 methanol/oil molar ratio, which is the solubility limit of methanol for this system. Based on this finding, Du et al. (2007) tested different proportions of oleic acid and refined oil as substrates, varying the concentration of oleic acid to refined oil mass from 5% to 100%. They observed that the tolerance of the C. antarctica lipase to the methanol was higher when

TABLE 2 System

Examples of Enzymatic Biodiesel Production Employing Low-Cost Raw Materials in Solvent-Free

Alcohol Type

Reaction Time (h)

Reuse of Lipase (cycles)

Raw Material

Lipase

Conversion (%)

Soybean oil deodorizer distillate (28% FFA)

C. antarctica

95

Methanol

10

–

Du et al. (2007)

Acid oil (78% FFA) þ refined oil

C. antarctica

97 (two steps)

Methanol

24 each step

>100 each step

Watanabe et al. (2007a)

Acid oil hydrolysate (92% FFA)

C. antarctica

99 (two steps)

Methanol

24 each step

40 each step

Watanabe et al. (2007b)

Waste fatty acids from tuna oil (100% FFA)

C. antarctica

98 (two steps)

Methanol

24 each step

>45

Watanabe et al. (2002a)

Rice bran oil dewaxed/ degummed (85% FFA)

C. antarctica or R. miehei

96

Methanol

6

14

Lai et al. (2005)

Madhuca indica acid oil (
20% FFA)

P. cepacia

99

Ethanol

2.5

–

Kumari et al. (2007)

Grease (8.5% FFA)

P. cepacia

96

Ethanol

18

–

Hsu et al. (2001)

Reference

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13. BIOTECHNOLOGICAL METHODS TO PRODUCE BIODIESEL

the oleic acid concentration was higher. For instance, when the substrate was 100% oleic acid, a 30:1 methanol/oil molar ratio yielded around 90% biodiesel, while a substrate with just 30% oleic acid deactivated the lipase and no biodiesel was produced. The improved enzyme stability was probably brought about by the greater solubility of the methanol in the presence of the FFAs (Du et al., 2007). It has also been reported that the esterification of FFAs with methanol catalyzed by a C. antarctica lipase is quicker than the methanolysis of TAG and requires a lower lipase concentration (Lai et al., 2005; Shimada et al., 1999; Watanabe et al., 2002a,b; Watanabe et al., 2005). It can be concluded that a reaction system that consists primarily of the esterification of FFAs may reduce total lipase costs and reaction times. The importance of removing water (a product of esterification) during biodiesel production has been demonstrated by some authors using a C. antarctica lipase and methanol in a solvent-free system. When Du et al. (2007) used soybean oil deodorizer distillate (25-35% FFAs) as a substrate, they obtained a higher conversion into biodiesel (95%) when they added an adsorbent to the reaction medium to control the water content. Meanwhile, Watanabe et al. (2005) used mixtures with 50-90% FFA in the TAG as a substrate, observing that the FFAs from the mixture were efficiently esterified with methanol, but the water produced by this process significantly inhibited the methanolysis of the TAGs, when a 1:1 methanol/FA molar ratio was used. The use of large quantities of methanol may be one way of overcoming methanolysis inhibition by water, and high biodiesel conversions may be obtained when the reaction equilibrium is shifted toward the production of methyl esters. Thus, the presence of water has less of an impact on esterification than on transesterification (Watanabe et al., 2005). Watanabe et al. (2007b) used glycerol to absorb the water produced during the esterification of the acid oil hydrolysate (92% FFA). The glycerol removed the water from the medium, resulting in a higher FAME yield, without any increase in the partial glyceride content being detected during the reaction. It has been noted that the yield of biodiesel from low-cost raw materials is usually lower than it is from refined materials. This could be caused by the other compounds in these materials, such as phospholipids found in crude oils which often inhibit lipase activity. When crude rice bran oil (20% FFAs) was used as a substrate, 56% FAME was obtained after 12 h. This concentration rose to 88% when dewaxed/degummed rice bran oil (20% FFAs) was used as the substrate (Lai et al., 2005). Watanabe et al. (2007b) detected low lipase stability in a mixture of acid oil hydrolysate with 1-2 mol methanol, which was assumed to have been caused by some inhibitors contained in the acid oil hydrolysate, since in a previous work by the same group (Watanabe et al., 2005) the lipase had been found to be stable in a mixture of pure FFAs with 1-2 mol methanol. This inhibition was controlled by the addition of 5-7 mol methanol. The authors related two hypotheses: one where the concentration of inhibitors was reduced by dilution with methanol, the other where the inhibitors adhered to the lipase in the presence of 1-2 mol methanol (low polarity) were released in the presence of 5-7 mol (high polarity; Watanabe et al., 2007b). Studies of lipases other than the C. antarctica lipase have been carried out using a solvent in the reaction medium. The Penicillium expansum lipase was used in FAME production from waste oil (
20% FFA) in the presence of tert-amyl alcohol. The water produced during the reaction was removed by the addition of adsorbents, resulting in a higher conversion

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325

into FAME (93%; Li et al., 2009). Likewise, the use of adsorbents by Deng et al. (2003) in the esterification of oleic acid and methanol catalyzed by the Candida sp. lipase in the presence of petroleum ether resulted in over 90% conversion. Wang et al. (2006) used a combination of lipases from C. antarctica and T. lanuginosus to catalyze biodiesel production from soybean oil deodorizer distillate in a medium with tert-butanol. Both the FFAs and the glycerides were converted into biodiesel simultaneously and reached a 97% conversion with the addition of an adsorbent with no obvious loss in lipase activity even after 120 cycles. Li et al. (2007b) used whole cells of R. oryzae and tert-butanol as a solvent, observing that the increase from 0% to 20% in FFAs in the oil resulted in higher conversion into biodiesel.

4 HYDROESTERIFICATION Hydroesterification is a process that combines two basic processes, hydrolysis and esterification, in sequential reactions in order to produce biodiesel. This methodology allows the use of raw materials with high concentrations of free fatty acids and water (as normally occurs with waste raw materials) without pre-treatment, since water is one of the reagents and high concentrations of fatty acids is the expected product of the hydrolysis reaction.

4.1 Enzymatic Hydrolysis The hydrolysis of oil and fat is an important industrial process. The products (FFAs and glycerol) are basic raw materials for a whole host of applications. Noor et al. (2003) studied the hydrolysis of palm oil in a stirred tank bioreactor by lipase-SP398, produced by Novo Nordisk S/A. Almost all the palm oil was hydrolyzed in 90 min, and the addition of gum arabic, which operated as a surfactant, increased the hydrolysis rate. Meanwhile, Talukder et al. (2010a) studied the hydrolysis of crude (unrefined) palm oil by the C. rugosa lipase, followed by the esterification of the FFAs from this oil with methanol by the C. antarctica lipase. The oil was completely hydrolyzed in 4 h in the presence of isooctane. The biocatalysts were reused for up to 10 cycles in hydrolysis and 50 cycles in esterification, with no significant loss of activity. Watanabe et al. (2007b) studied the enzymatic conversion of acid oils (byproduct of the refining of vegetable oils) into FFAs catalyzed by C. rugosa lipase and obtained an oil with 92% FFAs. The second step encompassed the esterification reaction catalyzed by C. antarctica lipase that obtained conversion of 96% after 24 h reaction. The final product contained 91 wt% methyl esters. Both steps could be repeated for 40 cycles without reduction of reaction conversion. Pugazhenthi and Kumar (2004) studied the hydrolysis of olive oil by the pancreatic lipase immobilized on poly methyl methacrylate-ethylene glycol dimethacrylate. In this study, the immobilized enzyme was used in reactions for over 50 h during 25 days. In a study by Gan et al. (1998), sunflower oil was completely hydrolyzed by a C. cylindracea lipase in an integrated reaction system involving an agitated tank reactor

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coupled to an ultrafiltration system, which provided the simultaneous separation of the product during the enzymatic hydrolysis of the oil. They found that the continuous separation of the reaction product (glycerol) and the recirculation of the free lipase in the system enhanced the production of FFAs. There is also increasing interest in the use of membrane technology to combine reactions involving lipases with separation systems in the processing of oils and fats for use in lipid refining (Koike et al., 1992), separation (Raman et al., 1996), discoloration (Reddy et al., 1996), and decontamination (Vavra and Koseuglu, 1994).

4.2 Hybrid Catalysis Saifuddin et al. (2009) developed a hybrid catalysis process for biodiesel production using waste cooking oil with high acidity (low quality) as a raw material. The lipase used, from Candida rugosa, hydrolyzed 88% of the cooking oil in 5 h at 40  C. The hydrolysate was then used in an esterification reaction catalyzed by sulfuric acid (2.5%) at a 1:15 raw material/methanol molar ratio, yielding up to 83% biodiesel in 1 h. Ting et al. (2008) studied the use of a C. rugosa lipase immobilized on chitosan in the hydrolysis step. The authors obtained 88% of the soybean oil hydrolysis after 5 h of reaction. The hydrolysate was esterified with methanol at a 1:15 molar ratio by acid catalysis (2.5% sulfuric acid), obtaining 99% conversion into biodiesel after 12 h at 50  C. Talukder et al. (2010b) studied the use of cooking oil for biodiesel production by enzymatic hydrolysis accompanied by chemical esterification. The C. rugosa lipase completely hydrolyzed the oil after 10 h. The FFAs were converted into biodiesel by chemical esterification using Amberlyst 15 (acidic styrene divinylbenzene) and a 99% conversion into biodiesel was obtained after 2 h. In this work, there was a loss of enzyme activity and the hydrolysis yield fell to 92% after five runs. Cavalcanti-Oliveira et al. (2011) studied the use of a T. lanuginosus lipase (TL 100 L) in the hydrolysis of soybean oil in a hydroesterification process. The lipase hydrolyzed 89% of the oil after 48 h. This stage was followed by the esterification of the FFAs with methanol, which was catalyzed by niobic acid in pellets. They obtained 92% conversion of the FFAs into FAMEs after 1 h. Sousa et al. (2010) studied the Physic nut lipase (Jatropha curcas L.) for the hydrolysis of different raw materials for biodiesel production using hydroesterification. The best conversions were obtained using soybean oil and jatropha oil, obtaining up to 98% FFA after 2 h. The esterification of the FFAs from the jatropha oil with methanol was catalyzed by niobic acid in pellets, obtaining up to 97% conversion into biodiesel after 2 h. The biodiesel obtained from this process fulfilled all the legal requirements for its commercial use.

5 REACTOR CONFIGURATIONS One of the problems to be overcome when biocatalysis is used for obtaining biodiesel on a large scale is the right setup and operation of the bioreactor, given that both factors, as well as the form of the biocatalyst (whether free or immobilized), have a direct impact on the stability of the enzyme and whether it can be reused, which are crucial for reducing costs in enzyme

5 REACTOR CONFIGURATIONS

327

catalysis. The set-up of the bioreactor for free or immobilized enzyme preparations should take into account how the biocatalyst will be reused from the product stream (biodiesel and glycerol). When free biocatalysts are used, ultrafiltration or centrifugation units may be coupled to the system. However, there are more process options and bioreactors to choose from if the biocatalyst is immobilized. The most widely used reactors for enzymatic biodiesel production are packed-bed reactors (PBRs) and stirred-tank reactors (STRs). However, as biodiesel is a chemical commodity, its production in continuous-flow systems would certainly reduce the operational costs of its production. As a result, these reactors are the most widely used in continuous operations with heterogeneous catalysts, such as immobilized enzymes. Even so, there are other reactor setups that are worth investigating, such as fluidized-bed reactors (FBRs), expanded bed reactors and membrane reactors (Fijerbaek et al., 2009). Several authors have investigated the use of PBRs operating continuously and in batches using enzymes immobilized on supports or whole cells as a biocatalyst. Table 3 summarizes the main works in the literature that use PBRs to obtain biodiesel using enzymes. Generally speaking, biodiesel production using continuous-flow PBRs has attained good enzyme stability and conversions, both with and without the use of solvents. Solvents may add to the overall production cost, but on a commercial scale, the absence of solvents may incur a marked drop in pressure on the bed, causing serious operational problems. PBRs should operate at low flow rates or using larger biocatalyst particle sizes to minimize such a drop in pressure. Fijerbaek et al. (2009) noted a drop in the effectiveness factor (Z) as the particle size of commercial biocatalysts increased. This was the equivalent of a 34% drop in the reaction rate due to the increased particle diameter and the correspondingly larger pore diffusion distance. These factors should therefore be taken into account when the bioreactor/ system is being chosen. FBRs have certain features that help overcome these problems, but imply in designing equipment that efficiently separates and recovers the biocatalyst. Another problem to be overcome in continuous-flow PBRs is the adsorption of the glycerol formed during the reaction on the immobilized biocatalyst bed, causing the inhibition of enzyme activity. Jachmania´n et al. (2010), in their investigation of the composition of the substrate, adjusted the ratio between the oil, alcohol, and solvent in such a way as to prevent the separation of alcohol from the substrate and/or glycerol from the product mixture, leading to optimal enzyme performance and productivity in continuous PBRs. Other procedures may help keep up enzyme activity, such as adding silica gel to the bed, using solvents, or using semicontinuous-flow processes that provide the opportunity for the biocatalyst to be washed periodically. The use of STRs has also been investigated on a smaller scale. They generally yield high conversion rates to begin with because of the high dispersion rate of the alcohol in the oil. However, some problems arising from physical damage to the biocatalyst caused by shear stress have been reported. Hama et al. (2007) noted that biodiesel production in batchstirred-tank reactor (BSTR) (150 rpm) using whole R. oryzae cells immobilized in polyurethane foam initially resulted in similar conversions to those obtained from PBRs. However, after 10 operation cycles, the conversion dropped to less than 10% of the initial value because of cell exfoliation. Meanwhile, Ognjanovic et al. (2009) obtained high conversions (transesterification of sunflower oil and methanol) using a commercial enzyme, Novozyme 435, in a BSTR equipped with a six-blade turbine impeller. This system provided good dispersion

328

TABLE 3 Enzymatic Biodiesel Production in Packed-Bed Reactors (PBR) Lipase Source

Conditions

Conversion Ratio

Stability/ Operation Time

Waste oil and methanol

Candida antarctica (Novozyme 435)

3 bioreactors continuously operated in series with addition of 1 molar equivalent alcohol for each bed (tR ¼  2.7 h each bed, T ¼ 30  C). Cosolvent-free system and glycerol removal

90%

100 d

Watanabe et al. (2001)

Vegetable oil and methanol

Candida antarctica (Novozyme 435)

3 bioreactors in series with addition of 1 molar equivalent alcohol for each bed (T ¼ 30  C). Cosolvent-free system and glycerol removal

90%

100 d

Shimada et al. (2002)

Cotton seed oil and methanol

Candida antarctica (Novozyme 435)

1 bioreactor continuously operated with 4.2:1 oil/alcohol ratio. 32,5 vol% tert-butanol as cosolvent.

95%

500 h

Royon et al. (2007)

Soybean oil and methanol

Candida antarctica (Novozyme 435)

1 bioreactor continuously operated (tR ¼ 30-40 min, T ¼ 52  C) with 4.3:1 oil/alcohol ratio. n-hexane:tert-butanol (9:1, v/v) as cosolvent.

75%

–

Shaw et al. (2008)

Soy bean oil and ethanol

Candida antarctica (Novozyme 435)

1 bioreactor continuously operated (tR ¼ 6 h). 1:12 oil/alcohol ratio at 70  C. Pressurized propane (60 bar, 7:1 ratio propane/oil) as cosolvent.

70-75%

24 h

Rosa et al. (2009)

Sun flour oil and methyl methanol

Candida antarctica (Novozyme 435)

1 bioreactor batch operated (tRT ¼ 8-10 h, T ¼ 45  C) with 3:1 alcohol/grease molar ratio. Cosolvent-free system

93-96%

72 h

Ognjanovic et al. (2009)

Waste cooking palm oil and methanol

Candida antarctica (Novozyme 435)

2 bioreactors operated in series. 1:4 oil/alcohol ratio (tRT ¼ 4 h, T ¼ 40  C). Tert-butanol (1/1 v/ v of oil) as cosolvent.

80%

120 h

Halim et al. (2009)

Soy bean oil and isopropanol

Candida antarctica (Novozyme 435)

1 bioreactor continuously operated (tR ¼  1 h, T ¼ 51.5  C). 1:4 oil/alcohol ratio. Cosolventfree system.

75%

168 h

Chang et al. (2009)

Reference

13. BIOTECHNOLOGICAL METHODS TO PRODUCE BIODIESEL

Oil/Fat Source Alcohol

Candida antarctica (Novozyme 435) plus pieces of loofa

1 bioreactor batch operated (tRT ¼ 72 h, T ¼ 38  C). 1:4.3 oil/alcohol. Cosolvent-free system

97%

432 h

Hajar et al. (2009)

Sunflower oil and isopropanol

Candida antarctica (Novozyme 435)

1 bioreactor continuously operated (T ¼ 50  C) with oil/alcohol/isopropyl ester weight ratio of 35:35:30.

90%

210 h

Jachmania´n et al. (2010)

Waste oil and methanol

Candida sp immobilized lipase in Cotton membrane

3 bioreactors continuously operated in series with 3 stepwise additions of alcohol (24 h) (tR ¼  100 min, T ¼ 40  C). Petroleum ether (3/2, v/v of oil) as cosolvent and glycerol removal by hydrocyclone

92%

500 h (32% conversion ratio)

Nie et al. (2006)

Waste cooking oil and methanol

Candida sp immobilized lipase in textile cloth

3 bioreactors in series with addition of 1 molar equivalent alcohol for each bed (T ¼ 45  C). Hexane/oil weight ratio of 15:100 and glycerol removal each step.

91%

30 h (91%)

Chen et al. (2009)

Restaurant grease and ethanol

Burkholderia cepacia immobilized lipase in Phyllosilicate sol-gel

1 bioreactor operated in batch mode (tRT ¼ 48 h, T ¼ 50  C) with 4:1 alcohol/grease molar ratio. Cosolvent-free system

96%

72 h

Hsu et al. (2004)

Soy bean oil and ethanol

Burkholderia cepacia— lyophilized and delipidated fermented solid

1 bioreactor operated in batch mode (tRT ¼ 46 h, T ¼ 50  C) with 2 stepwise additions of alcohol (3:1 alcohol/oil molar ratio). Cosolvent-free system

95%

140 h

Salum et al. (2010)

Soybean oil and methanol

Rhizopus oryzae whole cell immobilized in polyurethane foam

1 bioreactor operated in batch mode (tRT ¼ 50 h, room temperature) with 3 stepwise additions of alcohol. Cosolvent-free system, water/oil emulsification

80-90%

600 h

Hama et al. (2007)

100 h (77%)

5 REACTOR CONFIGURATIONS

Canola oil and methanol

tRT, reaction time; tR, residential time; Novozyme 435, Candida antarctica immobilized lipase.

329

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13. BIOTECHNOLOGICAL METHODS TO PRODUCE BIODIESEL

of the biocatalyst, reduced mass transfer resistance, and increased the overall reaction rate. The same authors also studied the agitation rate and method, finding that these variables were extremely important for obtaining high conversions and good enzyme stability. Sanches and Vasudevan (2006) also used Novozyme 435 in a BSTR (60  C, 100 rpm). They observed a slight drop-off after the initial activity level, but even so it did not drop under 95% during the first five batches, and remained above 70% after as many as eight batch cycles. The use of continuous and batch-stirred tank reactors has been investigated in some studies and patents, which indicate that the more efficient use of the enzyme preparation as the main advantage, in view of the fact that the tanks can operate with enzymes of different ages/ activities and units can be installed between the reactors to separate out the glycerol formed during the reaction. Bassheer et al. (2009) patented an enzymatic biodiesel production system using two continuous-stirred-tank reactors (CSTRs), with a bottom sintered glass filter, operating in series. The authors used a multi-enzyme preparation (enzymes immobilized in different microorganisms) in a cosolvent-free system. Separation equipment was installed between the reactors to remove the glycerol and excess water formed during the reaction. The system operated at high conversions (98%) over a short space of time (4 h), and the enzymes were reused in over 100 consecutive batch cycles. It can therefore be concluded that the mechanical resistance of the support, the set-up of the reactor (agitation, use of separators), the process conditions (temperature, type of alcohol/oil, use of solvents) and the way the process is conducted are the main points that must be assessed when a reactor is being selected. The immiscibility of the lipid and alcohol phases causes mass transfer problems in PBRs and CSTRs. This should be addressed when the design and optimization of the method for producing biodiesel is being prepared. One potential option that is not yet economically feasible would be to use bioreactors coupled to membranes to simultaneously separate out the product and recover the biocatalyst. The choice of the most suitable membrane would depend on what kind of biocatalyst was being used (free or immobilized).

5.1 Larger-Scale Reactors Many articles have been published about enzymatic biodiesel production on a bench scale, yet pilot-scale operations are fundamental for developing and consolidating the enzymebased technology for producing this commodity. Brenneis et al. (2004) described biodiesel production from used cooking oil and 2-ethyl-1hexanol using a liquid preparation and a commercially available thermostable lipase from Candida antarctica (Lipase A) called Novozym 735. They used a 3000-L STR at 500 rpm (disk-type agitator) and 50-57  C. The authors reported that alcoholysis was completed after about 7-10 h, when they used a TAG and 2-ethyl-1-hexanol solution (molar ratio of 1:3-1:3.1) and a lipase solution (1 wt% in relation to TAG). Park et al. (2008) produced methyl biodiesel on a pilot scale (50 L) by the transesterification of a waste material (activated bleaching earth) from the oil refining industry. They used a BSTR equipped with a filter press for separation of the FAME and solvent mixture. The biocatalyst used was a C. cylindracea lipase (added as a powder). Diesel oil was used as a cosolvent with the aim of obtaining a mixture with the biodiesel formed during the transesterification reaction. This mixture can be used to make biodiesel fuel, if it is blended

6 ECONOMIC EVALUATION OF ENZYMATIC BIODIESEL PRODUCTION

331

with diesel oil at an appropriate ratio. They obtained 97% conversion after 12 h at 25  C and 30 rpm, when 1% (w/w) lipase was added to the waste. The two biggest drawbacks noted by the authors were the difficulty of recovering the biodiesel from the waste, given that the cake of vegetable oil-free waste activated bleaching earth contained approximately 14% FAME and 16% solvent on a weight basis. To recover 100% FAME from the waste, the activated bleaching earth would require additional processing, that is, extraction using n-hexane. Another disadvantage was that it was impossible to separate the lipase from the final filter cake. In their review, Tan et al. (2010) cited the operation of two industrial-scale biodiesel production plants in China. In 2007, Lvming Co. Ltd. established an enzymatic production line with 10,000-ton capacity in Shanghai, with immobilized Candida sp. 99-125 lipase as a catalyst. The plant uses very acidic used cooking oil and methanol as substrates. The process is conducted in STRs and a centrifuge is used to separate out the glycerol and the water produced during the reaction. The authors reported 90% yields under optimal conditions.

6 ECONOMIC EVALUATION OF ENZYMATIC BIODIESEL PRODUCTION It seems to be a consensus in the literature that the cost of enzymes will have to fall before the process will be economically feasible. Alternatively, very high yields will have to be achievable, as already obtained by some authors (Chen and Wu, 2003; Shimada et al., 2002; Watanabe et al., 2002a), in which case the lipase can be recycled in a batch system or a continuous-flow process. Nielsen et al. (2008) analyzed studies from the literature to calculate the minimum yield in terms of kg biodiesel to kg enzyme. They calculated the maximum cost of the lipase, assuming that it should be the same as that of a chemical catalyst (25 USD/ton biodiesel). They found that enzymes costing 12-185 USD/kg could be feasible, depending on the process productivity. Fijerbaek et al. (2009) also calculated productivity from studies in the literature in order to compare them against an alkaline catalyst (NaOH; 1 wt% based on the mass of oil and complete conversion), presenting a yield of around 100 kg biodiesel per kilo of catalyst. According to their calculations, the lipases obtained yields that were up to 74 times higher. The average purchase price of 1000 US$ per kg for Novozym 435, compared to just 0.62 US$ (Haas et al., 2006) for NaOH, when offset against their respective yields, puts the cost of the enzyme at 0.14 US$ per kg of ester as against 0.006 US$ per kg of ester for NaOH. If the acquisition cost of the enzyme dropped to 44 US$ per kg or the enzyme could be reused for around 6 years, enzymes would become economically feasible from the perspective of process productivity alone. Nevertheless, it is no easy task to give a precise answer as to how cheap enzymes would have to be to compete with chemical catalysts. They are hard to compare because the chemical and enzyme processes are so different. Clearly, it is unsatisfactory to merely compare the cost of the catalysts in isolation, but a full economic analysis of enzyme versus chemical catalysts for biodiesel production would require whole host of assessments, including cost of oil (the use of low-cost oils with a high FFA concentration can have a major impact on overall process costs); cost of alcohol; cost of pretreatment stages; process yield; cost of waste treatment; commercial value of glycerol; and cost of downstream stages. Enzyme technology has a positive

332

13. BIOTECHNOLOGICAL METHODS TO PRODUCE BIODIESEL

impact on several of these factors: its feasibility for use with raw material of varying quality; process with fewer stages; better quality glycerol; better phase separation (with no emulsion caused by soap formation); less energy consumption; and less wastewater production (Nielsen et al., 2008). Sotoft et al. (2010) simulated the processes used in different enzymatic biodiesel production plants and evaluated them economically, using data taken from experiments by Shimada et al. (1999) and Li et al. (2006), who did excellent studies into solvent-free systems and systems using tert-butanol, respectively. They assessed continuous-flow biodiesel production plants that used high-quality rapeseed oil and methanol. They investigated two production scales (8 and 200 M.kg of biodiesel/year) and two enzyme prices (current prices of 762.71 €/kg enzyme and a lower future price of 7.627 €/kg enzyme). The economic analysis showed that the process that used solvent was more expensive to the point of being unfeasible, while the solvent-free process was found to be feasible on a larger production scale (200 M.kg of biodiesel/year) at today’s lipase price. At the projected future price of the enzyme, the smalland large-scale production processes were found to be feasible using a solvent-free medium. The total capital investment (TCI) was found to be lower for the solvent-free system than for the system using a solvent on both the scales studied. The equipment cost was cheaper for the plant using a solvent, but when the cost of installing the solvent recovery column was added, the total cost was higher, even taking into account the extra reactors and settling tanks required for the solvent-free set-up (Sotoft et al., 2010). As for production costs, the main contributory factors in all the scenarios studied were raw material costs and the sale price of the byproduct. The biggest single factor to affect raw material costs in the solvent-free system was the cost of the enzyme; its influence was less in the system using a solvent because the lipase was more productive in this system. The cost of the solvent, tert-butanol, was not significant, as it is reused, while the oil was the most expensive single element in the system using a solvent. The sale of the glycerol was found to be equally important in all the scenarios. The cost of utility bills was found to be very significant in the operation of the plants using a solvent because of the amount of energy required; indeed, this was one of the factors that made this process economically unfeasible. Meanwhile, the electricity costs of the solvent-free process were low (Sotoft et al., 2010). The economic feasibility study showed that at current lipase prices, the only plant that would be cost effective was the large-scale solvent-free plant, with a very short payback period of 0.25 year (assuming 1.12 €/kg as the sale price of the biodiesel) or a minimum product price of 0.73 €/kg. For the other plants, the minimum product price stood at 1.49 €/kg for the small-scale solvent-free plant, 2.38 €/kg for the small-scale plant using solvent, and 1.70 €/kg for the large-scale plant using solvent, all of which put the product price higher than 1.12 €/kg. Even when the projected future price of lipase was used, the plants using solvents were not deemed cost effective on either scale. The large solvent-free plant was considered very feasible, with a payback period of 0.09 year and a minimum product price of 0.05 €/kg. The product price is low because of the sale of the glycerol. The small-scale solvent-free plant gave a minimum product price of 0.75 €/kg, which is lower than the market price, but its payback period would be 3.59 years, which is borderline for projects of this kind. Generally speaking, the payback period for feasible processes should be under 2 years for high-risk projects; anything over 4 years is considered unfeasible (Seider et al., 2004 cited by Sotoft et al., 2010). Given the uncertainties inherent to the new and as yet volatile biofuel market,

REFERENCES

333

this kind of project is inherently high risk. The results of the economic feasibility analysis are very promising and enzymatic biodiesel production seems to be bordering on becoming a truly feasible industrial-scale option. A comparison with feasibility studies from the literature of processes using traditional catalysts shows that enzymatic biodiesel production is more expensive, but if the lifespan and yield of the lipases can be improved, plus the major improvement in environmental impacts when this technology is used, then the enzymatic production of biodiesel is sure to become a very attractive prospect (Sotoft et al., 2010).

7 CONCLUSIONS There are a few process conditions that should be taken into account before enzymatic technology can be feasibly designed for producing a commodity like biodiesel: (i) correlation between enzyme and raw material types and costs; mass transfer and reaction conditions; and product recovery when choosing whether to use a solvent; (ii) the choice of whether to use a free or immobilized biocatalyst should be dictated by weighing the cost of the support against the biocatalyst reuse capacity; (iii) long-term continuous-flow or batch experiments should be undertaken.

References Al-Zuhair, S., 2007. Production of biodiesel: possibilities and challenges. Biofuel Bioprod. Bior. 1, 57–66. Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis—key factors affecting efficiency of the process. Renew. Energy 34, 1185–1194. Bajaj, A., Lohan, P., Jha, P.N., Mehrotra, R., 2010. Biodiesel production through lipase catalyzed transesterification: an overview. J. Mol. Catal. B Enzym. 62, 9–14. Balat, M., Balat, H., 2010. Progress in biodiesel processing. Appl. Energy 87, 1815–1835. Ban, K., Kaieda, M., Matsumoto, T., Kondo, A., Fukuda, H., 2001. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem. Eng. J. 8, 39–43. Ban, K., Hama, S., Nishizuka, K., Kaieda, M., Matsumoto, T., Kondo, A., et al., 2002. Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production. J. Mol. Catal. B Enzym. 17, 157–165. Bassheer, S., Haj, M., Kaiyal, M.A., 2009. Robust multi-enzyme preparation for the synthesis of fatty acid alkyl ester. Patent WO 2009/O69116, PCT/IL2008/001497. Bisen, P.S., Sanodiya, B.S., Thakur, G.S., Baghel, R.K., Prasad, G.B.K.S., 2010. Biodiesel production with special emphasis on lipase-catalyzed transesterification. Biotechnol. Lett. 32, 1019–1030. Brenneis, R., Baeck, B., Kley, G., 2004. Alcoholysis of waste fats with 2-ethyl-1-hexanol using Candida antarctica lipase A in large-scale tests. Eur. J. Lipid Sci. Technol. 106, 809–814. Cavalcanti-Oliveira, E.D., Silva, P.R.R., Ramos, A.P., Aranda, D.A.G., Freire, D.M.G., 2011. Study of soybean oil hydrolysis catalyzed by Thermomyces lanuginosus lipase and its application to biodiesel production via hydroesterification. Enzym. Res. in press. 2011, 1–8. Chang, C., Chen, J.H., Chang, C.M.J., Wu, T.T., Shieh, C.J., 2009. Optimization of lipase-catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor using response surface methodology. New Biotechnol. 26, 187–192. Chen, J.W., Wu, W.T., 2003. Regeneration of immobilized Candida antarctica lipase for transesterification. J. Biosci. Bioeng. 95, 466–469. Chen, G., Ying, M., Li, W., 2006. Enzymatic conversion of waste cooking oils into alternative fuel-biodiesel. Appl. Biochem. Biotechnol. 132, 911–921. Chen, Y., Xiao, B., Chang, J., Fu, Y., Lv, P., Wang, X., 2009. Synthesis of biodiesel from waste cooking oil using immobilized lipase in fixed bed reactor. Energy Convers. Manage. 50, 668–673.

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List of Abbreviations NaOH Sodium Hydroxide KOH Potassium Hydroxide TAG Triacylglycerols DAG Diacylglycerols MAG Monoacylglycerols FFA Free Fatty Acid FAME Fatty Acid Methyl Esters FAEE Fatty Acid Ethyl Esters CLEA Cross-linked Enzyme Aggregates PCMC Protein-coated Microcrystals BSTR Batch-Stirred-Tank Reactor CSTR Continuous-Stirred-Tank Reactor PBR Packed-Bed Reactor MKg 106.Kg