**2. Enzymatic transesterification**

In transesterification reaction, one ester is converted into another ester. This conversion occurs as transfer of an acyl group. The acyl group transfer can take place between one ester and another ester (interesterification), an ester and an acid (acidolysis), or an ester and an alcohol (alcoholysis). In broad terms, the transesterification reaction between TAGs and alcohol to produce biodiesel is a sequence of three consecutive and reversible reactions, by which diacylglycerol (DAG) and monoacylglycerol (MAG) are formed as intermediates. Enzymatic synthesis of biodiesel has been usually performed at moderate temperature between 20 and 60 o C. When transesterification process is completed, the by-product glycerol (lower phase) is simply separated from the biofuel (upper phase) and neither neutralization nor deodorization of the product is necessary. However, an overdose of alcohol provides higher yield of biodiesel [26]. Biocatalysis has been considered a trend for sustainable synthesis technology due to biologic origin of the catalyst, selectivity and the possibility of reusing agro-industrial residues for biocatalyst production, which classifies the method as a green process [27]. Enzymatic catalysis has been applied for biodiesel which starts its industrial scale operation in China [28]. However, some factors such as substrate type, solvent type, alcohol type, water content of reaction medium, the reaction temperature, immobilization type and the lipase concentration influence the conversion of enzymatic transesterification reaction. In the literature, different lipases have been used upto now for biodiesel synthesis but it is hard to make any generali‐ zations about the optimal reaction conditions. This is because, lipases obtained from different sources tend to respond differently to changes in the reaction medium [29-39]. Costs of chemical biodiesel production have still been lower than those of the enzymatic processes, however, if the pollution of natural environment is also taken into consideration, these costs are comparable. In the enzyme-catalyzed biodiesel production, the high enzyme cost signifi‐ cantly impacts the process profitability. The cost of commercial products for industrial use of enzymes is approximately 1, 000 \$/kg which is significantly higher than that of the alkali catalyst (0.62 \$/kg). Biodiesel fuel is expensive in comparison with petroleum-based fuel as 60-80% of the cost is associated with the feedstock oil [40]. Production of cheaper, robust lipase preparations and development of systems providing the long-term, iterative use of these biocatalysts can give rise to the replacement of chemical processes with enzymatic ones [28]. Currently, the high cost of biodiesel is the biggest obstacle to commercialization. The main reason is highly purified straight vegetable oil (SVO) used as a feedstock and this problem can be overcome by using used/waste vegetable oils that is much cheaper than SVO. Another obstacle in biodiesel production is the high food prices for oil. Both problems can be solved by using waste/used oil thereby gaining cost advantage. In addition, evaluation of the waste oil in terms of biodiesel can help to solve the problem of waste oil disposal. However, high free fatty acid (FFA) content of feedstock is the main problem encountered when using alkali catalyst. On the other hand, enzymatic transesterification does not have this limitation and hence can be used with waste/used oil. Moreover, almost all FFAs present in the waste/used oil can be converted to biodiesel in high yield using this approach [41].

### **2.1. Alcohol**

in the raw material [10]. So far, many attempts have been made to develop enzymatic process by using either extracellular or intracellular lipase as a biocatalyst [1, 11]. Lipases (EC 3.1.1.3), also defined as triacylglycerol acylhydrolases, catalyze the hydrolysis of ester bonds in long chain triacylglycerols (TAGs) to produce free fatty acids (FFAs) and glycerol. In general, the active site of lipases is formed by serine, aspartic (or glutamic) acid and histidine amino acid groups. Interfacial activation, which is unique to the class of lipases for its use in transesteri‐ fication of fats and oils, takes place in presence of a substrate and lipase active site structure. Lipases are used in a wide range of fields due to their ability in utilizing all mono, di, and triglycerides as well as the FFA, low product inhibition, high activity and yield in non-aqueous media, low reaction time, temperature and alcohol resistance, but the high cost of enzyme remains a barrier for its industrial applications [10]. In order to decrease the cost of the process, the enzyme can be immobilized on a suitable carrier and reused many times. So far, many techniques and different carriers have been employed for immobilization of lipases to produce biodiesel. They have been successfully immobilized on porous kaolinite particle, biomass support particles, macroporous resin, gel-entrapped, celite, silica, and Eupergit C250L [12-16]. Several oils have been catalyzed with lipase enzymes until now. Lipase catalyzed production of biodiesel from soybean oil, sunflower oil, palm oil, kernel oil, coconut oil, rice bran oil, mixture of vegetable oils, grease and tallow oil, microbial oil, and waste oil containing vegetable oils have been reported in the past decades [17-25]. In this chapter, focus will be given toward enzymatic biodiesel production from various vegetable oils. *Thermomyces lanuginosus* lipase and *Candida antarctica* lipase A were immobilized on cotton cloth which is a low cost carrier. Transesterification of sunflower, canola, and waste cooking oil with methanol and ethanol was carried out by continuous operation system. The essential aim of this study was to investigate the production of biodiesel from vegetable oils by enzymatic transesterification with immobilized lipases on fibrous matrix by polyethyleneimine in a

In transesterification reaction, one ester is converted into another ester. This conversion occurs as transfer of an acyl group. The acyl group transfer can take place between one ester and another ester (interesterification), an ester and an acid (acidolysis), or an ester and an alcohol (alcoholysis). In broad terms, the transesterification reaction between TAGs and alcohol to produce biodiesel is a sequence of three consecutive and reversible reactions, by which diacylglycerol (DAG) and monoacylglycerol (MAG) are formed as intermediates. Enzymatic synthesis of biodiesel has been usually performed at moderate temperature between 20 and

C. When transesterification process is completed, the by-product glycerol (lower phase) is simply separated from the biofuel (upper phase) and neither neutralization nor deodorization of the product is necessary. However, an overdose of alcohol provides higher yield of biodiesel [26]. Biocatalysis has been considered a trend for sustainable synthesis technology due to biologic origin of the catalyst, selectivity and the possibility of reusing agro-industrial residues for biocatalyst production, which classifies the method as a green process [27]. Enzymatic catalysis has been applied for biodiesel which starts its industrial scale operation in China [28].

packed bed bioreactor at industrial scale.

22 Biofuels - Status and Perspective

**2. Enzymatic transesterification**

60 o

Various types of acyl acceptors, alcohols, primary short-chain alcohols like methanol, ethanol, propanol, and butanol, as well as secondary alcohols like isopropanol and 2-butanol, straight and branched-chain, esters can be employed in transesterification using lipases as catalysts [42]. The prerequisites for selecting the alcohol for industrial-scale biodiesel production are that it must be cheap and in plentiful supply. Due to their price and availability, methanol and ethanol have been the most used alcohols for industrial biodiesel production. Currently, only methanol and ethanol, meet these two requirements. Ethanol is renewable and less toxic than methanol but methanol is preferred in biodiesel production because it is less expensive and more readily available in most countries than ethanol [30, 42]. However, these two alcohols are the stronger denaturing agents than longer aliphatic alcohols and inactivate enzymes. Besides, the rate of lipase-catalyzed transesterification reaction usually increases with the length of hydrocarbon chain of alcohol [30]. Meanwhile, the short alcohol chain causes lipase deactivation. It is believed that this is because the essential water layer around them which is essential for the optimum conformation of the enzyme is stripped off [43]. Most of the refined plant oils can be converted into fatty acid methyl esters to meet the specifications of biodiesel standard by stepwise alcohol addition to prevent an irreversible lipase inactivation [44]. Shimada et al. (1999) reported that the lipase from *Candida antarctica* (Novozym 435) in a solvent-free system was deactivated irreversibly when the methanol concentration exceeded its solubility level. They found that stepwise addition of methanol prevented lipase deactiva‐ tion. A three-step addition process converted 98.4% of the oil to its corresponding methyl esters in 48 h and the immobilized lipase was set to be reused for 50 batches [1]. Watanabe et al. (2000) investigated the influence of methanol using a two-step strategy on biodiesel synthesis. It has been shown that one-third of the alcohol adding at the beginning of the reaction caused slow conversion into biodiesel (10 h reaction time). After that, the rest of the alcohol was added in a single step and biodiesel conversion increased by the presence of the biodiesel since its solubility increased [21]. In another study [45] two lipases from *Pseudomonas fluorescens* and *Pseudomonas cepacia* (now *Burkholderia cepacia*) were used and they provided 58% and 37% conversion in the presence of 1:8 oil/methanol molar ratio in a solvent-free system, respectively. However, they have been shown to be completely inactive for another six lipases tested under these conditions. It is clear that the excess alcohol above and beyond the stoichiometric ratio increases the reaction rate, but too much alcohol may also deactivate the enzyme [30]. 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. To prevent the alcohol deactivating the enzyme, many researchers have used organic solvents in the reaction medium to increase the solubility of the alcohol and reduce its concentration [12, 16, 18, 46].

#### **2.2. Water content**

The effect of water content is essential for enzymatic reactions due to formation of hydrogen bonds which are fundamental in the interactions for maintaining the conformation of the enzymes. Water has strong influence on the catalytic activity and stability of the lipase. Therefore, the transesterification yields depend on the size of interfacial area which can be increased by the addition of certain amounts of water as well as the availability of an oil-water interface. However, lipases increase the hydrolysis reaction in aqueous medium and excess water causes the decrease of the transesterification yield by promoting the hydrolysis reaction [42]. 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. Water content in reaction mixture can be determined by either water activity or as weight percentage of feedstock oil. Water activity is the ratio of vapor pressure of a given system [38]. Optimum water content for the transesterification reaction is very important. The optimum water content in the reaction depends upon the lipase type and feedstock, immobilization technique and solvent type [47]. 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 *Pseudomonas fluorescens* lipase, and 1-2% for *Pseudomonas cepacia* lipase [35]. Deng et al. (2005) studied several immobilized commercially available lipases and reported that the conversion obtained from the transesterification reaction with all the other lipases (*Thermomyces lanuginosus, Rhizomucor miehei, Pseudomonas cepacia,* and *Pseudomonas fluorescens*) with the exception of *Candida antarctica* was higher when anhydrous ethanol was replaced with hydrous ethanol (4% water) [48]. It is also very important to take into consideration the amount 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 [48]. Some authors have suggested that adding water into the enzymatic reaction medium can protect lipases against deactivation in the presence of short-chain alcohols [13, 19].

### **2.3. Organic solvent use**

solvent-free system was deactivated irreversibly when the methanol concentration exceeded its solubility level. They found that stepwise addition of methanol prevented lipase deactiva‐ tion. A three-step addition process converted 98.4% of the oil to its corresponding methyl esters in 48 h and the immobilized lipase was set to be reused for 50 batches [1]. Watanabe et al. (2000) investigated the influence of methanol using a two-step strategy on biodiesel synthesis. It has been shown that one-third of the alcohol adding at the beginning of the reaction caused slow conversion into biodiesel (10 h reaction time). After that, the rest of the alcohol was added in a single step and biodiesel conversion increased by the presence of the biodiesel since its solubility increased [21]. In another study [45] two lipases from *Pseudomonas fluorescens* and *Pseudomonas cepacia* (now *Burkholderia cepacia*) were used and they provided 58% and 37% conversion in the presence of 1:8 oil/methanol molar ratio in a solvent-free system, respectively. However, they have been shown to be completely inactive for another six lipases tested under these conditions. It is clear that the excess alcohol above and beyond the stoichiometric ratio increases the reaction rate, but too much alcohol may also deactivate the enzyme [30]. 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. To prevent the alcohol deactivating the enzyme, many researchers have used organic solvents in the reaction medium to increase the solubility of the alcohol and

The effect of water content is essential for enzymatic reactions due to formation of hydrogen bonds which are fundamental in the interactions for maintaining the conformation of the enzymes. Water has strong influence on the catalytic activity and stability of the lipase. Therefore, the transesterification yields depend on the size of interfacial area which can be increased by the addition of certain amounts of water as well as the availability of an oil-water interface. However, lipases increase the hydrolysis reaction in aqueous medium and excess water causes the decrease of the transesterification yield by promoting the hydrolysis reaction [42]. 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. Water content in reaction mixture can be determined by either water activity or as weight percentage of feedstock oil. Water activity is the ratio of vapor pressure of a given system [38]. Optimum water content for the transesterification reaction is very important. The optimum water content in the reaction depends upon the lipase type and feedstock, immobilization technique and solvent type [47]. 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 *Pseudomonas fluorescens* lipase, and 1-2% for *Pseudomonas cepacia* lipase [35]. Deng et al. (2005) studied several immobilized commercially available lipases and reported that the conversion obtained from the transesterification reaction with all the other lipases (*Thermomyces lanuginosus, Rhizomucor miehei, Pseudomonas cepacia,* and *Pseudomonas fluorescens*) with the exception of *Candida antarctica* was higher when anhydrous ethanol was replaced with hydrous ethanol (4% water) [48]. It is also very important to take into consideration the amount 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

reduce its concentration [12, 16, 18, 46].

**2.2. Water content**

24 Biofuels - Status and Perspective

The use of organic solvents in enzymatic biodiesel synthesis improves mutual solubility of hydrophobic compounds (e.g. TAG and biodiesel), triglycerides and hydrophilic compounds (e.g. alcohols and glycerol). Organic solvents also protect enzymes for denaturation resulted high concentrations of alcohols [42]. Solvents also serve to reduce the viscosity of the reaction medium, enabling a higher diffusion rate to be achieved and reducing mass transfer problems. Therefore, a suitable solvent must be found, which both enhances the catalytic activity of the enzyme and keeps it stable. Thus, the presence of a solvent renders a high yield and reduces the enzyme inhibition by alcohol [47]. The most suitable non-polar hydrophobic organic solvents such as n-heptane, petroleum ether, isooctane, n-hexane and cyclohexane were used for enzymatic biodiesel synthesis and immobilized lipases showed high degree of efficiency in the presence of non-polar solvents. But when using hydrophobic solvents, glycerol is insoluble and remains in the reactor and it is adsorbed to the immobilized lipase. The polar hydrophilic organic solvents are much less useful in enzyme-catalyzed biodiesel production as they strongly interact with the essential water microlayer around the enzyme molecules influencing its native structure, thereby, leading to denaturation [42]. Recently, processes of transesterification, which is well known for its compatibility with lipases, have been also conducted in less conventional solvents, e.g. in supercritical gases like butane (C4H10) and carbon dioxide (CO2). CO2 is also regarded as a green solvent owing to its low toxicity, nonflammability, and its environmentally good-natured character [30].

### **2.4. Biocatalysis type**

Recently, lipases have been studied for biodiesel production as whole-cell immobilized lipases. Each type of biocatalyst has its strengths and weaknesses when it comes to reducing the contributionofthebiocatalystinthe final costofthebiodiesel.Recent studieshavebeenfocusing on improving catalysis performance and stability of the enzyme with the aim to reduce the lipase cost in the biodiesel conversion process. Different approaches have been developed for application mode of lipases. Solid state fermentation, whole-cell biocatalyst and immobilized lipase in different supports are the main studied modes. The application of solid state fermen‐ tation was createdforreducing costin lipase production and couldbe used as a catalystin batch andcontinuous operation.The solidstate fermentationof agriculturalresiduespermits for costefficient production and low-price when compared to commercial enzymes. Since solid state fermentation avoids the extraction, purification, and immobilization steps in enzyme produc‐ tion with satisfactory catalytic results in transesterification reaction [27].

### *2.4.1. Free biocatalysis*

Microbial lipases have gained wide industrial importance and they now share about 5% of the world enzyme market after proteases and carbohydrases. Lipases of microbial origin are more stable than plant and animal lipases and are available in bulk at lower cost compared to lipases of other origin. Yeasts lipases are easy to handle and grow compared to bacterial lipases. Among the yeast lipases, *Candida rugosa* has gained good commercial importance. The most commonly used biocatalyst for biodiesel production are the microbial lipases that are pro‐ duced by a number of fungal, bacterial, and yeast species [40]. 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) [49].

### *2.4.2. Immobilized biocatalysis*

Immobilization of lipases was carried out using entrapment, physical adsorption, ion ex‐ change, and crosslinking. Carriers for lipase immobilization include polyurethane foam, silica, sepabeads, cellulosic nanofibers. Based on the criteria for selecting the immobilization technique and carrier dependings on the source of lipase, the type of reaction system (aqueous, organic solvent or two-phase system), and the bioreactor type (batch, stirred tank, membrane reactor, column and plug-flow) can be designed. The literature is replete with various lipase producing microorganisms, enzyme immobilization methods, and physical carriers. The challenge will be to select a carrier and immobilization technique that will allow maximum lipase activity, retention, and stability on the oil substrate. Among the immobilization method, adsorption technique is the simplest and most widely used technique for lipase immobiliza‐ tion. Adsorption method consists of bonding the lipase to the immobilization support surface through weak forces such as van der Waals or hydrophobic interactions. However, the main disadvantage of this technique is enzyme desorption from the support due to low bond strength between the enzyme and the support [40].

### *2.4.3. Whole-cell biocatalysis*

In recent years, whole-cell immobilized lipases have been studied for biodiesel production. This method is cheaper as it does not require the enzyme purification and isolation steps from fermentation broth. The efficiency of the transesterification process could be increased by using microbial cells that produce intra-cellular lipase as whole-cell biocatalysts [40, 46]. Filamentous fungi have been identified as robust whole-cell biocatalysts for biodiesel production: among these *Rhizopus* and *Aspergillus* have been most widely used [42]. There are several recent works reporting the utilization of bacteria, yeast and fungi as whole-cell biocatalysts in biodiesel process [27].
