Alcoholysis of vegetable oils is an important reaction to produce fatty acid alkyl esters which are excellent substitutes for diesel fuel and valuable intermediates in oleochemistry [1, 2]. Fatty acid methyl esters, a mixture of mono-alkyl esters are also known as biodiesel, obtained from both vegetable oils such as sunflower oil, canola oil, soyabean oil, jatropha oil, palm oil, rapeseed oil, peanut oil, cotton seed oil and animal fats such as beef tallow, and lard. Biodiesel can also be produced from other sources such as waste cooking oil, algae, and greases . Biodiesel production has attracted considerable attention in the past two decades because of biodegradable, renewable, non-toxic, and environmentally friendly and socially responsible fuel . Biodiesel can be produced by several methods: direct use or blending, microemulsion, thermal cracking (pyrolysis), and transesterification including acid-catalyzed processes, base-catalyzed processes, lipase-catalyzed processes, non-ionic base-catalyzed processes, and heterogeneously catalyzed processes [5, 6]. Among these methods, alkali catalyzed process including an alkali catalyst (usally NaOH, KOH, or sodium methoxide) has been accepted industrially due to its high conversion of triglycerides to methyl esters in a short reaction time and high reaction rates. In spite of these advantages of chemical transesterification process, it also possesses some disadvantages such as the need to eliminate the catalyst and salt from the biodiesel phase, to remove saponification products, the difficulty of recycling glycerol, and their energy-intensive nature, leading to development of alternative processes [7-9]. Alcoholysis is also carried out under acidic conditions, but this process requires higher reaction temperatures. In order to overcome these drawbacks, recently, enzymatic transesterification has attracted much attention for biodiesel production since it produces high purity product and provides an easy separation from the by-product glycerol. The use of enzymes (lipases) as catalysts in biodiesel production overcomes the problems inherent to alkali catalysts. It is reported that the enzymatic reactions are insensitive to free fatty acid (FFA) and water content in the raw material . 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 18.104.22.168), 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 transesterification 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 . 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.
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 oC. 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 . 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 . Enzymatic catalysis has been applied for biodiesel which starts its industrial scale operation in China . 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 generalizations 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 significantly 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 . 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 . 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 .
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 . 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 . 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 . 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 . Shimada et al. (1999) reported that the lipase from
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 . 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 . 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 . For example, Kaieda et al. 2001 found that the water concentrations that resulted in the best conversions were 8-20% for
2.3. Organic solvent use
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 . 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 . 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 . 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, non-flammability, and its environmentally good-natured character .
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 contribution of the biocatalyst in the final cost of the biodiesel. Recent studies have been focusing 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 fermentation was created for reducing cost in lipase production and could be used as a catalyst in batch and continuous operation. The solid state fermentation of agricultural residues permits for cost-efficient production and low-price when compared to commercial enzymes. Since solid state fermentation avoids the extraction, purification, and immobilization steps in enzyme production with satisfactory catalytic results in transesterification reaction .
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,
2.4.2. Immobilized biocatalysis
Immobilization of lipases was carried out using entrapment, physical adsorption, ion exchange, 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 immobilization. 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 .
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
Commercial powder lipase from
3.2. Experimental procedure
3.2.1. Lipase immobilization
Figure 1 shows multi-layer immobilization of
3.2.2. Biodiesel production with immobilized lipase-catalyzed transesterification
Production of biodiesel by enzymatic catalyzed transesterification from various vegetable oils was studied in a packed bed reactor (Figure 2). A small piece of immobilized cotton cloth (1 g) was placed in the glass column reactor (1 cm diameter x 12 cm height) with a water jacket maintained at a constant temperature (30 oC). Substrate mixture (oil and alcohol) was continuously recirculated throughout the immobilized enzyme reactor with a peristaltic pump at a flow rate of 50 mL/min by adding of alcohol in three-steps. Immobilized cotton cloths were washed by
4. Results and discussion
4.1. Lipase screening
Firstly, lipase screening was performed to find the lipase that has the best catalytic activity in the transesterification of sunflower oil. The most active lipase was then used in further transesterification studies. Two lipases,
4.2. Effect of reaction parameters on transesterification
4.2.1. Effect of alcohol type
It is well known that excessive short-chain alcohols such as methanol might inactivate lipase seriously. However, at least three molar equivalents of methanol are required to complete conversion to its corresponding methyl esters of the oil [26, 30]. Experiments were performed to determine the yield of methyl or ethyl esters by varying the alcohol type using
4.2.2. Effect of water concentration
The effect of water content was examined in the range of 0-2 g and at constant molar ratio of oil to methanol with sunflower oil. The reactions were carried out according to the reaction setup described earlier. The results presented in Figure 6 indicated that water was not required to activate the
4.2.3. Effect of reaction temperature
Experiments were performed to determine the effect of temperature on catalytic activity of immobilized
4.2.4. Effect of oil type
The results depicted in Figure 8 shows that sunflower oil provided the highest methyl ester yield (91.3%) in reactions with methanol, among sunflower, canola, and waste cooking oil. However, the initial reaction rate was higher for canola oil and waste cooking oil than sunflower oil. Free fatty acids formed soaps with alkali salts when alkali-catalyzed process was used to produce biodiesel from waste cooking oils. Use of waste cooking oil in the production of biodiesel with immobilized lipase to cotton cloth has been effective enough in providing substantial methyl ester yield. Since hydrophilic feature of carrier used in immobilization process may adsorb the water on cotton cloth in the reaction medium. Fatty acid methyl ester yields from canola and waste cooking oil were 79.9% and 81%, respectively.
4.2.5. Effect of washing with tert-butanol
The effect of washing with
The method of enzyme immobilization involving polyethyleneimine (PEI)-enzyme aggregate formation was developed and glutaraldehyde was used as a cross-linking agent between free amine groups increasing the enzyme stability. In the present study, a high biodiesel yield was obtained with
The study was supported by the TUBITAK, The Scientific and Technological Research Council of Turkey (Project No: MAG-107M487).
Shimada Y, Watanable Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 1999;76: 789-93.
Köse Ö, Tüter M, Aksoy HA. Immobilized Candida antarctica lipase-catalyzed alcoholysis of cotton seed oil in a solvent-free medium. Bioresour Technol 2002;83: 125-9.
Pearl GG. Animal fat potential for bioenergy use. Bioenergy 2002. The Tenth Biennial Bioenergy Conference, 22-26 Sept 2002, Boise, ID.
Lai C, Zullaikah S, Vali SR, Ju Y. Lipase-catalyzed production of biodiesel from rice bran oil. J Chem Tech Biotechnol 2005;80: 331-7.
Vargas RM, Sercheli R, Schuchardt U. Transesterification of vegetable oils: a review. J Braz Chem Soc 1998;9(1): 199-10.
Hanna MA, Ma F. Biodiesel production: A review. Bioresour Technol 1999;70: 1-15.
Zhang Y, Dubé MA, McLean DD, Kates M. Biodiesel production from waste cooking oil. 1. Process design and technological assessment. Bioresour Technol 2003;89: 1-16.
Hama S, Yamaji H, Fukumizu T, Tamalampudi S, Kondo A, Noda H, Fukuda H. Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 2007;34: 273-8.
Chiu CW, Dasari MA, Sutterlin WR, Suppes GJ. Removal of residual catalyst from simulated biodiesel’s crude glycerol for glycerol hydrogenolysis to propylene glycol. Ind Eng Chem Res 2006;45: 791-5.
Aarthya M, Saravananb P, Gowthamana MK, Rosea C, Kaminia NR. Enzymatic transesterification for production of biodiesel using yeast lipases: An overview. Chem Eng Res Design 2014;92: 1591-601.
Matsumoto T, Takahashi S, Ueda M, Tanaka A, Fukuda H, Kondo A. Preparation of high activity yeast whole cell biocatalysts by optimization of intracellular production of recombinant Rhizopus oryzae lipase. J Mol Catal-B: Enzym 2002;17: 143-9.
Ivanov AE, Schneider MP. Methods for the immobilization of lipases and their use for ester synthesis. J Mol Catal-B: Enzym 1997;3: 303-9.
Iso M, Chen B, Eguchi M, Kudo T, Shrestha S. Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase. J Mol Catal-B: Enzym 2001;16: 53-8.
Noureddini H, Gao X, Philkana RS. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol 2005;96: 769-7.
Fukuda H, Hama S. Tamalampudi S, Fukumizu T, Miura K, Yamaji H, Kondo A. Lipase localization in Rhizopus oryzae cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel-fuel production. J Biosci Bioeng 2006;101(4): 328-3.
Wang F, Gao Y, Tan TW, Nie KL. Immobilization of lipase on macroporous resin and its application in synthesis of biodiesel in low aqueous media. Chin J Biotechnol 2006; 22(1): 114-8.
Mittelbach M. Lipase catalyzed alcoholysis of sunflower oil. J Am Oil Chem Soc 1990;61(3): 168-70.
Okuba M, Takahashi M. Production of submicron-size monodisperse polymer particles having aldehyde groups by the seeded aldol condensation polymerization of glutaraldehyde (II). Colloid Polym Sci 1994;272: 422-6.
Nelson LA, Foglia TA, Marmer WN. Lipase-catalyzed production of biodiesel. J Am Oil Chem Soc 1996;73: 1191-95.
Kaieda M. Samukawa T, Matsumoto T, Ban K, Kondo A., Shimada Y., Noda H, Nomoto F, Ohtsuka K, Izumoto E, Fukuda H. Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J Biosci Bioeng 1999; 88(6): 627-31.
Watanable Y, Shimada Y, Sugihara A, Noda H, Fukuda H, Tominaga Y. Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase. J Am Oil Chem Soc 2000;77(4): 355-9.
Abigor RD, Uadia PO, Foglia TA, Haas, KC, Okpefa JE, Obibuzor JU. Lipase-catalyzed production of biodiesel fuel from some Nigerian lauric oils. Biochem Soc Trans 2000;28: 979-81.
Kamini NR, Lefuji H. Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Cryptoccusspp. S-2. Process Biochem 2001;37: 405-10.
Lara-Pizarro AV, Park EY. Lipase-catalyzed production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth. Process Biochem 2003;38: 1077-82.
Albayrak N, Yang ST. Production of galacto-oligosaccharides from lactose by Aspergillus oryzae β-galactosidase immobilized on cotton cloth. Biotechnol Bioeng 2002;77(1): 8-19.
Shimada Y, Watanable Y, Sugihara A, Tominaga Y. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal-B: Enzym 2002;17: 133-42.
Teixeira CB, Junior VM, Macedo GA. Biocatalysis combined with physical technologies for development of a green biodiesel process. Renew Sustain Energ Rev 2014;33: 333-43.
Tan T, Lu J, Nie K, Zhang H, Deng L, Wang F.. Progress on biodiesel production with enzymatic catalysis in China. Sheng Wu Gong Cheng Xue Bao 2010;26(7): 903-6. Review. Chinese.
Bajaj A, Lohan P, Jha PN, Mehrotra R. Biodiesel production through lipase catalyzed transesterification: an overview. J Mol Catal B Enzym 2010; 62: 9-14.
Antczak MS, Kubiak A, Antczak T, Bielecki S. Enzymatic biodiesel synthesis-key factors affecting efficiency of the process. Renew Energ 2009;34: 1185-94.
Salis A, Pinna M, Monduzzi M, Solinas V. Biodiesel production from triolein and short chain alcohols through biocatalysis. J Biotechnol 2005;119: 291-9.
Balcio VM, Paiva AL, Malcata FX. Bioreactors with immobilized lipases: State of the art. Enzyme Microb Tech 1996;18(5): 392-416.
Wehtje E, Adlercreutz P. Lipases have similar water activity profiles in different reactions. Biotechnol Lett 1997;19: 537-40.
Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Fukuda H. Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from pant oil. J Biosci Bioeng 2000;90(2): 180-3.
Kaieda M, Samukawa T, Kondo A, Fukuda H. Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J Biosci Bioeng 2001;91: 12-5.
Dossat V, Combes D, Marty A. Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production. Enzyme Microb Tech 1999;25: 194-200.
Du W, Xu Y, Liu D, Zeng J. Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors. J Mol Catal-B: Enzym 2004;30: 125-9.
Abdulla R, Ravindra P. Immobilized Burkholderia cepacia lipase for biodiesel production from crude Jatropha curcas L. oil. Biomass Bioenergy 2013;56: 8-13.
Ondul E, Dizge N, Albayrak N. Immobilization of Candida antarctica A and Thermomyces lanuginosus lipases on cotton terry cloth fibrils using polyethyleneimine. Colloids Surf B 2012;95: 109-14.
Christopher PL, Kumar H, Zambare VP. Enzymatic biodiesel: Challenges and opportunities. Appl Energ 2014;119: 497-520.
Al-Zuhair S, Almenhali A, Hamad I, Alshehhi M, Alsuwaidi N, Mohamed S. Enzymatic production of biodiesel from used/waste vegetable oils: Design of a pilot plant. Renew Energ 2011;36: 2605-14.
Gog A, Roman M, Tos M, Paizs C, Irimie FD. Biodiesel production using enzymatic transesterification: Current state and perspectives. Renew Energ 2012;39: 10-6.
Zeng J, Du W, Liu X, Liu D, Dai L. Study on the effect of cultivation parameters and pretreatment on Rhizopus oryzae cell-catalyzed transesterification of vegetable oils for biodiesel production. J Mol Catal B: Enzym 2006;43: 15-1.
Hamaa S, Kondo A. Enzymatic biodiesel production: An overview of potential feedstocks and process development. Bioresour Technol 2013;135: 386-95.
Salis A, Pinna M, Monduzzi M, Solinas V. Comparison among immobilised lipases on macroporous polypropylene toward biodiesel synthesis. J Mol Catal B: Enzym 2008;54: 19-26.
Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel. Bioresour Technol 2008;99: 3975-81.
Guldhe A, Singh B, Mutanda T, Permaul K, Bux F. Advances in synthesis of biodiesel via enzyme catalysis: Noveland sustainable approaches. Renew Sustain Energ Rev 2015;41: 1447-64.
Deng L, Xu X, Haraldsson GG, Tan T, Wang F. Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols. J Am Oil Chem Soc 2005;82: 341-7.
Nielsen PM, Brask J, Fjerbaek L. Enzymatic biodiesel production: Technical and economical considerations. Eur J Lipid Sci Technol 2008;110: 692-700.
Turkan A, Kalay S. Monitoring lipase-catalyzed methanolysis of sun flower oil by reversed-phase high-performance liquid chromatography: Elucidation of mechanisms of lipases. J Chromatogr A 2006;1127: 34-44.