An overview on the recent advances in the transesterification of vegetable oils for biodiesel production using chemical and biocatalysts

Biodiesel, chemically defined as monoalkyl esters of long chain fatty acids, are derived from renewable feed stocks like vegetable oils and animal fats. It is produced by both batch and continuous transesterification processes in which, oil or fat is reacted with a monohydric alcohol in the presence of a catalyst. The conventional method of producing biodiesel involves acid and base catalysts to form fatty acid alkyl esters. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods and alternative substrates. Enzymatic reactions involving lipases can be an excellent alternative to produce biodiesel through a process commonly referred to as alcoholysis, a form of transesterification reaction or through an interesterification reaction. In order to increase the cost effectiveness of the process, the enzymes are immobilized using a suitable matrix. The use of immobilized lipases and whole cells may lower the overall cost, while presenting less downstream processing problems. Main focus of this paper is to discuss the important parameters that affect the biodiesel yield, various immobilization techniques employed, mechanisms and kinetics of transesterification reaction and the recent advances in continuous transesterification processes.     

Biodiesel production by transesterification using immobilized lipase

Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.     

Biodiesel production from Jatropha curcas: a critical review

The fuel crisis and environmental concerns, mainly due to global warming, have led researchers to consider the importance of biofuels such as biodiesel. Vegetable oils, which are too viscous to be used directly in engines, are converted into their corresponding methyl or ethyl esters by a process called transesterification. With the recent debates on “food versus fuel,” non-edible oils, such as Jatropha curcas, are emerging as one of the main contenders for biodiesel production. Much research is still needed to explore and realize the full potential of a green fuel from J. curcas. Upcoming projects and plantations of Jatropha in countries such as India, Malaysia, and Indonesia suggest a promising future for this plant as a potential biodiesel feedstock. Many of the drawbacks associated with chemical catalysts can be overcome by using lipases for enzymatic transesterification. The high cost of lipases can be overcome, to a certain extent, by immobilization techniques. This article reviews the importance of the J. curcas plant and describes existing research conducted on Jatropha biodiesel production. The article highlights areas where further research is required and relevance of designing an immobilized lipase for biodiesel production is discussed.     

脂肪酶协同催化猪油合成生物柴油工艺研究

探讨了以乙酸甲酯为酰基受体两种脂肪酶协同催化猪油转酯合成生物柴油的工艺条件。首先利用单因子试验确定2种固定化脂肪酶Novozym435、Lipozyme TLIM单独作为催化剂时的最佳酶用量为40％,反应温度为50℃,乙酸甲酯用量为14（相对于油的摩尔比）。在此基础上,采用3因素5水平和3个中心点的中心组分旋转设计法研究了上述2种脂肪酶协同使用时脂肪酶用量（g/g）、混合酶的配比（%/%）以及乙酸甲酯用量诸因素共同作用对转酯反应转化率的影响。优化后的反应条件为：总酶用量为40％,混合酶配比为50/50,乙酸甲酯用量为14,在该条件下甲酯得率可达97.6％,比同质量的Novozym435、Lipozyme TLIM的催化活性分别高出7.6％、22.3％。表明脂肪酶协同催化猪油合成生物柴油工艺可以较好地提高甲酯得率,并且节约生产成本。     

An overview of enzymatic production of biodiesel

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.     

Biodiesel production with immobilized lipase: A review

Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is “greener”. This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99–125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored.     

Dynamic modeling of biodiesel production from simulated waste cooking oil using immobilized lipase

The effectiveness of lipase immobilized on ceramic beads, in the production of biodiesel from simulated waste cooking oil in organic solvent system, was compared to that of free lipase. Experimental determination of the effect of concentrations of methanol on the rate of the enzymatic transesterification was experimentally determined. In addition, the effectiveness of lipases from bacterial and yeast sources for biodiesel production from simulated waste cooking oil was compared. A kinetic model was developed to describe the system, taking into consideration the mass transfer resistances of the reactants. Inhibition effects by both substrates on the interfacial reaction were also considered. The experimental results were used to determine the kinetic parameters of the proposed model and to determine the effect of mass transfer. On the other hand, it was shown that biodieasel can be produced in considerable amounts, with yield reaching 40%, in absence of organic solvent using immobilized lipase from P. cepacia on ceramic beads.     

Study on acyl migration in immobilized lipozyme TL-catalyzed transesterification of soybean oil for biodiesel production

During enzymatic transesterification of soybean oils with methanol for biodiesel production, it was supposed that the maximum biodiesel yield was only 66% since lipozyme TL was a typical lipase with a strict 1,3-positional specificity. However, it has been observed that over 90% biodiesel yield could be obtained. It was therefore assumed, and subsequently demonstrated, that acyl migration occurred during the reaction process. Different factors which may influence the acyl migration were explored further and it has been found that the silica gel acting as the immobilized material contributes significantly to the promotion of acyl migration in the transesterification process. The final biodiesel yield was only 66% when 4% lipozyme TL used, while about 90% biodiesel yield could be achieved when combining 6% silica gel with 4% lipozyme TL, almost as high as that of 10% immobilized lipase used for the reaction.     

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system

In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with the support surface, resulting in activation of the enzyme catalytic center. Due to the interest in biodiesel production by mild enzymatic transesterification, this heterogeneous biocatalyst has been used in transesterification of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Biocatalysis: Towards ever greener biodiesel production

The cost of lipases and the relatively slower reaction rate remain as the major obstacles for enzymatic production of biodiesel as opposed to the conventional chemical processes. This paper reviews the starting oils usually employed in biodiesel production, the processes for transforming them to biodiesel placing particular emphasis on enzymatic transesterification. The pros and cons of the lipase-based process, the key operational variables and the technological alternatives for attenuating lipase deactivation are also discussed. Finally, suggestions are made for future studies, paying particular attention to the use of whole cell immobilization in the production process, as this methodology may reduce both the cost of the biocatalyst and dependence on lipase manufacturers.     

Biodiesel fuel production via transesterification of oils using lipase biocatalyst

Biodiesel has gained widespread importance in recent years as an alternative, renewable liquid transportation fuel. It is derived from natural triglycerides in the presence of an alcohol and an alkali catalyst via a transesterification reaction. To date, transesterification based on the use of chemical catalysts has been predominant for biodiesel production at the industrial scale due to its high conversion efficiency at reasonable cost. Recently, biocatalytic transesterification has received considerable attention due to its favorable conversion rate and relatively simple downstream processing demands for the recovery of by-products and purification of biodiesel. Biocatalysis of the transesterification reaction using commercially purified lipase represents a major cost constraint. However, more cost-effective techniques based on the immobilization of both extracellular and intracellular lipases on support materials facilitate the reusability of the catalyst. Other variables, including the presence of alcohol, glycerol and the activity of water can profoundly affect lipase activity and stability during the reaction. This review evaluates the current status for lipase biocatalyst-mediated production of biodiesel, and identifies the key parameters affecting lipase activity and stability. Pioneer studies on reactor-based lipase conversion of triglycerides are presented.     

Production of Biodiesel Using Immobilized Lipase—A Critical Review

Increase in volume of biodiesel production in the world scenario proves that biodiesel is accepted as an alternative to conventional fuel. Production of biodiesel using alkaline catalyst has been commercially implemented due to its high conversion and low production time. For the product and process development of biodiesel, enzymatic transesterification has been suggested to produce a high purity product with an economic, environment friendly process at mild reaction conditions. The enzyme cost being the main hurdle can be overcome by immobilization. Immobilized enzyme, which has been successfully used in various fields over the soluble counterpart, could be employed in biodiesel production with the aim of reducing the production cost by reusing the enzyme. This review attempts to provide an updated compilation of the studies reported on biodiesel production by using lipase immobilized through various techniques and the parameters, which affect their functionality.     

超声波辅助下脂肪酶催化高酸值废油脂制备生物柴油

探讨了超声波辅助条件下脂肪酶催化高酸值废油脂转化为生物柴油的反应。来源于Aspergillus oryzae和Candida antarctica的固定化脂肪酶,在超声波辅助下,对高酸值废油脂转化为生物柴油具有高的催化活性。以来自于C.antarctica的固定化脂肪酶Novozym435为催化剂,以酸价为157mg KOH/g的高酸值废油脂为原料在超声波辅助下与丙醇反应,在脂肪酶用量为油质量的8%、初始醇油摩尔比为3∶1、反应温度控制在40~45℃、超声波频率和功率分别采用28kHz和100W的条件下,反应50min转化率达到94.86%。在此条件下,不同碳原子数(C1~C5)的直链和支链醇均有较高的转化率,在短链醇的选择上具有宽广的适应性。超声波还减少了反应产物和反应体系中其他黏性杂质在固定化脂肪酶表面的吸附,回收的Novozym435相较单纯机械搅拌条件下回收的外观干净、分散良好无结块现象、易于洗涤和再次利用,具有良好的操作稳定性。     

Biotechnological production of biodiesel fuel using biocatalysed transesterification: A review

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

脂肪酶催化合成生物柴油的研究

生物柴油是用动植物油脂或长链脂肪酸与甲醇等低碳醇合成的脂肪酸甲酯,是一种替代能源。这里探讨了生物法制备生物柴油的过程,采用脂肪酶酯化和酯交换两条工艺路线进行催化合成。深入研究制备过程中,不同脂肪酶、酶的用量和纯度、有机溶剂、低碳醇的抑制作用、吸水剂的作用、反应时间和进程、底物的特异性和底物摩尔比等参数对酯化过程的影响。试验结果表明,采用最佳酯化反应参数和分批加入甲醇并用硅胶作脱水剂的工艺过程,酯化率可以达到92％,经分离纯化后的产品GC分析的纯度可达98％以上,固定化酶的使用半衰期可达到360h。同时对酯交换制备生物柴油过程中,甲醇的用量和甲醇的加入方式对脂肪酶催化过程的影响作了初步研究,优化后的酯交换率可达到83％。     

Selection and Characterization of Biofuel-Producing Environmental Bacteria Isolated from Vegetable Oil-Rich Wastes

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Development of an amphiphilic matrix for immobilization of <Emphasis Type="Italic">Candida antartica</Emphasis> lipase B for biodiesel production

Biodiesel has been greatly interested as an alternative fuel and is produced by a transesterification reaction of oil with alcohol. Recently, microbial lipases have been used for biodiesel production. Among the microbial lipase, immobilized Candida antartica lipase B (CALB) is the most widely used. However, CALB is unstable and shows low catalytic efficiency in the reaction media because the reaction media contains a high concentration of methanol and the lipase is also inhibited by the by-product glycerol. In this study, to overcome these limitations, we developed an amphiphilic matrix to immobilize CALB. The immobilized lipase in an amphiphilic matrix with 80% ethyltrimethoxysilane (ETMS) in tetramethoxysilane (TMOS) and pretreated with oil showed the highest specific activity and biodiesel conversion ratio; about 90% biodiesel conversion in 24 h at an initial molar ratio of 1: 1 (oil: methanol) with stepwise methanol feeding in order to adjust the net molar ratio to be 1: 3.     

Production of biodiesel by lipase-catalyzed transesterification of vegetable oils: a kinetics study

Kinetics of production of biodiesel by enzymatic methanolysis of vegetable oils using lipase has been investigated. A mathematical model taking into account the mechanism of the methanolysis reaction starting from the vegetable oil as substrate, rather than the free fatty acids, has been developed. The kinetic parameters were estimated by fitting the experimental data of the enzymatic reaction of sunflower oil by two types of lipases, namely, Rhizomucor miehei lipase (RM) immobilized on ion-exchange resins and Thermomyces lanuginosa lipase (TL) immobilized on silica gel. There was a good agreement between the experimental results of the initial rate of reaction and those predicted by the proposed model equations, for both enzymes. From the proposed model equations, the regions where the effect of alcohol inhibition fades, at different substrate concentrations, were identified. The proposed model equation can be used to predict the rate of methanolysis of vegetable oils in a batch or a continuous reactor and to determine the optimal conditions for biodiesel production.