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.     

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.     

A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium

A new enzymatic route for biodiesel production from soybean oil was developed using methyl acetate as a novel acyl acceptor. Novozym 435 (immobilized Candida antarctica lipase) gave the highest methyl ester (ME) yield of 92%. The optimum conditions of the transesterification were 30% enzyme based on oil weight; a molar ratio of methyl acetate/oil of 12:1; temperature 40 °C and reaction time 10 h. Since no glycerol was produced in the process, this method is very convenient for recycling the catalyst and by-product triacetylglycerol showed no negative effect on the fuel property.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.     

Improved triglyceride transesterification by circular permuted Candida antarctica lipase B

Lipases represent a versatile class of biocatalysts with numerous potential applications in industry including the production of biodiesel via enzyme‐catalyzed transesterification. In this article, we have investigated the performance of cp283, a variant of Candida antarctica lipase B (CALB) engineered by circular permutation, with a series of esters, as well as pure and complex triglycerides. In comparison with wild‐type CALB, the permutated enzyme showed consistently higher catalytic activity (2.6‐ to 9‐fold) for trans and interesterification of the different substrates with 1‐butanol and ethyl acetate as acyl acceptors. Differences in the observed rates for wild‐type CALB and cp283 are believe to be related to changes in the rate‐determining step of the catalytic cycle as a result of circular permutation. Biotechnol. Bioeng. 2010;105: 44–50. © 2009 Wiley Periodicals, Inc.     

酶法合成生物柴油工业化研究进展

介绍了北京化工大学近年来酶法合成生物柴油工业化研究的结果。主要内容包括以下几个方面:高产脂肪酶菌株的选育、脂肪酶发酵工艺优化及放大、脂肪酶固定化方法、酶反应器放大、生物柴油分离精制及副产物甘油综合利用。该脂肪酶假丝酵母Candida sp.99-125在5 m3罐发酵活力不低于8 000 IU/mL,然后将该脂肪酶吸附固定在织物膜上并进行表面改性,用于搅拌罐式反应器生产每吨甲酯的需酶量仅为4.2 kg,产品经分离精制调质后,其各项指标完全符合德国生物柴油生产标准。副产物甘油可用于1,3-丙二醇发酵,30 L发酵罐中1,3-丙二醇的产量可达到76.1 g/L。     

Mechanistic modeling of biodiesel production using a liquid lipase formulation

In this article, a kinetic model for the enzymatic transesterification of rapeseed oil with methanol using Callera? Trans L (a liquid formulation of a modified Thermomyces lanuginosus lipase) was developed from first principles. We base the model formulation on a Ping‐Pong Bi‐Bi mechanism. Methanol inhibition, along with the interfacial and bulk concentrations of the enzyme was also modeled. The model was developed to describe the effect of different oil compositions, as well as different water, enzyme, and methanol concentrations, which are relevant conditions needed for process evaluation, with respect to the industrial production of biodiesel. The developed kinetic model, coupled with a mass balance of the system, was fitted to and validated on experimental results for the fed‐batch transesterification of rapeseed oil. The confidence intervals of the parameter estimates, along with the identifiability of the model parameters were presented. The predictive capability of the model was tested for a case using 0.5% (wt. Enzyme/wt. Oil), 0.5% (wt. Water /wt. Oil) and feeding 1.5 times the stoichiometric amount of methanol in total over 24 h. For this case, an optimized methanol feeding profile that constrains the amount of methanol in the reactor was computed and the predictions experimentally validated. Monte‐Carlo simulations were then used to characterize the effect of the parameter uncertainty on the model outputs, giving a biodiesel yield, based on the mass of oil, of 90.8 ± 0.55 mass %. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1277–1290, 2014     

间歇及连续式固定化酶反应生产生物柴油

探讨了利用本实验室自制的Candida sp99．125脂肪酶转酯化合成生物柴油的过程。在利用间歇式反应得到最佳反应条件的情况下利用固定床反应器生产生物柴油,经过初步优化的试验结果表明,在采用分级流加甲醇下,生物柴油的转化率可以达到93％左右,并且固定化酶的使用寿命超过480h。     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Biocatalytic transesterification of triglycerides and alcohols for the production of biodiesel using cutinase in organic media

Abstract

The world's energy supply is mainly composed of fossil fuels, which are a non-renewable source of energy that is rapidly running out. To overcome this concern, industry has been focusing on the production of biofuels such as biodiesel. A range of approaches has been considered to transform oils into applicable biodiesel: dilutions, microemulsifications, pyrolysis and transesterification. The latter method consists of the conversion of triglycerides to a mixture of alkyl esters and glycerol, in the presence of an acyl acceptor and a catalyst. Due to high selectivity when using enzymes as catalysts, and mild operating conditions, biocatalytic transesterification has proven to be an efficient method. Cutinase, from the superfamily α/β hydrolases, is an enzyme with lipolytic activity that effectively catalyses transesterification reactions. This article highlights the use of cutinase microencapsulated in bis(2ethylhexyl) sodium sulfosuccinate (AOT)-reversed micelles to perform the biocatalytic transesterification of triglycerides, with low chain-length alcohols (e.g. methanol), in organic media to produce biodiesel.     

脂肪酶催化合成生物柴油的瓶颈问题及其对策研究进展

生物柴油,一种新型的清洁能源燃料,具有可再生、可生物降解、环境友好等优良的品性,可部分或全部替代石化柴油。碱催化法、脂肪酶催化法及超临界法是合成生物柴油的主要工艺,其中脂肪酶催化法是一种节能型、环保型工艺,在节能和环保方面,有着碱催化法无可比拟的优越性,具有良好的工业应用前景。但目前在实现产业化的进程中仍存在如酶成本高、稳定性较差、甲醇对酶的失活效应及反应时间长等瓶颈问题。通过固定化技术和全细胞催化剂的采用、甲醇流加方式的改进、溶剂工程的改善及酰基受体和耐醇酶的开发等技术手段,结合固定床生物反应器,较好地解决了这些瓶颈问题,从而推进了酶催化法合成生物柴油的工业化进程。本文主要对酶法合成生物柴油工艺存在的主要问题及相应对策研究进展进行概括介绍,并对其工业化发展前景进行讨论。     

Performance of a cutinase membrane reactor for the production of biodiesel in organic media

The enzymatic transesterification of oils with an alcohol, using recombinant cutinase of Fusarium solani pisi microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reversed micelles, was performed in a membrane bioreactor (MBR). A tubular ceramic membrane with a nominal molecular weight cut off of 15,000 Da was used to retain the enzyme, and characterized in terms of rejection coefficients of the reaction components by transmission experiments. The performance of the MBR in a total recirculation-batch mode was compared with results obtained in a stirred batch tank reactor. The continuous operation of the MBR was also evaluated and the influence of the alcohol type and permeate flow rate on conversion degree and productivity (up to 500 g(product) /day/g(enzyme) was attained) were analyzed. Cutinase wild type and mutant T179C were tested for this process and the high long-term operational stability of the cutinase mutant demonstrated its potential as biocatalyst for the enzymatic continuous production of biodiesel.     

固定化脂肪酶催化制备L-薄荷醇

用大孔树脂NKA固定高选择性的脂肪酶,催化有机相中转酯化反应,从而拆分八异构体消旋薄荷醇来制备L-薄荷醇。研究pH、载体与酶比例对固定化酶制备的影响及固定化酶的反应稳定性;考察温度、转酯化过程醇酯比例、及底物醇异构组成变化对拆分效果的影响。结果表明:固定化酶的最适pH为8,载体与酶的比例为5∶1时,所得固定化酶的反应稳定性比游离酶的反应稳定性提高了约50%;转酯化反应的最优温度为40℃,醇酯比例为1.5∶1时最佳,改进八异构体消旋薄荷醇组分比例后,非对映体选择率dep达到了95.1%。     

Large-scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors

An integrated approach of biodiesel production from heterotrophic Chlorella protothecoides focused on scaling up fermentation in bioreactors was reported in this study. Through substrate feeding and fermentation process controls, the cell density of C. protothecoides achieved 15.5 g L(-1) in 5 L, 12.8 g L(-1) in 750 L, and 14.2 g L(-1) in 11,000 L bioreactors, respectively. Resulted from heterotrophic metabolism, the lipid content reached 46.1%, 48.7%, and 44.3% of cell dry weight in samples from 5 L, 750 L, and 11,000 L bioreactors, respectively. Transesterification of the microalgal oil was catalyzed by immobilized lipase from Candidia sp. 99-125. With 75% lipase (12,000 U g(-1), based on lipid quantity) and 3:1 molar ratio of methanol to oil batch-fed at three times, 98.15% of the oil was converted to monoalkyl esters of fatty acids in 12 h. The expanded biodiesel production rates were 7.02 g L(-1), 6.12 g L(-1), and 6.24 g L(-1) in 5 L, 750 L, and 11,000 L bioreactors, respectively. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with the US Standard for Biodiesel (ASTM 6751). These results suggest that it is feasible to expand heterotrophic Chlorella fermentation for biodiesel production at the industry level.     

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.     

Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium

tert-Butanol, as a novel reaction medium, has been adopted for lipase-catalyzed transesterification of rapeseed oil for biodiesel production, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to catalyze the methanolysis and the highest biodiesel yield of 95% could be achieved under the optimum conditions (tert-butanol/oil volume ratio 1:1; methanol/oil molar ratio 4:1; 3% Lipozyme TL IM and 1% Novozym 435 based on the oil weight; temperature 35 °C; 130 rpm, 12 h). There was no obvious loss in lipase activity even after being repeatedly used for 200 cycles with tert-butanol as the reaction medium. Furthermore, waste oil was also explored for biodiesel production and it has been found that lipase also showed good stability in this novel system.