Interesterification of fats and oils by immobilized fungus at constant water concentration

The kinetics of enzymatic interesterification of oils and fats, using acetone-dried cells of Rhizopus chinensis immobilized on biomass support particles as a lipase catalyst, were investigated in batch operations at several constant water concentrations.Even under microaqueous (i.e., low-water-content) conditions, not only interesterification but also hydrolysis occured, and the water content in the reaction system decreased. The reaction rates of interesterification and hydrolysis at constant water concentrations were determined.For the reactions between olive oil and methyl stearate at several water concentrations, the parameters involved in the reaction model were determined by a trial-and-error method so as to make the calculated results correlate with the experimental data. The relationship between the parameters obtained and water concentration were examined.The rate constants involved in the reaction model of both interesterification and hydrolysis increased or decreased monotonically with the increasing water content, while the apparent activity of the lipase catalyst for interesterification had a maximum value at a water concentration of about 50 ppm. This suggests that when the water content is excessive the hydrolysis activity of lipase is accelerated more than its interesterification activity, and that when the water content is too little lipase activity can not be activated for either hydrolysis or interesterification.     

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.     

超临界甲醇酯交换法制备生物柴油研究进展

超临界甲醇法制备生物柴油是动、植物油脂与超临界甲醇发生酯交换反应生成脂肪酸甲酯的工艺。与传统的酸、碱催化法以及酶催化法等技术相比,超临界酯交换反应具有不需要催化剂、反应速度快、产物分离简单等突出特点。缺点在于反应温度和压力条件不够温和,对设备要求较高,操作费用可观。如何从系统工程的角度发挥其优点、克服缺点,则是未来该项技术能否实现工业化应用的关键。回顾了该技术的研究进展,重点对过程的影响因素进行了分析讨论。     

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％。     

Rhizopus oryzae IFO 4697 whole cell catalyzed methanolysis of crude and acidified rapeseed oils for biodiesel production in tert-butanol system

Whole cell Rhizopus oryzae (R. oryzae) IFO4697 immobilized within biomass support particles (BSPs) was used as catalyst for biodiesel production in tert-butanol, in which the stability of the catalyst could be enhanced significantly. Different feedstocks (refined, crude and acidified rapeseed oils) were adopted further for biodiesel production in tert-butanol system and it was found that when acidified rapeseed oil was used as feedstocks, the reaction rate and final methyl ester (ME) yield were significantly higher than that of refined and crude rapeseed oil. Major differences among the aforementioned oils were found to be the contents of free fatty acid (FFA), water and phospholipids, which showed varied influences on whole cell mediated methanolysis for biodiesel production. The reaction rate increased with the increase of free fatty acid content in oils; water content had varied influence on reaction rate and biodiesel yield; using adsorbent to remove excessive water could increase biodiesel yield significantly (from 73 to 84%); it was also found interestingly that phospholipids contained in oils could increase the reaction rate to a certain extent.     

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme–substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.     

Enzyme catalysed production of vegetable oils containing omega-3 polyunsaturated fatty acid

Summary The application of enzymatic interesterification for production of vegetable oils containing omega-3 polyunsaturated fatty acids was investigated. Six veteable oils were used as substrates, together with omega-3 polyunsaturated fatty acid, and reactions were catalysed by immobilized Mucor miehei lipase in organic solvent. The degree of incorporation of eicosapentaenoic acid and docosahexaenoic acid into corn oil, sunflower oil, peanut oil, olive oil and soybean oil were 17.71, 17.59, 16.79, 14.89, 13.91 and 10.48%, respectively, after a 12 h incubation period.     

Effects of water on biodiesel fuel production by supercritical methanol treatment

In the conventional transesterification of fats/vegetable oils for biodiesel production, free fatty acids and water always produce negative effects, since the presence of free fatty acids and water causes soap formation, consumes catalyst and reduces catalyst effectiveness, all of which result in a low conversion. The objective of this study was, therefore, to investigate the effect of water on the yield of methyl esters in transesterification of triglycerides and methyl esterification of fatty acids as treated by catalyst-free supercritical methanol. The presence of water did not have a significant effect on the yield, as complete conversions were always achieved regardless of the content of water. In fact, the present of water at a certain amount could enhance the methyl esters formation. For the vegetable oil containing water, three types of reaction took place; transesterification and hydrolysis of triglycerides and methyl esterification of fatty acids proceeded simultaneously during the treatment to produce a high yield. These results were compared with those of methyl esters prepared by acid- and alkaline-catalyzed methods. The finding demonstrated that, by a supercritical methanol approach, crude vegetable oil as well as its wastes could be readily used for biodiesel fuel production in a simple preparation.     

A kinetic model of the Amberlyst-15 catalyzed transesterification of methyl stearate with n-butanol

An attractive approach to improving cold flow properties of biodiesel is to transesterify fatty acid methyl esters with higher alcohols such as n-butanol or with branched alcohols such as isopropanol. In this study, the reaction kinetics of Amberlyst-15 catalyzed transesterification of methyl stearate, a model biodiesel compound, with n-butanol have been examined. After identifying conditions to minimize both internal and external mass transfer resistances, the effects of catalyst loading, temperature, and the mole ratio of n-butanol to methyl stearate in the transesterification reaction were investigated. Experimental data were fit to a pseudo-homogeneous, activity-based kinetic model with inclusion of etherification reactions to appropriately characterize the transesterification system.     

Kinetic modeling of interesterification reactions catalyzed by immobilized lipase

Kinetic data for lipase-catalyzed interesterification reactions between free fatty acids and triglycerides were collected and the dynamics of the interesterification reactions were successfully modeled using tow rate experssions requiring a total of five adjustable parameters. One rate expression describes the disappearance of the free fatty acid (octanoic or linolenic acid), and the second describes the rate of release of fatty acid residues from the triglycerides (olive oil or milkfat). This model is able to account for the effects of the concentration of all chemical species participating in interesterification throughout the entire reaction. When the data for both milkfat and olive oil were subjected to nonlinear regression analyses using the same mathematical model, the parameter estimates for both systems were comparable. In addition to reproducing the tendencies observed experimentally, simulations of the interesterification system under a variety of initial conditions provided insight into the effects of several reaction variables which could not be examined experimentally. Among the most significant findings of the simulation work are (1) there is a limit beyond which increasing the initial concentration of water produces no further increase in the initial rate of the interesterification reaction; (2) an increase in the initial concentration of lower glycerides produces a concomitant increase in the rate of the interesterification reaction; (3) the free fatty acids inhibit the rate of hydrolysis of the fatty acid residues of the triglycerides; (4) there is a limit beyond which increasing the initial concentration of triglycerides produces no significant increase in the rate of either the hydrolysis reaction or the interesterification reaction. (c) 1994 John Wiley & Sons, Inc.     

Lipase-catalyzed acidolysis of menhaden oil with conjugated linoleic acid: effect of water content

The effect of the water content on the lipase-catalyzed (Candida rugosa) interesterification (acidolysis) of menhaden oil with conjugated linoleic acid was studied for amounts of added water ranging from 0-4% (w/w). The rate of the acidolysis reaction increased with increasing water content, but the corresponding percentage of n-3 fatty acids liberated also increased. The implications of water content for minimization of the release of n-3 fatty acid residues while maximizing incorporation of CLA are discussed.     

Biodiesel production—current state of the art and challenges

Biodiesel is a clean-burning fuel produced from grease, vegetable oils, or animal fats. Biodiesel is produced by transesterification of oils with short-chain alcohols or by the esterification of fatty acids. The transesterification reaction consists of transforming triglycerides into fatty acid alkyl esters, in the presence of an alcohol, such as methanol or ethanol, and a catalyst, such as an alkali or acid, with glycerol as a byproduct. Because of diminishing petroleum reserves and the deleterious environmental consequences of exhaust gases from petroleum diesel, biodiesel has attracted attention during the past few years as a renewable and environmentally friendly fuel. Since biodiesel is made entirely from vegetable oil or animal fats, it is renewable and biodegradable. The majority of biodiesel today is produced by alkali-catalyzed transesterification with methanol, which results in a relatively short reaction time. However, the vegetable oil and alcohol must be substantially anhydrous and have a low free fatty acid content, because the presence of water or free fatty acid or both promotes soap formation. In this article, we examine different biodiesel sources (edible and nonedible), virgin oil versus waste oil, algae-based biodiesel that is gaining increasing importance, role of different catalysts including enzyme catalysts, and the current state-of-the-art in biodiesel production. JIMB 2008: BioEnergy—special issue.     

Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.     

Lipase-Catalyzed Reactions for Modification of fats and other Lipids

Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed. At present, the application of lipases in biotechnological processes seems to be economically feasible and appropriate mainly for the preparation of specific products of high commercial value, which cannot be prepared conveniently by chemical synthesis. For example, polyunsaturated fatty acids that can be used in dietetic products are prepared under mild conditions by hydrolysis of marine oils and certain plant oils with non-specific triacylglycerol lipases. Very long chain monounsaturated fatty acids (gadoleic, erucic and nervonic) that are of value in oleochemical industry can be prepared by partial hydrolysis of cruciferous oils with sn-1,3-specific lipases. Lipase-catalyzed esterification yields a variety of products, such as monoacylglycerols that are used as emulsifiers, and wax esters resembling jojoba oil which is used in cosmetics industry. Interesterification of fats with sn-1,3-specinc lipases affords specialty products, such as cocoa butter substitutes which are used in confectionary products and medium chain triacylglycerols that can be used in dietetic products. Phospholipase-catalyzed exchange of acyl moieties or bases of glycerophos-pholipids yields several products of biomedical interest.     

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.     

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in α-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary α-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.     

Process engineering and optimization of glycerol separation in a packed-bed reactor for enzymatic biodiesel production

A process model for efficient glycerol separation during methanolysis in an enzymatic packed-bed reactor (PBR) was developed. A theoretical glycerol removal efficiency from the reaction mixture containing over 30% methyl esters was achieved at a high flow rate of 540 ml/h. To facilitate a stable operation of the PBR system, a batch reaction prior to continuous methanolysis was conducted using oils with different acid values and immobilized lipases pretreated with methyl esters. The reaction system successfully attained the methyl ester content of over 30% along with reduced viscosity and water content. Furthermore, to obtain a high methyl ester content above 96% continuously, long-term lipase stability was confirmed by operating a bench-scale PBR system for 550 h, in which the intermediates containing methyl esters and residual glycerides were fed into the enzyme-packed columns connected in series. Therefore, the developed process model is considered useful for industrial biodiesel production.     

Lipase-Catalyzed Reactions for Modification of fats and other Lipids

Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed. At present, the application of lipases in biotechnological processes seems to be economically feasible and appropriate mainly for the preparation of specific products of high commercial value, which cannot be prepared conveniently by chemical synthesis. For example, polyunsaturated fatty acids that can be used in dietetic products are prepared under mild conditions by hydrolysis of marine oils and certain plant oils with non-specific triacylglycerol lipases. Very long chain monounsaturated fatty acids (gadoleic, erucic and nervonic) that are of value in oleochemical industry can be prepared by partial hydrolysis of cruciferous oils with sn-1,3-specific lipases. Lipase-catalyzed esterification yields a variety of products, such as monoacylglycerols that are used as emulsifiers, and wax esters resembling jojoba oil which is used in cosmetics industry. Interesterification of fats with sn-1,3-specinc lipases affords specialty products, such as cocoa butter substitutes which are used in confectionary products and medium chain triacylglycerols that can be used in dietetic products. Phospholipase-catalyzed exchange of acyl moieties or bases of glycerophos-pholipids yields several products of biomedical interest.     

Use of re-esterified palm oils,differing in their acylglycerol structure,in weaning-piglet diets

Re-esterified oils are new fat sources obtained from chemical esterification of acid oils with glycerol (both economically interesting by-products from oil refining and biodiesel industries, respectively). The different fatty acid (FA) positional distribution and acylglycerol composition of re-esterified oils may enhance the apparent absorption of saturated fatty acids (SFA) and, thus, their overall nutritive value. The aim of the present study was to investigate the potential use of re-esterified palm oils, in comparison with their corresponding acid and native oils, and also with an unsaturated fat source in weaning-piglet diets. The parameters assessed were: FA apparent absorption, acylglycerol and free fatty acid (FFA) composition of feces, and growth performance. One-hundred and twenty weaning piglets (average weight of 8.50±1.778 kg) were blocked by initial BW (six blocks) and randomly assigned to five dietary treatments, resulting in four piglets per pen and six replicates per treatment. Dietary treatments were a basal diet supplemented with 10% (as-fed basis) of native soybean oil (SN), native palm oil (PN), acid palm oil (PA), re-esterified palm oil low in mono- (MAG) and diacylglycerols (DAG) (PEL), or re-esterified palm oil high in MAG and DAG (PEH). Results from the digestibility balance showed that SN reached the greatest total FA apparent absorption, and statistically different from PN, PA and PEL (P<0.05). There were no statistical differences among palm-oil sources (P>0.05), but PEH achieved the greatest total FA apparent absorption. Animals fed PEL, despite the fact that PEL oil contained more sn-2 SFA, did not show an improved absorption of SFA (P>0.05). Animals fed PA and PN showed similar apparent absorption coefficients (P>0.05), despite the high FFA content of PA oil. The acylglycerol and FFA composition of feces was mainly composed of FFA. There were no significant differences in growth performance (P>0.05). Results of the present study suggest that, despite the different acylglycerol structure of re-esterified oils, there were no significant differences in digestibility or performance with respect to their corresponding PN and PA oils in weaning-piglet diets.