Lipase-mediated Transformation of Vegetable Oils into Biodiesel using Propan-2-ol as Acyl Acceptor

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 °C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7^th cycle when methanol was used as an acyl acceptor, under standard reaction conditions. Revisions requested 22 December 2005; Revisions received 26 January 2006     

Lipase catalyzed transesterification of soybean oil using ethyl acetate,an alternative acyl acceptor

Methanol, the acyl acceptor usually used in the commercial process of biodiesel production, is associated with some problems such as immiscibility with oils and lipase deactivation. To overcome these barriers, ethyl acetate was proposed as an alternative acyl acceptor for the production of biodiesel from soybean oil using an immobilized lipase, Novozym 435, Ethyl acetate mixed well with soybean oil, and only slightly inhibited the lipase activity by 5%. The effects of various environmental parameters, such as the composition of soybean oil and ethyl acetate, lipase content, and reaction temperature, were investigated to determine the optimal conditions for biodiesel production. As a result, the highest biodiesel production yield, 63.3 (±0.6)%, was obtained by using an ethyl acetate and soybean oil mixture with a 6∶1 molar ratio, with 8% of the immobilized lipase based on the weight of oil added at 70°C and 600 rpm.     

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.     

Study on the kinetics of enzymatic interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor

A new route for biodiesel production using methyl acetate instead of methanol as the acyl acceptor was proposed in our previous research, and it has been found that this novel route could enhance the stability of the immobilized lipase greatly. In this paper, the kinetics of lipase-catalyzed interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor was further studied. First, a simplified model based on Ping Pong Bi Bi with substrate competitive inhibition mechanism was proposed to describe the reaction kinetics of the interesterification. During our further study, it was observed that three consecutive and reversible reactions occurred in the interesterification of triglycerides and methyl acetate. So, a kinetic model based on mass balance of three second-order reversible reactions was developed and the reaction rate constant, k, was determined by solving the differential rate equations of the reaction system. The results showed that k_DG–MG (0.1124) and k_MG–TA (0.1129) were much higher than k_TG–DG (0.0311), which indicated that the first step reaction was the limit step for the overall interesterification.     

Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

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。     

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.     

Biodiesel Production from Non-edible Oils of Jatropha and Karanj for Utilization in Electrical Generator

The biodiesel processor was developed for the production of biodiesel from non-edible oil of jatropha and karanj. The newly developed biodiesel processor is suitable for farmers in village level biodiesel production. The biodiesel processor was capable of producing 15 kg biodiesel per batch in 1.5 h at reaction temperature of 60°C. The biodiesel was produced from raw jatropha and karanj oil, and its blends with diesel were tested for power generation in a 7.5-kVA diesel engine generator set. The fuel properties, namely, kinematic viscosity and specific gravity, were found within the limits of Bureau of Indian Standards specifications. The overall efficiency of the generator for 4,500 W loading condition of jatropha- and karanj-biodiesel-blended fuel were recorded in the range of 21–23% and 24–27%, respectively. The overall efficiency of the generator for 6,000 W loading conditions was improved for jatropha and karanj biodiesel blends and were found in the range of 31–33% and 33–39%, respectively. Biodiesel blends B80 and pure biodiesel of karanj produced more power, and maximum overall efficiency was recorded as compared with diesel fueled generator. The overall efficiency on jatropha-biodiesel-blended fuel were found less than the diesel-fueled generator. The biodiesel processor based on alkali-catalyzed transesterification process can be used for quality biodiesel production from edible and non-edible vegetable oils. This processor can be integrated with rural energy system for domestic and small-scale industrial unit for biodiesel production.     

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.     

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.     

Resolution of d,l-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea

Summary Interesterification in isooctane with triacetin as an acyl donor was found to be a new and effective method of racemic resolution of d,l-menthol, when using the free and immobilized lipase of Candida cylindracea. No water was produced by this highly stereoselective type of reaction in contrast to ester synthesis with acetic acid as an acyl donor. Even with diacetin no possible back reaction occurred and the enzyme was easily separated from the reaction solution as opposed to ester hydrolysis in aqueous systems. Inhibition of interesterification was caused by increasing concentrations of the acyl donor triacetin by more than 10 mmol·l^-1 on the one hand, and especially by diacetin on the other hand. The reaction product menthyl acetate had no influence. By adding water the interesterification activity of the lipase was reduced significantly. An alteration of the acyl donor triacetin to longerchained triglycerides caused changes in higher specific activities but poor enantioselectivities of the products, as in the case of ester synthesis starting from longer-chained organic acids.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 60th birthday     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Enzymic interesterification of fats: The effect of non-lipase material on immobilized enzyme activity

An immobilized lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) suitable for fat interesterification has been prepared by precipitation onto diatomaceous earth (Celite) with acetone of a crude lipase preparation from an Aspergillus. Non-lipase material present in the preparation which precipitated at high acetone concentrations or ovalbumin added prior to the immobilization reduced the measured interesterification activity without affecting lipolytic activity. The non-lipase material reduced the interesterification activity by as much as 50%. The interesterification activity of immobilized preparations was enhanced by the use of higher concentrations of the crude lipase or, more substantially, by admixture of purified lipase.     

Effect of moisture on enzymatic reaction in supercritical carbon dioxide

The continuous acidolysis of triolein and stearic acid was carried out by an immobilized lipase to elucidate the characteristics of supercritical carbon dioxide (SC-CO₂) as a reaction medium. At first, an effect of temperature and pressure on the water adsorption to the immobilized lipase in the SC-CO₂ was examined. Then, the continuous interesterification of triolein and stearic acid by the moist immobilized lipase was examined. The amount of water adsorption to the immobilized lipase in the supercritical carbon dioxide measured under the condition of a different temperature and pressure has been expressed by a correlation equation of Freundlich type by using relative water standardized with the solubility of water in each condition. Optimum operating conditions of the interesterification by immobilized lipase in the SC-CO₂ was 323 K, 16.9 MPa and adsorbed-water concentration of 2 wt%. The production rate obtained by enzymatic acidolysis in the SC-CO₂ was found to be about 0.03 mmol/h2g-immobilized enzyme, leaving 74% residual triglyceride at the optimum operating conditions.     

Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. (c) 1993 John Wiley & Sons, Inc.     

Enzymic interesterification of fats: Laboratory and pilot-scale studies with immobilized lipase from Rhizopus arrhizus

An immobilized lipase suitable for fat interesterification has been prepared by precipitation with acetone of a commercial lipase from Rhizopus arrhizus onto diatomaceous earth. As observed previously with a less active enzyme from Aspergillus sp., the interesterification activity was enhanced by addition of purified lipase or by high loadings of commercial enzyme. The interesterification activities reached maximum values in both cases. For immobilized preparations with purified enzyme, interesterification activity was also enhanced by the presence of a precoat of glutaraldehyde cross-linked commercial lipase. A 2.9-L column of immobilized lipase was used to interesterify batches of shea oleine (67 kg) and shea oil (40 kg). Little activity was lost processing shea oleine, but slow poisoning of the bed occurred when shea oil was fed to the column.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

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.     

One‐pot synthesis of (R)‐1‐(pyridin‐4‐yl)ethyl acetate using tandem catalyst prepared by co‐immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling

The synthesis of (R )‐1‐(pyridin‐4‐yl)ethyl acetate was achieved over tandem palladium‐lipase catalyst with 100% selectivity using 4‐acetyl pyridine as a reactant. The 2% w /w palladium and lipase catalyst was successfully co‐immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4‐acetyl pyridine to form 1‐(pyridin‐4‐yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R )‐1‐(pyridin‐4‐ yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1‐(pyridin‐4‐yl)ethanol) was 51.38%. The final product yield of ((R )‐1‐(pyridin‐4‐yl)ethyl acetate) was 48.62%.