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.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Synthesis of biodiesel in column fixed-bed bioreactor using the fermented solid produced by Burkholderia cepacia LTEB11

We produced a lipase from Burkholderia cepacia in solid-state fermentation and used it to catalyze the synthesis of biodiesel in a fixed-bed reactor. In the solid-state fermentation step, a 50:50 (by mass) mixture of sugarcane bagasse and sunflower seed meal gave 234 units of pNPP-hydrolyzing activity per gram of dry solids at 96 h. This fermented solid was lyophilized and delipidated, packed into a column and used to catalyze the synthesis of biodiesel through the ethanolysis of soybean oil in a medium free of co-solvents, with the reaction mixture being continuously circulated through the column. The best conversion was 95% after 46 h, which was obtained at 50 °C, with an alcohol:oil molar ratio of 3:1, alcohol addition in two steps and the addition of 1% of (m/m) water to the reaction medium. This system has potential to decrease the costs of enzyme-catalyzed transesterification reactions.     

Effects of environmental conditions and methanol feeding strategy on lipase-mediated biodiesel production using soybean oil

Methanol is a commonly used acyl acceptor for lipase-driven biodiesel production, but a high concentration of methanol is detrimental for lipase activity. To overcome this drawback, a simple fed-batch process was developed by optimization of the methanol feeding strategy and reaction conditions. For the feeding strategy, an equal volume of pure methanol was fed twice with specified time intervals into a reactor initially containing a 1:1 molar ratio of soybean oil to methanol in order to adjust the net molar ratio of the oil to methanol to 1:3. In contrast with the batch reaction, a higher agitation speed in the fed-batch process elevated the conversion yield of soybean oil to biodiesel. An agitation speed of 600 rpm and a reaction temperature of 70°C were chosen as the optimal environmental conditions. Residual lipase activities for the fed-batch operation at 40 ∼ 70°C and 600 rpm were 7.1 ± 1.4 times higher than that of the batch method at 40°C with the same agitation speed, indicating that methanol feeding can prevent significant deactivation of lipase. Finally, two times feeding methanol at 2 and 6 hr resulted in a biodiesel productivity of 10.7%/h and 94.9% final conversion yield under the optimal conditions.     

Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435

The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production.     

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 biodiesel production from soybean oil deodorizer distillate with absorbent present in tert-butanol system

Lipase-catalyzed alcoholysis of soybean oil deodorizer distillate (SODD) for biodiesel production was studied. During this system both free fatty acids and glycerides could be converted to biodiesel simultaneously. tert-Butanol has been adopted as the reaction medium, in which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated completely. There was no obvious loss in lipase activity even after being repeatedly used for 120 cycles. Fine-pored silica gel and 3 Å molecular were found to be effective to control by-product water concentration and much higher biodiesel yield could be achieved with those adsorbents present in the reaction system. The highest biodiesel yield of 97% could be achieved with 3 Å molecular sieve as the adsorbent.     

Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids

Penicillium expansum lipase (PEL) was used to catalyze biodiesel production from corn oil in [BMIm][PF₆]¹ (an ionic liquid, IL) and tert-butanol. Both systems were optimized in terms of MeOH/oil molar ratio, reaction temperature, enzyme loading, solvent volume, and water content. The high conversion obtained in the IL (86%) as compared to that in tert-butanol (52%) demonstrates that the IL is a superior solvent for PEL-catalyzed biodiesel production. Poor yields were obtained in a series of hydrophilic ILs. Addition of salt hydrates affected biodiesel production predominantly through the specific ion (Hofmeister) effect. The impact of methanol on both activity and stability of PEL in the IL and in hexane was investigated, in comparison to the results obtained by two commonly used lipases, Novozym 435 and Lipozyme TLIM. The results substantiate that while different lipases show different resistance to methanol in different reaction systems, PEL is tolerant to methanol in both systems.     

Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system

Biocatalytic synthesis is a promising environmentally friendly process for the production of biodiesel, a sustainable alternative fuel from renewable plant resources. In order to develop an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs) were prepared from a commercial enzyme preparation from a recombinant Aspergillus strain expressing Thermomyces lanuginosus lipase and used for synthesis of biodiesel from palm olein by ethanolysis. Reaction parameters, including catalyst loading, temperature, and oil/alcohol molar ratio have been systematically optimized. Addition of tert-butanol was found to markedly increase the biocatalyst activity and stability resulting in improved product yield. Optimized reactions (20%, w/w PCMC-lipase to triacylglycerol and 1:4 fatty acid equivalence/ethanol molar ratio) led to the production of alkyl esters from palm olein at 89.9% yield on molar basis after incubation at 45 °C for 24 h in the presence of tert-butanol at a 1:1 molar ratio to triacylglycerol. Crude palm oil and palm fatty acid distillate were also efficiently converted to biodiesel with 82.1 and 75.5% yield, respectively, with continual dehydration by molecular sieving. Operational stability of PCMC-lipase could be improved by treatment with tert-butanol allowing recycling of the biocatalyst for at least 8 consecutive batches with only slight reduction in activity. This work thus shows a promising approach for biodiesel synthesis with microcrystalline lipase which could be further developed for cost-efficient industrial production of biodiesel.     

Use of experimental design to investigate biodiesel production by multiple-stage Ultra-Shear reactor

This work presents biodiesel production from soybean oil and bioethanol by multiple-stage Ultra-Shear reactor (USR). The experiments were carried out in the following conditions: reaction time from 6 to 12 min; catalyst concentration from 0.5% to 1.5% by weight of soybean oil; ethanol: soybean oil molar ratio from 6:1 to 10:1. The experimental design was used to investigate the influence of process variables on the conversion in biodiesel. The best ethyl ester conversion obtained was 99.26 wt.%, with ethanol:soybean oil molar ratio of 6:1, catalyst concentration of 1.35% and with 12 min of reaction time.     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

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.     

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.     

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.     

Biocatalytic methanolysis activities of cross-linked protein-coated microcrystalline lipase toward esterification/transesterification of relevant palm products

Biocatalysis by immobilized lipase is an efficient alternative process for conversion of crude vegetable oil with high free fatty acid content to biodiesel, which is the limit of the conventional alkaline-catalyzed reaction. In this study, influences of solid-state organic and inorganic buffer core matrices with different pK_a on catalytic performance of cross-linked protein coated microcrystalline biocatalysts prepared from Thermomyces lanuginosus lipase (CL-PCMC-LIP) toward esterification of palmitic acid (PA), transesterification of refined palm oil (RPO), and co-ester/transesterification of crude palm oil (CPO) to fatty acid methyl ester (FAME) was studied. Glycine, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid) were shown to be potent core matrices for these reactions. The optimal reaction contained 4:1 [methanol]/[fatty acid] molar equivalence ratio with 20% (w/w) CL-PCMC-LIP on glycine in the presence of tert-butanol as a co-solvent. Deactivation effect of glycerol on the biocatalyst reactive surface was shown by FTIR, which could be alleviated by increasing co-solvent content. The maximal FAME yields from PA, RPO, and CPO reached 97.6, 94.9, and 95.5%, respectively on a molar basis under the optimum conditions after incubation at 50 °C for 6 h. The biocatalyst retained >80% activity after recycling in five consecutive batches. The work demonstrates the potential of CL-PCMC-LIP on one-step conversion of inexpensive crude fatty acid-rich feedstock to biodiesel.     

Kinetic resolution of α-lipoic acid via enzymatic differentiation of a remote stereocenter

Kinetic resolution of α-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-α-lipoic acid, leaving the target product (R)-α-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50°C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of α-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.