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ABSTRACT: 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.
Biotechnology advances 04/2009; 27(4):398-408. · 8.25 Impact Factor
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ABSTRACT: An enzymatic method that involves seed oil hydrolysis and selective esterification of free fatty acids (FFAs) has been essayed for screening of lipases in order to concentrate γ-linolenic acid (GLA; 18:3n-6). This procedure has been essayed by using the seed oil of two plant species, Borago officinalis and Echium fastuosum. No GLA enrichment was observed in the FFA fractions resulting from the enzymatic hydrolysis performed by all essayed lipases. However, starting from the FFA fraction of B. officinalis seed oil, and after esterfication with Candida rugosa lipase, in the fatty acid (FA) unesterified fraction a 91% GLA purity was obtained after two successive esterifications, yielding 59% GLA. GLA from E. fastuosum seed oil was also esterified; GLA was concentrated in the FA unesterified fraction by using Mucor javanicus lipase, obtaining 64% purity and 68% GLA yield.
Journal of Food Lipids 11/2006; 13(4):362 - 374. · 1.27 Impact Factor
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ABSTRACT: The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol
using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best
lipase was maximization of the dimensionless effectiveness factor (FAE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after
esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate FAE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate
ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase.
The maximum FAE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate
EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison
of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended
on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.
Journal of Oil & Fat Industries 02/2006; 83(3):215-221. · 1.77 Impact Factor
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ABSTRACT: Structured triacylglycerols with caprylic acid at the sn-1 and sn-3 positions of the glycerol backbone and eicosapentaenoic acid (EPA) at the position sn-2 were synthesised by acidolysis of a commercially available EPA-rich oil (EPAX4510, Pronova Biocare) and caprylic acid catalysed by the 1,3-specific immobilised lipase Lipozyme IM. The reaction was carried out in an immobilised lipase packed-bed reactor by recirculating the reaction mixture through the bed. The exchange equilibrium constants between caprylic acid and the native fatty acids of EPAX4510 were determined. The n-3 polyunsaturated fatty acids (PUFAs), EPA and docosohexaenoic acid (DHA), were the most easily displaced by the caprylic acid. The exchange equilibrium constants were 3.68 and 3.06 for EPA and DHA, respectively. The influence of the flow rate of the reaction mixture through the packed-bed and the substrate concentration in the reaction rate were studied. For flow rates between 74 and 196 cm3 h−1 (bed of 6.6 mm internal diameter and 0.46 porosity) and triacylglycerol concentrations between 0.036 and 0.108 M, the data fitted well to an empirical kinetic model which allowed representative values of the apparent kinetic constant to be obtained. Hence, the average reaction rates and kinetic constants of exchange of caprylic acid and native fatty acids of EPAX4510 could be calculated. In the conditions indicated, the parameter (lipase mass × time/triacylglycerol mass, mLt/V[TG]0) constituted the intensive variable of the process for use in predicting the composition of structured triacylglycerols at different reaction times. At equilibrium, the structured triacylglycerol produced had the following composition: caprylic acid 59.5%, EPA 9.6%, DHA 2.2% and oleic acid 11.8%. Copyright © 2004 Society of Chemical Industry
Journal of Chemical Technology & Biotechnology 12/2004; 80(1):35 - 43. · 2.17 Impact Factor
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ABSTRACT: The influence of the molar ratio caprylic acid/triolein, enzyme concentration and water content on the kinetics of the interesterification reaction of triolein (TO) and caprylic acid (CA) were studied. The enzyme used was the 1,3-specific Rhizomucor miehei lipase. Data modelling was based on a simple scheme in which the acid was only incorporated in positions 1 and 3 of the glyceride backbone. In addition, it was assumed that positions 1 and 3 of the triglycerides were equivalent and that the events at position 1 did not depend on the nature of the fatty acid in position 3 and vice versa. Monoglycerides and diglycerides were not detected during the experiments. This was attributed to the low water content of the immobilised enzyme particles. The value of the equilibrium constant, K, for the exchange of caprylic and oleic acids was 2.7, which indicated that the incorporation of caprylic acid into triglycerides was favoured compared with the incorporation of oleic acid. Simple first order kinetics could describe the interesterification reaction. Using this model and the calculated equilibrium constant, the apparent kinetic constants were calculated. The model fitted all the experimental data except for the CA/TO molar ratios larger than 6. Moreover, the interesterification reaction rate had a maximum value at CA/TO molar ratios of 4–6 mol mol−1.Copyright © 2003 Society of Chemical Industry
Journal of Chemical Technology & Biotechnology 03/2003; 78(4):461 - 470. · 2.17 Impact Factor
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ABSTRACT: Fatty acids in the microalga Phaeodactylum tricornutum were isolated using an optimized three-step method: extraction of crude fatty acid potassium salts made by direct saponification
of lipids in the microalgal biomass with KOH/ethanol (96%, vol/vol), separation of unsaponifiable lipids by extraction with
hexane, and final purification of fatty acids by acidification of the alcoholic solution of potassium soaps followed by extraction
of fatty acid into hexane. Direct saponification was carried out in ethanol (96%, vol/vol) using 2.09 mL ethanol (96%) per
gram of wet biomass (10 mL/g of dry biomass) mixed with 0.4 g KOH/g of biomass. Under these conditions the fatty acid yield
was 87%. The optimal water content of the alcoholic solution for extraction of the unsapononifiables was established as 40%,
w/w. Data on equilibrium carotenoid distribution between the alcoholic (40%, w/w water) and hexane phases were determined.
These data allow prediction of the carotenoid yields with different volumes of hexane in several extraction steps. The optimal
pH of the alcoholic solution before extracting the purified fatty acid was established as pH 6, and the equilibrium fatty
acid distribution between the alcoholic and hexane phases was determined. This optimized method permited a 20% reduction in
the production costs of highly purified eicosapentaenoic acid (EPA) in the three-step preparative process (extraction of fatty
acid, concentration of polyunsaturated fatty acids by the urea method, and EPA fractionation through preparative high-performance
liquid chromatography) previously developed by the authors.
Journal of Oil & Fat Industries 11/1998; 75(12):1735-1740. · 1.77 Impact Factor
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ABSTRACT: Eicosapentaenoic acid (FPA, 20:5n-3) and arachidonic acid (AA, 20:4n-3)were obtained from the microalga Porphyridium cruentum by a three-stepprocess: fatty acid extraction by direct saponification of biomass,polyunsaturated fatty acid (PUFA) concentration by urea inclusion complexingand EPA isolation by high-performance liquid chromatography (HPLC). Twosolvents were tested for direct saponification of lipids in biomass. Themost efficient solvent, ethanol (96% v/v), extracted 75% ofthe fatty acids. PUFAs concentration by urea inclusion employed a urea/fattyacid ratio of 4:1 wt/wt at the crystallization temperatures of 4C and28C. Concentration factors were similar at both temperatures, but theEPA and AA recoveries were higher at 28C (67.7% and 61.8%for the two acids, respectively). EPA and AA were purified from this PUFAconcentrate using analytical scale HPLC and the best results of thisseparation were scaled up to preparative level (4.7 i. d. 30 cmcompression radial cartridge). A 94.3% pure EPA fraction and a81.4% pure AA fraction were obtained. Suitability of severalmicroalgae (Porphyridium cruentum, Phaeodactylum tricornutum and Isochrysisgalbana) and cod liver oil as sources of highly pure PUFAs, mainly EPA, wascompared.
Bioseparation 02/1998; 7(2):89-99.
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ABSTRACT: This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with
glycerol. A PUFA concentrate obtained from cod liver oil was used to optimize the reaction to favor triglyceride synthesis
with lipases. The type and amount of lipase and organic solvent, glycerol content, temperature, water content, and amount
and time of addition of molecular sieves were studied. The optimal reaction mixture and conditions were: 9 mL hexane, 60°C,
0.5% (vol/vol) water, 1 g molecular sieves added after 24 h of reaction, glycerol/fatty acid molar ratio 1:3 and 100 mg of
Novozym 435 (Novo Nordisk A/S) lipase. Under these conditions, an enriched triglyceride yiedl of 84.7% containing 27.4% eicosapentaenoic
acid and 45.1% docosahexaenoic acid was obtained from a cod liver oil PUFA concentrate.
Journal of Oil & Fat Industries 01/1998; 75(10):1329-1337. · 1.77 Impact Factor
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ABSTRACT: This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. The lipase Novozym 435 (Novo Nordisk, A/S) from Candida antarctica was used to catalyze this reaction. The main factors influencing the degree of esterification and triglyceride yield were the amount of enzyme, water content, temperature and glycerol/fatty acid ratio. The optimum reaction conditions were established as: 100 mg of lipase; 9 ml hexane; 50°C; glycerol/PUFA concentrate molar ratio 1.2:3; 0% initial water; 1 g molecular sieves added at the start of reaction; and an agitation rate of 200 rpm. Under these conditions, a triglyceride yield of 93.5% was obtained from cod liver oil PUFA concentrate; the product contained 25.7% eicosapentaenoic acid and 44.7% docosahexaenoic acid. These optimized conditions were used to study esterification from a PUFA concentrate of the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. With the frst, a triglyceride yield of 96.5%, without monoglycerides and very few diglycerides, was obtained after 72 h of reaction; the resulting triglycerides had 42.5% eicosapentaenoic acid. A triglyceride yield of 89.3% was obtained from a P. cruentum PUFA concentrate at 96 h of reaction, which contained 43.4% arachidonic acid and 45.6% EPA. These high triglyceride yields were also achieved when the esterification reaction was scaled up 5-fold.
Progress in Industrial Microbiology.
