Enzymatic Synthesis of Biodiesel from Transesterification Reactions of Vegetable Oils and Short Chain Alcohols

Biodiesel synthesis by alcoholysis of three vegetable oils (soybean, sunflower and rice bran) catalyzed by three commercial lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM), and the optimization of the enzymes stability over repeated batches is described. The effects of the molar ratio of alcohol to oil and the reaction temperature with methanol, ethanol, propanol and butanol were also studied. All three enzymes displayed similar reaction kinetics with all three oils and no significant differences were observed. However, each lipase displayed the highest alcoholysis activity with a different alcohol. Novozym 435 presented higher activity in methanolysis, at a 5:1 methanol:oil molar ratio; Lipozyme TL-IM presented higher activity in ethanolysis, at a 7:1 ethanol:oil molar ratio; and Lipozyme RM-IM presented higher activity in butanolysis, at a 9:1 butanol:oil molar ratio. The optimal temperature was in the range of 30–35 °C for all lipases. The assessment of enzyme stability over repeated batches was carried out by washing the immobilized enzymes with different solvents (n-hexane, water, ethanol, or propanol) after each batch. When washing with n-hexane, approximately 90% of the enzyme activity remained after seven synthesis cycles.     

Optimization of the synthesis of lower glycerides rich in unsaturated fatty acid residues obtained via enzymatic ethanolysis of sesame oil

The lipases Novozym 435, Lipozyme TL IM and Lipozyme RM IM were employed in the production of lower acylglycerols (LG), i.e. mono‐ (MAG) and diacylglycerols (DAG), rich in unsaturated fatty acids from sesame oil in batch reactors. The effect of the molar ratio of ethanol to fatty acids on the reusability of these immobilized lipases was studied in detail. The effects of pretreatment on lipase activity for ethanolysis were investigated. Glycerol had a strong product inhibition effect on the ethanolysis reaction, and a relatively large excess of ethanol was necessary to remove the glycerol adsorbed on these biocatalysts. The enzymatic activity was drastically reduced by addition of water to the reaction medium. The presence of organic solvents (hexane and acetone) did not favor the production of LG. For the Novozym 435‐catalyzed reaction, optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 15 wt‐% lipase and 50 °C. For Lipozyme TL IM, the optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 20 wt‐% biocatalyst, and 30 °C. Novozym 435 and Lipozyme TL IM produced LG with molar ratios of unsaturated to saturated fatty acids of 20.4 in 1 h and 25.3 in 5 h, respectively. In the original oil, this ratio was 5. For trials conducted under optimum conditions, the products from the Novozym 435 trials contained 21.8 wt‐% triacylglycerols (TAG), 24 wt‐% DAG and 54.2 wt‐% MAG. The products of the Lipozyme TL IM trials consisted of 12.9 wt‐% DAG and 87.1 wt‐% MAG. No TAG species were detected.     

Immobilized lipase-catalyzed ethanolysis of sunflower oil

The ethanolysis of sunflower oil catalyzed by an immobilized 1,3-specific porcine pancreatic lipase in a medium composed solely of substrates was investigated. The effects of the oil/ethanol molar ratio, temperature, amount of added water, and amount of enzyme used [respectively, 1∶3, 45°C, 0% (vol/vol), and 0.5 g of lipase, i.e., 10% w/w of total substrate]. To investigate the reusability of the lipase, the four-step ethanolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentage of conversion after the first usage decreased markedly.     

Immobilized lipase-catalyzed ethanolysis of sunflower oil in a solvent-free medium

The ethanolysis of sunflower oil (SFO) with Lipozyme (immobilizéd 1,3-specific Mucor miehei lipase) in a medium solely composed of substrates was investigated. The effects of oil/ethanol molar ratio, temperature, added water content, and amount of enzyme were analyzed. The optimal values were, respectively, 1:3, 50°C, 0% (vol/vol), and 0.4 g of Lipozyme per 5.7 mmol of SFO. The use of immobilized lipase made the reuse of enzyme feasible, and the enzyme could be recovered easily from the reaction mixture and recycled to reduce the cost of catalyst. In the last three consecutive runs of enzyme reuse, the final conversion yield of SFO from ethanolysis with added silica gel support was higher than that obtained from ethanolysis under standard conditions. The lipase-catalyzed alcoholytic reaction is potentially useful in the production of alkyl esters of specific interest.     

Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase

Biodiesel derived from vegetable oils has drawn considerable attention with increasing environmental consciousness. We attempted continuous methanolysis of vegetable oil by an enzymatic process. Immobilized Candida antarctica lipase was found to be the most effective for the methanolysis among lipases tested. The enzyme was inactivated by shaking in a mixture containing more than 1.5 molar equivalents of methanol against the oil. To fully convert the oil to its corresponding methyl esters, at least 3 molar equivalents of methanol are needed. Thus, the reaction was conducted by adding methanol stepwise to avoid lipase inactivation. The first step of the reaction was conducted at 30°C for 10 h in a mixture of oil/methanol (1:1, mol/mol) and 4% immobilized lipase with shaking at 130 oscillations/min. After more than 95% methanol was consumed in ester formation, a second molar equivalent of methanol was added and the reaction continued for 14 h. The third molar equivalent of methanol was finally added and the reaction continued for 24 h (total reaction time, 48 h). This three-step process converted 98.4% of the oil to its corresponding methyl esters. To investigate the stability of the lipase, the three-step methanolysis process was repeated by transferring the immobilized lipase to a fresh substrate mixture. As a result, more than 95% of the ester conversion was maintained even after 50 cycles of the reaction (100 d).     

Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase

Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.     

Characteristics of menhaden oil ethanolysis by immobilized lipase in supercritical carbon dioxide

Ethanolysis of menhaden oil was performed with 1,3-regiospecific lipase to produce diglycerides and monoglycerides containing polyunsaturated fatty acids, and fatty acid ethyl esters. Immobilized lipases like lipozyme TL-IM (Thermomuces lanuginosa immobilized on silica gel) were used for enzymatic ethanolysis. Ethanolysis was carried out in different processes (solvent free, organic solvent and supercritical fluid system) to compare the reaction rate and yield obtained by menhaden oil ethanolysis. Organic solvent (hexane) and supercritical carbon dioxide (SC-CO₂) were used as reaction medium. The reaction products were analyzed by gas chromatography (GC), thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Higher amounts of ethanol as a substrate caused substrate inhibition which dramatically decreased the reaction rate of ethanolysis. To elucidate the effect of pressure, enzymatic ethanolysis was performed in SC-CO₂ at pressures ranging from 75 to 121 bar. Enzymatic ethanolysis of menhaden oil in SC-CO₂ decreased by substrate inhibition. Reaction rate and optimum amount of ethanol used were depended on SC-CO₂ density individually. Kinetic model with substrate inhibition (dead-end inhibition) by excess ethanol was set up to measure the reaction and inhibition rates.     

Effect of methanol content on enzymatic production of biodiesel from waste frying oil

The enzymatic production of biodiesel from waste frying oil with methanol has been studied using immobilized lipase Novozym 435 as catalyst. The effects of methanol to oil molar ratio, dosage of enzyme and reaction time were investigated. The optimum reaction conditions were methanol to oil molar ratio of 25:1, 10% of Novozym 435 based on oil weight and reaction period of 4 h at 50 °C obtaining a biodiesel yield of 89.1%. Moreover, the reusability of the lipase over repeated cycles was also investigated under standard conditions.     

Stability of Immobilized Candida sp. 99–125 Lipase for Biodiesel Production

The stability of the immobilized lipase from Candida sp. 99–125 during biodiesel production was investigated. The lipase was separately incubated in the presence of various reaction components such as soybean oil, oleic acid methyl ester, n‐hexane, water, methanol, and glycerol, or the lipase was stored at 60, 80, 100 and 120 °C. Thereafter the residual lipase activity was determined by methanolysis reaction. The results showed that the lipase was rather stable in the reaction media, except for methanol and glycerol. The stability study performed in a reciprocal shaker indicated that enzyme desorption from the immobilized lipase mainly contributed to the lipase inactivation in the water system. So the methanol and glycerol contents should be controlled more precisely to avoid lipase inactivation, and the immobilization method should be improved with regard to lipase desorption.     

Study on Free Lipase-Catalyzed Ethanolysis for Biodiesel Preparation in an Oil/Water Biphasic System

Using free lipase as the catalyst for biodiesel production has drawn increasing attention in recent years due to its advantages of lower cost and faster reaction rate compared to immobilized lipase. Our previous study showed that free lipase NS81006 could effectively catalyze the methanolysis of renewable oil. Ethanol, derived from renewable biomass, has a greater potential for biodiesel production. In this paper, ethanol was explored for the first time as the acyl acceptor for free lipase-mediated biodiesel preparation. The effect of stirring rate, water content, molar ratio of ethanol to oil and ethanol adding strategy was investigated systematically during the process of free lipase NS81006-catalyzed ethanolysis. An ethyl ester yield of 90% was obtained under the optimized conditions. Further study showed that the free lipase could be repeatedly used by simple separation of the water phase from the oil phase and there was no obvious loss in lipase activity after five repeated uses.     

Regiospecific analysis by ethanolysis of oil with immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

A mixture of oil/ethanol (1∶3, w/w) was shaken at 30°C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C₁₄−C₂₄ FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28–29 mol% based on the total amount of FA in the reaction mixture at 59–69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG.     

Synthesis of Fatty Acid Ethyl Ester from Acid Oil in a Continuous Reactor via an Enzymatic Transesterification

Synthesis of a fatty acid ethyl ester via the lipase‐catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by‐product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half‐life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.     

Enzymatic conversion of waste edible oil to biodiesel fuel in a fixed-bed bioreactor

The conversion of waste edible oil to biodiesel fuel in a fixed-bed bioreactor was investigated. Three-step methanolysis of waste oil was conducted using three columns packed with 3 g of immobilized Candida antarctica lipase. A mixture of waste oil and 1/3 molar equivalent of methanol against total fatty acids in the oil was used as substrate for the first-step reaction, and mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-step reactions, respectively. Ninety percent of waste oil was converted to the corresponding methyl esters (ME) by feeding substrate mixtures into the first, second, and third reactors at flow rates of 6, 6 and 4 mL/h, respectively. We also attempted one-step methanolysis of waste oil. When a mixture of waste oil and 90% ME-containing eluate (1∶3, wt/wt) and an equimolar amount of methanol against total fatty acids in the waste oil was fed into a reactor packed with 3 g of immobilized C. antarctica lipase at a flow rate of 4 mL/h, the ME content in the eluate reached 90%. The immobilized biocatalyst could be used for 100 d in the two reaction systems without significant decrease in its activity. Waste oil contained 1980 ppm water and 2.5% free fatty acids, but these contaminants had little influence on enzymatic production of biodiesel fuel.     

Production of FAME from acid oil model using immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Acid oil is a by-product in the neutralization step of vegetable oil refining and is an alternative source of biodiesel fuel. A model substrate of acid oil, which is composed of TAG and FFA, was used in experiments on the conversion to FAME by immobilized Candida antarctica lipase. FFA in the mixture of TAG/FFA were efficiently esterified with methanol (MeOH), but the water generated by the esterification significantly inhibited methanolysis of TAG. We thus attempted to convert a mixture of TAG/FFA to FAME by a two-step process comprising methyl esterification of FFA and methanolysis of TAG by immobilized C. antarctica lipase. The first reaction was conducted at 30°C in a mixture of TAG/FFA (1∶1, wt/wt) and 10 wt% MeOH using 0.5 wt% immobilized lipase, resulting in efficient esterification of FFA. The reaction mixture after 24 h was composed of 49.1 wt% TAG, 1.3 wt% FFA, 49.1 wt% FAME, and negligible amounts of DAG and MAG (<0.5 wt%). The reaction mixture was then dehydrated and used as a substrate for the second reaction, which was conducted at 30°C in a solution of the dehydrated mixture and 5.5 wt% MeOH using 6 wt% immobilized lipase. The activity of the lipase increased gradually when the reaction was repeated by transferring the enzyme to a fresh substrate mixture. The activity reached a maximum after 6 cycles, and the content of FAME achieved was >98.5 wt% after a 24-h reaction. The immobilized lipase was very stable in the first-and second-step reactions and could be used for >100 d without significant loss of activity.     

The Development of Flow-through Bio-Catalyst Microreactors from Silica Micro Structured Fibers for Lipid Transformations

This study demonstrates the utility of a flow-through enzyme immobilized silica microreactor for lipid transformations. A silica micro structured fiber (MSF) consisting of 168 channels of internal diameter 4–5 μm provided a large surface area for the covalent immobilization of Candida antartica lipase. The specific activity of the immobilized lipase was determined by hydrolysis of p-nitrophenyl butyrate and calculated to be 0.81 U/mg. The catalytic performance of the lipase microreactor was demonstrated by the efficient ethanolysis of canola oil. The parameters affecting the performance of the MSF microreactor, including temperature and reaction flow rate, were investigated. Characterization of the lipid products exiting the microreactor was performed by non-aqueous reversed-phased liquid chromatography (NARP-LC) with evaporative light scattering detector (ELSD) and by comprehensive two-dimensional gas chromatography (GC × GC). Under optimized conditions of 1 μL/min flow rate of 5 mg/mL trioleoylglycerol (TO) in ethanol and 50 °C reaction temperature, 2-monooleoylglycerol was the main product at >90% reaction yield. The regioselectivity of the Candida antartica lipase immobilized MSF microreactor in the presence of ethanol was found to be comparable to that obtained under conventional conditions. The ability of these reusable flow-through microreactors to regioselectively form monoacylglycerides in high yield from triacylglycerides demonstrate their potential use in small-scale lipid transformations or analytical lipids profiling.     

Immobilization of Candida antarctica lipase onto cellulose acetate-coated Fe2O3 nanoparticles for glycerolysis of olive oil

Candida antarctica lipase was covalently immobilized onto the surface of cellulose acetate-coated Fe₂O₃ nanoparticles. The characterizations of immobilized lipase were examined by Fourier transform infrared spectrophotometer (FTIR) and field emission gun-scanning electron microscopy (FEG-SEM). The immobilized lipase was assayed for production of monoglycerides (MG) and diglycerides (DG) by glycerolysis of olive oil in a solvent medium. The effect of various reaction conditions on the MG and DG production such as reaction time, temperature, the molar ratio of glycerol to oil and amount of immobilized lipase was investigated. The optimum condition for MG and DG production was found at 50 °C temperature and 0.025 g of lipase with the molar ratio of glycerol to oil 1.5: 1 in 5 h of reaction time. The effect of substrate concentration on enzymatic activity of the free and immobilized lipase showed the best fits to the Lineweaver-Burk plots. The K _m and V _max values of immobilized lipase were found to be 25mM and 0.58mM/min, whereas that for free lipase was 52.63mM and 1.75mM/min, respectively. The activation and deactivation energy was found to decrease for immobilization of lipase on cellulose acetate-coated Fe₂O₃ nanoparticles.     

Lipase‐catalyzed alcoholysis of vegetable oils

Fatty acid alkyl esters were produced from various vegetable oils by transesterification with different alcohols using immobilized lipases. Using n‐hexane as organic solvent, all immobilized lipases tested were found to be active during methanolysis. Highest conversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. In contrast, this lipase was almost inactive in a solvent‐free reaction medium using methanol or 2‐propanol as alcohol substrates. This could be overcome by a three‐step addition of methanol, which works efficiently for a range of vegetable oils (e.g. cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was very stable over 120 h. AK and RM lipases also showed acceptable conversion levels for cottonseed oil with ethanol, 1‐propanol, 1‐butanol and isobutanol (50‐65% conversion after 24 h) in solvent‐free conditions. Methyl and isopropyl fatty acid esters obtained by enzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuels and cosmetics industry, respectively.     

Optimization of enzymatic methanolysis of soybean oil by response surface methodology

Enzymatic methanolysis of refined soybean oil with methanol was investigated using Rhizomucor miehei lipase, Lipozyme RM IM, in n-hexane for reaction times of 30 min. Response surface methodology (RSM) based on three-level, three-factor (variable) face-centered cube design was used for the optimization of methanolysis. The independent variables that affect the methanolysis reaction conducted in n-hexane are temperature (°C), enzyme/oil weight ratio, and oil/methanol molar ratio. A good quadratic model was obtained for the methyl ester production by multiple regression and backward elimination. A linear relationship was observed between the observed and predicted values (R²−0.9635). The effects of temperature and enzyme amount, which affected methyl ester content of the product (response) positively, were significant (P<0.01). The quadratic term of temperature and the interaction term of enzyme amount with temperature affected the response negatively (P<0.01). The interaction term of enzyme amount with substrate mole ratio had a positive effect on the response (P<0.05). Critical conditions for the response at which methyl ester content of the product was 76.9% were determined to be 50°C, 2.37 methanol/oil mole ratio, and 0.09 enzyme/oil weight ratio.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99‐125

A lipase preparation developed from Candida sp. 99‐125 was used for fatty acid alkyl ester synthesis by both enzymatic esterification of fatty acids, and transesterification of oils and fats. The lipase preparation was chosen based on screening of lipases from commercial sources as well as those produced in the laboratory. The effects of enzyme dosage, solvent types, water absorbent additions, inhibition of short‐chain alcohols, alcohol and acid types, molar ratio of substrates, and reusability of the lipase preparation in esterification were studied. Degree of esterification between oleic acid and methanol under optimal conditions reached 92%. Purity of the methyl ester after washing with water and distillation was 98%. Half‐life of the lipase preparation was calculated to be approximately 340 h. For transesterification of rapeseed oil with the same lipase preparation, the amount of methanol and mode of methanol addition to the reaction were also conducted. Transesterification of the oil with stepwise methanol addition reached 83% after 36 h reaction time.