A review on biodiesel production using catalyzed transesterification

Biodiesel fuel production by transesterification of oils.

Biodiesel processing and production

Industrial applications of microbial lipases

https://seniordesign.engr.uidaho.edu/2007_2008/frenchfryfuel/Biodiesel%20from%20WVO%20Part%201.pdf

Biodiesel production from waste cooking oil: 1. Process design and technological assessment

Biodiesel fuel (fatty acid methyl esters; FAMEs) can be produced by methanolysis of waste edible oil with a lipase. The degree of methanolysis was low in reaction systems so far reported, and the lipase catalyst could not be reused in spite of using immobilized enzyme. We clarified this problem was due to the irreversible inactivation of the lipase by contact with insoluble methanol (MeOH). Based on this result, we developed a stepwise methanolysis system with immobilized Candida antarctica lipase. Two-step batch methanolysis was most effective for the production of biodiesel fuel from waste oil: the first-step reaction was conducted in the presence of 1/3 molar equivalent of MeOH for the stoichiometric amount, and the second-step reaction was performed by adding 2/3 molar equivalent of MeOH. If the immobilized carrier is destroyed by agitation in a reactor with impeller, three-step flow reaction will be available: the first-step substrates were waste oil and 1/3 molar equivalent of MeOH; the second-step, the first-step eluate and 1/3 molar equivalent of MeOH; the third-step, the second-step eluate and 1/3 molar equivalent of MeOH. The conversion of waste oil to biodiesel fuel reached >90% in the two reaction systems, and the lipase catalyst could be used for >100 days without decrease of the activity. The stepwise alcoholysis could successfully be applied to ethanolysis of tuna oil.

Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing

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).

Conversion of vegetable oil to biodiesel 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.

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

Crude soybean oil did not undergo methanolysis with immobilized Candida antarctica lipase but degummed oil did. Therefore, the substance that was removed in the degumming step was estimated to inhibit the methanolysis of soybean triacylglycerols (TAGs). The main components of soybean gum are phospholipids (PLs), and soybean PLs actually inhibited the methanolysis reaction. In addition, PLs were detected in chloroform/methanol (MeOH) extracts from the immobilized lipase preparation that had been used in the methanolysis of crude soybean oil. These results showed that PLs were at least one of the inhibitory substances in methanolysis of TAGs. The inhibition may due to the interference of the interaction of the lipase molecule with substrates by PLs bound on immobilized preparation. These findings indicated that degummed oil has to be used as a substrate for enzymatic methanolysis. Indeed, three-step methanolysis successfully converted 93.8% degummed soybean oil to its corresponding methyl esters, and the lipase could be reused for 25 cycles without any loss of the activity.

Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.

Yoshio Tominaga

Papers

Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing

Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase

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

Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase

Purification and Characterization of a Novel Thermostable Lipase from Bacillus sp.