Masaru Kaieda

Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4-30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80-90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.

Effect of Methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system

Methyl esters synthesized from plant oil and methanol by the methanolysis reaction are potentially important as a biodiesel fuel. The methanolysis of soybean oil by lipases from various microorganisms was investigated. Several of the lipases were found to catalyze methanolysis in a water-containing system without an organic solvent. The lipases from Candida rugosa, Pseudomonas cepacia, and Pseudomonas fluorescens displayed particularly high catalytic ability. The reaction rates of methanolysis catalyzed by the C. rugosa and P. fluorescens lipases decreased significantly when the water content was low, showing that water prevents the inactivation of these lipases by methanol. On the other hand, the methanolysis reaction rate catalyzed by the P. cepacia lipase remained high even under a low water content. In addition, the P. cepacia lipase gave high methyl ester contents in the reaction mixture up to 2 or 3 molar equivalents of methanol to oil, which is attributed to the P. cepacia lipase having substantial methanol resistance. For the same methanol content, the reaction rates of methanolysis catalyzed by the P. cepacia lipase increased with decreasing water content, and hence lipases strongly resistant to high methanol, such as that from P. cepacia, are desirable for use in methanolysis reaction processes.

Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles.

As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae) IFO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanolysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium. Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction mixture reached 90% - the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is considered to be promising for biodiesel fuel production in industrial applications.

Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil

The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.

Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production

Abstract With a view to utilizing Rhizopus oryzae cells immobilized within biomass support particles (BSPs) as a whole-cell biocatalyst for biodiesel fuel production, an investigation was made of the effect of cross-linking treatment with glutaraldehyde (GA) on the stability of lipase activity. Although the lipase activity of the BSP-immobilized cells decreased considerably in the presence of the methyl esters produced by methanolysis, the activity of cells treated with 0.1% GA solution showed no significant decrease during six batch cycles, with the methyl ester content of the reaction mixture reaching 70–83% in each cycle. In contrast, without GA treatment, activity decreased gradually with each cycle to give a methyl ester content of only 50% at the sixth batch cycle. These findings indicate that, given the simplicity of the lipase production process and the long-term stability of lipase activity, the use of whole-cell biocatalysts immobilized within BSPs and treated with GA solution offers a promising means of biodiesel fuel production for industrial application.