Shinji Hama

Whole-cell biocatalysts for biodiesel fuel production

Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.

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.

Enzymatic biodiesel production: An overview of potential feedstocks and process development

The increased global demand for biofuels has prompted the search for alternatives to edible oils for biodiesel production. Given the abundance and cost, waste and nonedible oils have been investigated as potential feedstocks. A recent research interest is the conversion of such feedstocks into biodiesel via enzymatic processes, which have considerable advantages over conventional alkali-catalyzed processes. To expand the viability of enzymatic biodiesel production, considerable effort has been directed toward process development in terms of biodiesel productivity, application to wide ranges of contents of water and fatty acids, adding value to glycerol byproducts, and bioreactor design. A cost evaluation suggested that, with the current enzyme prices, the cost of catalysts alone is not competitive against that of alkalis. However, it can also be expected that further process optimization will lead to a reduced cost in enzyme preparation as well as in downstream processes.

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.

Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

Enzymatic production of biodiesel from waste cooking oil in a packed-bed reactor: An engineering approach to separation of hydrophilic impurities

An engineering approach was applied to an efficient biodiesel production from waste cooking oil. In this work, an enzymatic packed-bed reactor (PBR) was integrated with a glycerol-separating system and used successfully for methanolysis, yielding a methyl ester content of 94.3% and glycerol removal of 99.7%. In the glycerol-separating system with enhanced retention time, the effluent contained lesser amounts of glycerol and methanol than those in the unmodified system, suggesting its promising ability to remove hydrophilic impurities from the oil layer. The PBR system was also applied to oils with high acid values, in which fatty acids could be esterified and the large amount of water was extracted using the glycerol-separating system. The long-term operation demonstrated the high lipase stability affording less than 0.2% residual triglyceride in 22 batches. Therefore, the PBR system, which facilitates the separation of hydrophilic impurities, is applicable to the enzymatic biodiesel production from waste cooking oil.

Development of an Aspergillus oryzae whole-cell biocatalyst coexpressing triglyceride and partial glyceride lipases for biodiesel production

An Aspergillus oryzae whole-cell biocatalyst which coexpresses Fusarium heterosporum lipase (FHL) and mono- and di-acylglycerol lipase B (mdlB) in the same cell has been developed to improve biodiesel production. By screening a number of transformants, the best strain was obtained when FHL gene was integrated into A. oryzae chromosome using sC selection marker while mdlB was integrated using niaD selection marker. The reaction system using the lipase-coexpressing whole-cells was found to be superior in biodiesel production to others such as lipase-mixing and two-step reactions, affording the highest reaction rate and the highest ME content (98%). Moreover, an ME content of more than 90% was maintained during 10 repeated batch cycles. The whole-cell biocatalyst developed in this work would be promising biocatalysts for efficient biodiesel production.

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed. Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification, r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalents of methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintaining the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALB can be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester content of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALB shows promise for biodiesel production from plant oil hydrolysates.

Production of d-lactic acid from hardwood pulp by mechanical milling followed by simultaneous saccharification and fermentation using metabolically engineered Lactobacillus plantarum

This study focused on the process development for the d-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure d-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5-15% loads) resulted in the lactic acid titers of 55.2-84.6g/L after 72h and increased productivities of 1.77-2.61g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (⩽10min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3g/L, an optical purity of 99.2%, and a yield of 0.879g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer d-lactic acid using the L. plantarum mutant.

Process engineering and optimization of glycerol separation in a packed-bed reactor for enzymatic biodiesel production.

A process model for efficient glycerol separation during methanolysis in an enzymatic packed-bed reactor (PBR) was developed. A theoretical glycerol removal efficiency from the reaction mixture containing over 30% methyl esters was achieved at a high flow rate of 540 ml/h. To facilitate a stable operation of the PBR system, a batch reaction prior to continuous methanolysis was conducted using oils with different acid values and immobilized lipases pretreated with methyl esters. The reaction system successfully attained the methyl ester content of over 30% along with reduced viscosity and water content. Furthermore, to obtain a high methyl ester content above 96% continuously, long-term lipase stability was confirmed by operating a bench-scale PBR system for 550 h, in which the intermediates containing methyl esters and residual glycerides were fed into the enzyme-packed columns connected in series. Therefore, the developed process model is considered useful for industrial biodiesel production.