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Production of biodiesel fuel in ionic liquids catalyzed by whole-cell biocatalysts
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S41–S56 BR-O4 Production of biodiesel fuel in ionic liquids catalyzed by whole-cell biocatalysts Kazunori Nakashima,1 Shogo Arai,1 Takanori Tanino,2 Chiaki Ogino,1 Akihiko Kondo,1 and Hideki Fukuda1 Kobe University, 1-1 Rokkoudaicho, Nada-ku, Kobe 657-8501, Japan 1 and Gunma University, Tenjincho 1-5-1, Kiryu-shi, Gunma 376-8515, Japan 2 The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in oil/ionic liquid biphasic systems. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 molar equivalents of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of wholecell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in ionic liquid for 72 h. The stability of w-ROL in ionic liquid was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as reaction media in biodiesel fuel production by whole-cell biocatalysts. doi:10.1016/j.jbiosc.2009.08.123
BR-O5 Development of efficient whole-cell biocatalysts for oxidative biotransformations Hyo-Seel Seo,1 Na-Rae Lee,1 Eun-Hee Doo,1 Sunghoon Park,2 and Jin-Byung Park1 Department. of Food Science and Engineering, Ewha Womans University, Seoul, Republic of Korea 1 and Department of Chemical Engineering, Pusan National University, Pusan, Republic of Korea 2 Selective oxidation is one of the most useful biotransformations for synthetic applications. Biooxidations are often catalyzed by oxygenases, which are usually cofactor dependent and consist of multicomponent enzymes. Thus, oxygenase-expressing whole cells particularly growing cells have been used as biocatalysts for practical application (1). The productivity of growing cell-based biocatalysts are steadily increasing, sometimes approaching 1000U/L (1). However, there are a number of problems in commercialization of oxygenation processes using growing cells as biocatalysts. For instance, the oxygenases should compete for molecular oxygen and cofactors (NAD(P)H) with
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metabolic enzymes in growing cell biocatalysts limiting their specific productivity and requiring vigorous reaction conditions. We have developed an efficient Escherichia coli-based biocatalyst for oxidation of styrene into (S)-styrene oxide. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL-GroES and DnaK-DnaJ-GrpE). The styrene monooxygenases were produced to ca. 40k of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn directed cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40k. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 106 U/g dry cells (6.4 mmol/g dry cells/h) and 1110 U/Laq (67 mM/h), respectively, under mild reaction conditions. These results indicated that the high productivity with high product yield on energy source and on molecular oxygen was driven by the high enzyme activity. Therefore, it is concluded that oxygenase activity of whole-cell biocatalysts is critical for their catalytic performance. Reference 1. Park, J.-B.: Oxygenase-based whole-cell biocatalysis in organic synthesis, J. Microbiol. Biotechnol., 17, 379–392 (2007).
doi:10.1016/j.jbiosc.2009.08.124
BR-O6 Biorefinery for effective utilization of agricultural wastes Jun-ichi Horiuchi Kiyoshi TadaKitami Institute of Technology, Kitami, Hokkaido, Japan A biorefinery is an integrated process for biomass conversion to produce multiple products including fuels and chemicals. The concept of biorefinery was learned from an existing oil refinery. The oil refinery, which consists of various unit operations, has been designed to utilize all components of crude oil and to maximize the values derived from crude oil with minimum energy consumption. This design policy needs to be passed to the successful development of biorefinery, that is, it is important to organize various unit operations concerning biorefinery to maximize profitability with low environmental impact. In this view, we experimentally examined the feasibility of biorefinery for producing multiple products using corn cobs containing cellulose and hemicellulose as a feed stock. The corn cobs were stepwisely decomposed to xylose and glucose and used to produce ethanol, xylitol and astaxanthin by biological processing. About 25 g xylose/l was obtained from hemicellulose contained in 100 g corn cobs/l by acid hydrolysis, which was then converted to 18.7 g xylitol/l using Candida magnoliae (1, 2). It was also found that the xylitol could be used as substrate for astaxanthin production, high value products using Xanthophyllomyces dentrohous. The residual cellulose in corn cobs was enzymatically hydrolyzed to glucose followed by successful utilization as substrate for ethanol or astaxanthin production. Finally total process evaluation will be presented from the view points of profitability, energy consumption and environmental impact.
BR-O5 Development of efficient whole-cell biocatalysts for oxidative biotransformations Hyo-Seel Seo,1 Na-Rae Lee,1 Eun-Hee Doo,1 Sunghoon Park,2 and Jin-Byung Park1 Department. of Food Science and Engineering, Ewha Womans University, Seoul, Republic of Korea 1 and Department of Chemical Engineering, Pusan National University, Pusan, Republic of Korea 2 Selective oxidation is one of the most useful biotransformations for synthetic applications. Biooxidations are often catalyzed by oxygenases, which are usually cofactor dependent and consist of multicomponent enzymes. Thus, oxygenase-expressing whole cells particularly growing cells have been used as biocatalysts for practical application (1). The productivity of growing cell-based biocatalysts are steadily increasing, sometimes approaching 1000U/L (1). However, there are a number of problems in commercialization of oxygenation processes using growing cells as biocatalysts. For instance, the oxygenases should compete for molecular oxygen and cofactors (NAD(P)H) with
S43
metabolic enzymes in growing cell biocatalysts limiting their specific productivity and requiring vigorous reaction conditions. We have developed an efficient Escherichia coli-based biocatalyst for oxidation of styrene into (S)-styrene oxide. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL-GroES and DnaK-DnaJ-GrpE). The styrene monooxygenases were produced to ca. 40k of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn directed cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40k. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 106 U/g dry cells (6.4 mmol/g dry cells/h) and 1110 U/Laq (67 mM/h), respectively, under mild reaction conditions. These results indicated that the high productivity with high product yield on energy source and on molecular oxygen was driven by the high enzyme activity. Therefore, it is concluded that oxygenase activity of whole-cell biocatalysts is critical for their catalytic performance. Reference 1. Park, J.-B.: Oxygenase-based whole-cell biocatalysis in organic synthesis, J. Microbiol. Biotechnol., 17, 379–392 (2007).
doi:10.1016/j.jbiosc.2009.08.124
BR-O6 Biorefinery for effective utilization of agricultural wastes Jun-ichi Horiuchi Kiyoshi TadaKitami Institute of Technology, Kitami, Hokkaido, Japan A biorefinery is an integrated process for biomass conversion to produce multiple products including fuels and chemicals. The concept of biorefinery was learned from an existing oil refinery. The oil refinery, which consists of various unit operations, has been designed to utilize all components of crude oil and to maximize the values derived from crude oil with minimum energy consumption. This design policy needs to be passed to the successful development of biorefinery, that is, it is important to organize various unit operations concerning biorefinery to maximize profitability with low environmental impact. In this view, we experimentally examined the feasibility of biorefinery for producing multiple products using corn cobs containing cellulose and hemicellulose as a feed stock. The corn cobs were stepwisely decomposed to xylose and glucose and used to produce ethanol, xylitol and astaxanthin by biological processing. About 25 g xylose/l was obtained from hemicellulose contained in 100 g corn cobs/l by acid hydrolysis, which was then converted to 18.7 g xylitol/l using Candida magnoliae (1, 2). It was also found that the xylitol could be used as substrate for astaxanthin production, high value products using Xanthophyllomyces dentrohous. The residual cellulose in corn cobs was enzymatically hydrolyzed to glucose followed by successful utilization as substrate for ethanol or astaxanthin production. Finally total process evaluation will be presented from the view points of profitability, energy consumption and environmental impact.