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Yeast immobilization on lignocellulosic materials for ethanol production used as fuel
S346
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
order to change substrate specificity of NXRG, five point mutations were introduced by site-directed mutagenesis. Mutated NXRG was placed under the control of a glyceraldehyde 3-phosphate dehydrogenase promoter and integrated into the genome of C. tropicalis K1. The resulting recombinant yeast, C. tropicalis KNV, showed XR activity of 80 mU/mg of proteins toward D-xylose, whereas activity toward L-arabinose was not detected. In fed batch fermentation for xylitol production, KNV showed lower arabitol/xylitol ratio (23%) than that of the control strain (33%).
[P-I.13] Induction and immobilization of NAD+ - dependent alcohol dehydrogenase with activity towards long chain aliphatic alcohols in thermophilic organisms Lorena Alvarez ∗ , Fernando Acevedo, Andres Illanes Pontificia Universidad Católica de Valparaíso, Chile Keywords: long chain aliphatic alcohols; polycosanols; alcohol dehydrogenase; immobilized enzyme
doi:10.1016/j.jbiotec.2010.09.378 [P-I.12] Yeast immobilization on lignocellulosic materials for ethanol production used as fuel ˜ L.M. Agudelo ∗ , M. Penuela Universidad de Antioquia, Colombia Keywords: immobilized; yeast; ethanol; lignocellulosic materials The continuous processes are an alternative to improve the efficiency and the performance in the fermentations undertaken to ethanol yield and reduce production cost. The continuous processes using immobilized cells show great advantages over continuous processes with free cells. Immobilization matrices have been the most widely used polymer gels such as alginate, where cells are immobilized by entrapment in the gel. However, this method of restraint is not viable on an industrial scale because f the cost of raw material and the relative complexity of preparation of biocatalysts with respect to the operational lifetime of the process. Recently, we begin to evaluate raw materials derived from agro- industrial waste and its application as promising carries for the immobilization of cells, in these carries cells are immobilized by adsorption technique that is simple and economically favorable. This study evaluated different lignocellulosic materials (wood shavings, bagasse, corncob and calyx) abundant in our environment, as potential matrices for immobilization of yeast by adsorption and subsequent employment in column bioreactors for production ethanol. The characterizations made it possible to establish the microscopic structural differences that present the material, as well as differences in composition of lignin, cellulose, hemicelluloses and ash. Protocol was established for the preparation of materials, we developed a protocol to establish the cell immobilization on these supports, modifying the technique for measuring dry weight. Using an experimental design, the effect size vs. flow for lignocellulosic materials, and established the size and flow rate have better operations stability for fermentation in packed bed bioreactor. Ethanol production by continuous fermentation using immobilized cells made in lignocellulosic media, have allowed confirming the potential of implementing the industrialscale development. doi:10.1016/j.jbiotec.2010.09.379
Long chain fatty acids are valuable compounds for the pharmaceutical, nutritional and cosmetic industry. Behenic and lignoceric acids can be obtained by oxidation of the corresponding polycosanols but neither scientific information nor patent claim on their production exist. We have developed a strategy for the production in thermophilic organisms of alcohol dehydrogenases with capacity to oxidize long-chain aliphatic alcohols by induction with alcohols and alkanes of 22 and 24 carbon atoms. Thermus thermophilus PRQ25 and Thermus AB1 strains were provided by Dr José Berenguer (Universidad Autónoma de Madrid). Batch fermentations were conducted for 96 hours in 500 ml shake flasks at 200 rpm, at 70◦ for PRQ25, and at 50 ◦ C for AB1. Induction was done by adding 0.5% of eicosanol for PRQ 25 and 0.5% docosanol for AB1 to Thermus culture medium (tryptone, yeast extract and mineral salts). Activity was measured based on the initial rate of NADH production with 0.2 mM substrates (butanol, docosanol and tetracosanol) in 100 mM phosphate buffer pH 7.0 with 0.6% diglyme. Immobilization on CNBr activated sepharose 4B (BrCN) and Glyoxyl agarose (Glx) was carried out by adding 1 g of each support to 10 ml of protein extract. After induction, the maximum specific activity in PRQ 25 was 16.4 IU/g protein, obtained when using docosanol as substrate, while maximum activity in AB1 was 42.9 IU/g proteins when using tetracosanol as substrate. Immobilization yield for AB1 was 50% in BrCN (0.46 IU/g support) and 79% in Glx (0.85 IU/g support). Immobilization yield for PRQ 25 was 76% (0.65 IU /g support) in BrCN and 100% in Glx (0.90 IU /g support). Glx biocatalysts maintained 80% of their initial activity after 48 hours of incubation at 60 ◦ C. Similar results were obtained with PRQ 25 when incubated at 70 ◦ C; nevertheless, AB1 residual activity was only 60%. doi:10.1016/j.jbiotec.2010.09.380 [P-I.14] Functional display of target proteins on Bacillus subtilis spore and its application JUNEHYUNG KIM Dong-A University, Korea, Republic of Keywords: Surface display; Beta-galactosidase; Bacillus subtilis; Spore Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore was developed. Among 11 spore coat proteins examined, cotE and cotG were
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
order to change substrate specificity of NXRG, five point mutations were introduced by site-directed mutagenesis. Mutated NXRG was placed under the control of a glyceraldehyde 3-phosphate dehydrogenase promoter and integrated into the genome of C. tropicalis K1. The resulting recombinant yeast, C. tropicalis KNV, showed XR activity of 80 mU/mg of proteins toward D-xylose, whereas activity toward L-arabinose was not detected. In fed batch fermentation for xylitol production, KNV showed lower arabitol/xylitol ratio (23%) than that of the control strain (33%).
[P-I.13] Induction and immobilization of NAD+ - dependent alcohol dehydrogenase with activity towards long chain aliphatic alcohols in thermophilic organisms Lorena Alvarez ∗ , Fernando Acevedo, Andres Illanes Pontificia Universidad Católica de Valparaíso, Chile Keywords: long chain aliphatic alcohols; polycosanols; alcohol dehydrogenase; immobilized enzyme
doi:10.1016/j.jbiotec.2010.09.378 [P-I.12] Yeast immobilization on lignocellulosic materials for ethanol production used as fuel ˜ L.M. Agudelo ∗ , M. Penuela Universidad de Antioquia, Colombia Keywords: immobilized; yeast; ethanol; lignocellulosic materials The continuous processes are an alternative to improve the efficiency and the performance in the fermentations undertaken to ethanol yield and reduce production cost. The continuous processes using immobilized cells show great advantages over continuous processes with free cells. Immobilization matrices have been the most widely used polymer gels such as alginate, where cells are immobilized by entrapment in the gel. However, this method of restraint is not viable on an industrial scale because f the cost of raw material and the relative complexity of preparation of biocatalysts with respect to the operational lifetime of the process. Recently, we begin to evaluate raw materials derived from agro- industrial waste and its application as promising carries for the immobilization of cells, in these carries cells are immobilized by adsorption technique that is simple and economically favorable. This study evaluated different lignocellulosic materials (wood shavings, bagasse, corncob and calyx) abundant in our environment, as potential matrices for immobilization of yeast by adsorption and subsequent employment in column bioreactors for production ethanol. The characterizations made it possible to establish the microscopic structural differences that present the material, as well as differences in composition of lignin, cellulose, hemicelluloses and ash. Protocol was established for the preparation of materials, we developed a protocol to establish the cell immobilization on these supports, modifying the technique for measuring dry weight. Using an experimental design, the effect size vs. flow for lignocellulosic materials, and established the size and flow rate have better operations stability for fermentation in packed bed bioreactor. Ethanol production by continuous fermentation using immobilized cells made in lignocellulosic media, have allowed confirming the potential of implementing the industrialscale development. doi:10.1016/j.jbiotec.2010.09.379
Long chain fatty acids are valuable compounds for the pharmaceutical, nutritional and cosmetic industry. Behenic and lignoceric acids can be obtained by oxidation of the corresponding polycosanols but neither scientific information nor patent claim on their production exist. We have developed a strategy for the production in thermophilic organisms of alcohol dehydrogenases with capacity to oxidize long-chain aliphatic alcohols by induction with alcohols and alkanes of 22 and 24 carbon atoms. Thermus thermophilus PRQ25 and Thermus AB1 strains were provided by Dr José Berenguer (Universidad Autónoma de Madrid). Batch fermentations were conducted for 96 hours in 500 ml shake flasks at 200 rpm, at 70◦ for PRQ25, and at 50 ◦ C for AB1. Induction was done by adding 0.5% of eicosanol for PRQ 25 and 0.5% docosanol for AB1 to Thermus culture medium (tryptone, yeast extract and mineral salts). Activity was measured based on the initial rate of NADH production with 0.2 mM substrates (butanol, docosanol and tetracosanol) in 100 mM phosphate buffer pH 7.0 with 0.6% diglyme. Immobilization on CNBr activated sepharose 4B (BrCN) and Glyoxyl agarose (Glx) was carried out by adding 1 g of each support to 10 ml of protein extract. After induction, the maximum specific activity in PRQ 25 was 16.4 IU/g protein, obtained when using docosanol as substrate, while maximum activity in AB1 was 42.9 IU/g proteins when using tetracosanol as substrate. Immobilization yield for AB1 was 50% in BrCN (0.46 IU/g support) and 79% in Glx (0.85 IU/g support). Immobilization yield for PRQ 25 was 76% (0.65 IU /g support) in BrCN and 100% in Glx (0.90 IU /g support). Glx biocatalysts maintained 80% of their initial activity after 48 hours of incubation at 60 ◦ C. Similar results were obtained with PRQ 25 when incubated at 70 ◦ C; nevertheless, AB1 residual activity was only 60%. doi:10.1016/j.jbiotec.2010.09.380 [P-I.14] Functional display of target proteins on Bacillus subtilis spore and its application JUNEHYUNG KIM Dong-A University, Korea, Republic of Keywords: Surface display; Beta-galactosidase; Bacillus subtilis; Spore Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore was developed. Among 11 spore coat proteins examined, cotE and cotG were