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Effect of the concentration of glycerol on the productivity of 1,3-propanediol using Clostridium butyricum
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
mediation process. Anaerobic bacteria are important members of microbial communities with economic, environmental and biotechnological interest. They can be found in a variety of environments such as soils, sediments and domestic, industrial and mining sludge and wastewaters. The studies of U(VI) bio-removal were carried in the presence of 20 mg/L of this metal and the influence of sulphate in metal removal was investigated. Among several environmental samples screened, a bacterial consortium resistant to uranium was obtained from a soil sample from Monchique thermal place, located in south Portugal (Algarve). The bacteria present in the consortium have the ability to remove 20 mg/L of U(VI) in 15 days, at room temperature. The results show that the presence of sulphate did not affect metal removal. Phylogenetic analysis of the 16S gene sequence revealed that this bacterial consortium contains species affiliated to Clostridium spp., Sporotalea colonica spp., and to Rhodocyclus spp. The mechanism of uranium removal from aqueous solution was also investigated and it was observed that U(VI) removal occurs only in the presence of living cells. The existence of dense precipitates, mainly within the periplasmatic region, was observed through Transmission Electron Microscopy (TEM). This result is usually associated to enzymatic reduction of uranium (VI) to uranium (IV). Future work will be focused on the characterization and isolation of the enzymes involved in this mechanism. Thus, this consortium shows potential for biotechnological applications. doi:10.1016/j.nbt.2009.06.378
2.1.132 Characterization of prorenin processing enzyme responsible for in situ active human renin production by baculovirus-infected Sf-9 cells and inhibition of excessive processing T. Gotoh 1,∗ , H. Awa 1 , S. Hikage 1 , K.I. Kikuchi 1 , S. Takahashi 2 1
2
Akita University, Akita, Japan Akita Research Institute of Food and Brewing, Japan
Expression of recombinant human (rh)-prorenin in Sf-9 insect cells using baculovirus vector system has advantageously led to the in situ production of active rh-renin by the action of intrinsic protease. However, the produced active rh-renin has been gradually degraded in the same time by excessive proteolysis and decreased in a very late phase of the infection culture. The present study was addressed to characterize the protease (prorenin processing enzyme: PPE) responsible for the active rh-renin production and to inhibit the excessive degradation of the produced rh-renin, exploring a new possible application of the baculovirus vector system, which can synthesize mature proteins from expressed precursor proteins. The PPE was partially purified by three steps of column chromatography and revealed to have a molecular weight of 32 kDa and belong to the cysteine proteases. The culture media of baculovirus-infected Sf-9 were supplemented with either protease inhibitors or BSA. The degradation of active rh-renin after the maximum accumulation was depressed in some extent by the supplementation of cysteine protease inhibitors. However, the S96
www.elsevier.com/locate/nbt
processing from inactive rh-prorenin to active rh-renin was also depressed. On the contrary, the supplementation of BSA successfully inhibited the degradation of active rh-renin without affecting the processing from inactive rh-prorenin to active rh-renin. doi:10.1016/j.nbt.2009.06.380
2.1.133 Effect of the concentration of glycerol on the productivity of 1,3-propanediol using Clostridium butyricum D. Olivares ∗ , P. Poirrier, J.C. Gentina, G. Aroca Universidad Católica de Valparaíso, Valparaiso, Chile
Among the different alternatives for using glycerol, as a by-product from biodiesel production by transesterification of triglycerides, the production of 1,3-propanediol (1,3-PD) is one that has great potential. In this work we report the effect of the feeding concentration of glycerol on the productivity of 1,3-PD, using an anaerobic continuous culture of C. butyricum at a dilution rate of 0.16 hour−1 . The experiments were carried out in a 1 l chemostat at pH 7, temperature of 37◦ C and 200 rpm of agitation rate. The medium used had glycerol 20 g l−1 , yeast extract 2 g l−1 , (NH4 )2 SO4 2 g l−1 , K2 HPO4 ·3H2 O 3.4 g l−1 , KH2 PO4 1.3 g l−1 , MgSO4 ·7H2 O 0.2 g l−1 , 20 ml of a mineral solution, resazurine 0.001 g l−1 , and cysteine 0.2 g l−1 . This medium was amplified by 2×, 3×, and 4.5× for reaching different steady states. Glycerol, 1,3-PD, and volatile fatty acids were quantified by gas chromatography. Biomass was estimated by optical density and dry weight. The results show an influence of the glycerol feeding concentration on the volumetric and specific productivity of 1,3-PD. At S0 = 20 g l−1 a volumetric productivity of 1.26 (g hour−1 l−1 ) and a specific productivity of 1.26 (g g−1 hour−1 ) were reached, whereas at S0 = 90 g l−1 a Qp = 4.54 (g hour−1 l−1 ) and qp = 2.24 (g g−1 hour−1 ) were reached. The increase in the specific productivity evidence a metabolic effect of the glycerol concentration on the 1,3-propanediol production, effect that is now under study. doi:10.1016/j.nbt.2009.06.382
2.1.134 Production and purification of lipase enzyme by some new fungal sources ¸i N. Cihangir ∗ , Ö. Kebabc Hacettepe University, Beytepe/Ankara, Turkey
Lipases are the enzymes which catalyze the hydrolysis of triglycerides. They are produced by various microorganisms, plants and animals. Most of the lipases for commercial use are synthesized by fungi and yeasts. Lipases are widely used enzymes in various plants, for example, dairy, food, detergent, cosmetic and tanning industries. In this study, fungi isolated from soil were screened for exogenous lipolytic activity. Some cultural parameters influencing the
ABSTRACTS
mediation process. Anaerobic bacteria are important members of microbial communities with economic, environmental and biotechnological interest. They can be found in a variety of environments such as soils, sediments and domestic, industrial and mining sludge and wastewaters. The studies of U(VI) bio-removal were carried in the presence of 20 mg/L of this metal and the influence of sulphate in metal removal was investigated. Among several environmental samples screened, a bacterial consortium resistant to uranium was obtained from a soil sample from Monchique thermal place, located in south Portugal (Algarve). The bacteria present in the consortium have the ability to remove 20 mg/L of U(VI) in 15 days, at room temperature. The results show that the presence of sulphate did not affect metal removal. Phylogenetic analysis of the 16S gene sequence revealed that this bacterial consortium contains species affiliated to Clostridium spp., Sporotalea colonica spp., and to Rhodocyclus spp. The mechanism of uranium removal from aqueous solution was also investigated and it was observed that U(VI) removal occurs only in the presence of living cells. The existence of dense precipitates, mainly within the periplasmatic region, was observed through Transmission Electron Microscopy (TEM). This result is usually associated to enzymatic reduction of uranium (VI) to uranium (IV). Future work will be focused on the characterization and isolation of the enzymes involved in this mechanism. Thus, this consortium shows potential for biotechnological applications. doi:10.1016/j.nbt.2009.06.378
2.1.132 Characterization of prorenin processing enzyme responsible for in situ active human renin production by baculovirus-infected Sf-9 cells and inhibition of excessive processing T. Gotoh 1,∗ , H. Awa 1 , S. Hikage 1 , K.I. Kikuchi 1 , S. Takahashi 2 1
2
Akita University, Akita, Japan Akita Research Institute of Food and Brewing, Japan
Expression of recombinant human (rh)-prorenin in Sf-9 insect cells using baculovirus vector system has advantageously led to the in situ production of active rh-renin by the action of intrinsic protease. However, the produced active rh-renin has been gradually degraded in the same time by excessive proteolysis and decreased in a very late phase of the infection culture. The present study was addressed to characterize the protease (prorenin processing enzyme: PPE) responsible for the active rh-renin production and to inhibit the excessive degradation of the produced rh-renin, exploring a new possible application of the baculovirus vector system, which can synthesize mature proteins from expressed precursor proteins. The PPE was partially purified by three steps of column chromatography and revealed to have a molecular weight of 32 kDa and belong to the cysteine proteases. The culture media of baculovirus-infected Sf-9 were supplemented with either protease inhibitors or BSA. The degradation of active rh-renin after the maximum accumulation was depressed in some extent by the supplementation of cysteine protease inhibitors. However, the S96
www.elsevier.com/locate/nbt
processing from inactive rh-prorenin to active rh-renin was also depressed. On the contrary, the supplementation of BSA successfully inhibited the degradation of active rh-renin without affecting the processing from inactive rh-prorenin to active rh-renin. doi:10.1016/j.nbt.2009.06.380
2.1.133 Effect of the concentration of glycerol on the productivity of 1,3-propanediol using Clostridium butyricum D. Olivares ∗ , P. Poirrier, J.C. Gentina, G. Aroca Universidad Católica de Valparaíso, Valparaiso, Chile
Among the different alternatives for using glycerol, as a by-product from biodiesel production by transesterification of triglycerides, the production of 1,3-propanediol (1,3-PD) is one that has great potential. In this work we report the effect of the feeding concentration of glycerol on the productivity of 1,3-PD, using an anaerobic continuous culture of C. butyricum at a dilution rate of 0.16 hour−1 . The experiments were carried out in a 1 l chemostat at pH 7, temperature of 37◦ C and 200 rpm of agitation rate. The medium used had glycerol 20 g l−1 , yeast extract 2 g l−1 , (NH4 )2 SO4 2 g l−1 , K2 HPO4 ·3H2 O 3.4 g l−1 , KH2 PO4 1.3 g l−1 , MgSO4 ·7H2 O 0.2 g l−1 , 20 ml of a mineral solution, resazurine 0.001 g l−1 , and cysteine 0.2 g l−1 . This medium was amplified by 2×, 3×, and 4.5× for reaching different steady states. Glycerol, 1,3-PD, and volatile fatty acids were quantified by gas chromatography. Biomass was estimated by optical density and dry weight. The results show an influence of the glycerol feeding concentration on the volumetric and specific productivity of 1,3-PD. At S0 = 20 g l−1 a volumetric productivity of 1.26 (g hour−1 l−1 ) and a specific productivity of 1.26 (g g−1 hour−1 ) were reached, whereas at S0 = 90 g l−1 a Qp = 4.54 (g hour−1 l−1 ) and qp = 2.24 (g g−1 hour−1 ) were reached. The increase in the specific productivity evidence a metabolic effect of the glycerol concentration on the 1,3-propanediol production, effect that is now under study. doi:10.1016/j.nbt.2009.06.382
2.1.134 Production and purification of lipase enzyme by some new fungal sources ¸i N. Cihangir ∗ , Ö. Kebabc Hacettepe University, Beytepe/Ankara, Turkey
Lipases are the enzymes which catalyze the hydrolysis of triglycerides. They are produced by various microorganisms, plants and animals. Most of the lipases for commercial use are synthesized by fungi and yeasts. Lipases are widely used enzymes in various plants, for example, dairy, food, detergent, cosmetic and tanning industries. In this study, fungi isolated from soil were screened for exogenous lipolytic activity. Some cultural parameters influencing the