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Fluorescence alternative of gram staining as a tool for metabolic switch recognition in butanol fermentation
S160
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
8% v/v ethanol using commercial reverse-osmosis spiral-wound membrane modules provided that ethanol rejection ranged from 0.5 to 0.9. doi:10.1016/j.jbiotec.2010.08.413 [P-B.61] Inhibitory effect of crude glycerol on ethanol production by Enterobacter aerogenes Sang Jun Lee 1,∗ , Sung Bong Kim 1 , Seong Woo Kang 1 , Sung Ok Han 1 , Chulhwan Park 2 , Seung Wook Kim 1 1
Korea university, Korea, Republic of Kwangwoon university, Korea, Republic of Keywords: Crude glycerol; Enterobacter aerogenes; Ethanol production; Inhibitory effect 2
Glycerol utilization has a significant role on biodiesel production since large amount of glycerol is obtained as a by-product. Therefore, the development of new methods to convert crude and waste glycerol into higher value products is an urgent need. Recently, the glycerol as feedstock has been used to produce various value added products such as 1,3-propanediol, dihydroxyacetone, ethanol etc. through the biological process. Waste glycerol, which is generated from biodiesel manufacturing process, contains various inhibitory compounds (NaCl, KCl, biodiesel residue, and unreacted oils) and pH range of waste glycerol is very broad (pH 3-11). In this study, ethanol production from pure glycerol using Enterobacter aerogenes (ATCC 29007, 13048, 35028) was evaluated in anaerobic culture condition. Inhibitory effects of osmotic pressure of glycerol, pH, and salt concentrations were investigated based on waste glycerol ingredients. The ingredients of waste glycerol were supplied from a biodiesel manufacturing company in Korea. Ethanol production was performed with pure glycerol concentration of 5 g/L ∼ 30 g/L to evaluate effects of substrate concentration and osmotic pressure. The consumed glycerol was 3 ∼ 5 g/L and conversion rate was more than 0.9 mol-ethanol/molglycerol after 24 h of cultivation. To evaluate inhibitory effects of salts (NaCl and KCl), experiments were performed under 0 g/L ∼ 20 g/L of only one of each salt. Inhibitory effects of salts were shown at the high salt concentration. And then, inhibitory effect of pH was performed in the pH range of 4 ∼ 10 and cell growth and ethanol production was higher at pH 5 ∼ 7 than the others. doi:10.1016/j.jbiotec.2010.08.414 [P-B.62] Ethanol production from lignocellulosic materials by S. cerevisiae and P. stipitis Hawon Lee 1 , Yong Hwan Kim 1 , Sang Jun Lee 2,∗ , Seung Wook Kim 2 , Tak-Hyun Kim 3 , Chulhwan Park 1 1
Kwangwoon University, Korea, Republic of Korea University, Korea, Republic of 3 Korea Atomic Energy Research Institute, Korea, Republic of Keywords: Ethanol production; Lignocellulosic materials; S. cerevisiae; P. stipitis 2
The hydrolysis process of lignocelllosic materials to convert polysaccharides to monosaccharides is required in lignocellulosic ethanol production. However, during hydrolysis process various toxic compounds, which can inhibit ethanol production, are also produced and released. We investigated the performance of Saccharomyces cerevisiae K35 and Pichia stipitis KCCM 12009 in
the synthetic medium including model compounds (acetic acid, furfural, 5-hydroxymethylfurfural (5-HMF), syringaldehyde, and pcoumaric acid) as well as in lignocellulosic hydrolysates (yellow poplar, waste wood, and rice hull hydrolysates). In the case of fermentation by S. cerevisiae K35 in synthetic medium, the cell growth was inhibited with the increase in the acetic acid concentration but the ethanol yield was over 96% of the theoretical yield. In the high concentration (over 3 g/L), furfural and 5-HMF significantly decreased cell growth and ethanol production rate but not ethanol yield. S. cerevisiae K35 did not grow and did not produce ethanol at 5 g/L syringaldehyde and p-coumaric acid. In the fermentation of hydrolysates containing a variety of toxic compounds by S. cerevisiae K35, there was no synergetic effect of multiple inhibitory compounds. S. cerevisiae K35 showed high resistance, while P. stipitis KCCM 12009 was sensitive to inhibitory compounds comparison to S. cerevisiae K35. As the concentration of inhibitory compounds increased, cell growth of and ethanol production by P. stipitis KCCM 12009 decreased. P. stipitis KCCM 12009 was especially inhibited in the high concentration (5 g/L) of furans and phenolics. In the case of hydrolysates except yellow poplar hydrolysates, the ethanol production of P. stipitis KCCM 12009 was similar to that of the reference culture containing no inhibitory compounds. Acknowledgements: The authors gratefully acknowledge the financial support provided by the Korea Ministry of Environment (Eco-Star Project). This research was also supported by a Research Grant from Kwangwoon University in 2010. doi:10.1016/j.jbiotec.2010.08.415 [P-B.63] Fluorescence alternative of gram staining as a tool for metabolic switch recognition in butanol fermentation M. Linhova ∗ , P. Patakova, J. Lipovsky, P. Fribert, M. Rychtera, K. Melzoch Institue of Chemical Technology Prague, Czech Republic Keywords: Acetone-butanol-ethanol fermentation; Clostridium; Flow cytometry; Gram staining 1-butanol, a possible bio-component of liquid fuels with more advantageous properties in comparison with ethanol, can be produced by various strains of the genera Clostridium at so-called acetone-butanol-ethanol (ABE) fermentation using a sacharidic substrate. A cultivation course is typically biphasic when growth of cells is connected with butyrate and acetate formations and solvents production is achieved at cells sporulation. Therefore, detailed knowledge of clostridia behaviour is necessary for fermentation control. Although, the process is nowadays studied very intensively a deep attention is devoted mainly to few solventogenic strains like C.acetobutylicum ATCC 824 or C.beijerinckii NCIMB 8052. A combination of dyes hexidium iodide and SYTO 13 together with fluorescent microscopy and flow cytometry was used for recognition of acidogenic and solventogenic metabolic phases using species C.pasteurianum NRRL B-598, C.acetobutylicum DSM 1731 and C.beijerinckii CCM 6218. Cells in acidogenic state, showing G+ response if stained according to Gram, fluoresced bright red while solventogenic cells, having G− response at traditional Gram staining, were of green-yellow colour. The method was used for cells labelling during cultivation using glucose cultivation medium. Labelling patterns were significantly diverse in case of different strains and corresponded to determined concentrations of liquid (acids and solvents) and gaseous (H2 and CO2 ) metabolites. doi:10.1016/j.jbiotec.2010.08.416
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
8% v/v ethanol using commercial reverse-osmosis spiral-wound membrane modules provided that ethanol rejection ranged from 0.5 to 0.9. doi:10.1016/j.jbiotec.2010.08.413 [P-B.61] Inhibitory effect of crude glycerol on ethanol production by Enterobacter aerogenes Sang Jun Lee 1,∗ , Sung Bong Kim 1 , Seong Woo Kang 1 , Sung Ok Han 1 , Chulhwan Park 2 , Seung Wook Kim 1 1
Korea university, Korea, Republic of Kwangwoon university, Korea, Republic of Keywords: Crude glycerol; Enterobacter aerogenes; Ethanol production; Inhibitory effect 2
Glycerol utilization has a significant role on biodiesel production since large amount of glycerol is obtained as a by-product. Therefore, the development of new methods to convert crude and waste glycerol into higher value products is an urgent need. Recently, the glycerol as feedstock has been used to produce various value added products such as 1,3-propanediol, dihydroxyacetone, ethanol etc. through the biological process. Waste glycerol, which is generated from biodiesel manufacturing process, contains various inhibitory compounds (NaCl, KCl, biodiesel residue, and unreacted oils) and pH range of waste glycerol is very broad (pH 3-11). In this study, ethanol production from pure glycerol using Enterobacter aerogenes (ATCC 29007, 13048, 35028) was evaluated in anaerobic culture condition. Inhibitory effects of osmotic pressure of glycerol, pH, and salt concentrations were investigated based on waste glycerol ingredients. The ingredients of waste glycerol were supplied from a biodiesel manufacturing company in Korea. Ethanol production was performed with pure glycerol concentration of 5 g/L ∼ 30 g/L to evaluate effects of substrate concentration and osmotic pressure. The consumed glycerol was 3 ∼ 5 g/L and conversion rate was more than 0.9 mol-ethanol/molglycerol after 24 h of cultivation. To evaluate inhibitory effects of salts (NaCl and KCl), experiments were performed under 0 g/L ∼ 20 g/L of only one of each salt. Inhibitory effects of salts were shown at the high salt concentration. And then, inhibitory effect of pH was performed in the pH range of 4 ∼ 10 and cell growth and ethanol production was higher at pH 5 ∼ 7 than the others. doi:10.1016/j.jbiotec.2010.08.414 [P-B.62] Ethanol production from lignocellulosic materials by S. cerevisiae and P. stipitis Hawon Lee 1 , Yong Hwan Kim 1 , Sang Jun Lee 2,∗ , Seung Wook Kim 2 , Tak-Hyun Kim 3 , Chulhwan Park 1 1
Kwangwoon University, Korea, Republic of Korea University, Korea, Republic of 3 Korea Atomic Energy Research Institute, Korea, Republic of Keywords: Ethanol production; Lignocellulosic materials; S. cerevisiae; P. stipitis 2
The hydrolysis process of lignocelllosic materials to convert polysaccharides to monosaccharides is required in lignocellulosic ethanol production. However, during hydrolysis process various toxic compounds, which can inhibit ethanol production, are also produced and released. We investigated the performance of Saccharomyces cerevisiae K35 and Pichia stipitis KCCM 12009 in
the synthetic medium including model compounds (acetic acid, furfural, 5-hydroxymethylfurfural (5-HMF), syringaldehyde, and pcoumaric acid) as well as in lignocellulosic hydrolysates (yellow poplar, waste wood, and rice hull hydrolysates). In the case of fermentation by S. cerevisiae K35 in synthetic medium, the cell growth was inhibited with the increase in the acetic acid concentration but the ethanol yield was over 96% of the theoretical yield. In the high concentration (over 3 g/L), furfural and 5-HMF significantly decreased cell growth and ethanol production rate but not ethanol yield. S. cerevisiae K35 did not grow and did not produce ethanol at 5 g/L syringaldehyde and p-coumaric acid. In the fermentation of hydrolysates containing a variety of toxic compounds by S. cerevisiae K35, there was no synergetic effect of multiple inhibitory compounds. S. cerevisiae K35 showed high resistance, while P. stipitis KCCM 12009 was sensitive to inhibitory compounds comparison to S. cerevisiae K35. As the concentration of inhibitory compounds increased, cell growth of and ethanol production by P. stipitis KCCM 12009 decreased. P. stipitis KCCM 12009 was especially inhibited in the high concentration (5 g/L) of furans and phenolics. In the case of hydrolysates except yellow poplar hydrolysates, the ethanol production of P. stipitis KCCM 12009 was similar to that of the reference culture containing no inhibitory compounds. Acknowledgements: The authors gratefully acknowledge the financial support provided by the Korea Ministry of Environment (Eco-Star Project). This research was also supported by a Research Grant from Kwangwoon University in 2010. doi:10.1016/j.jbiotec.2010.08.415 [P-B.63] Fluorescence alternative of gram staining as a tool for metabolic switch recognition in butanol fermentation M. Linhova ∗ , P. Patakova, J. Lipovsky, P. Fribert, M. Rychtera, K. Melzoch Institue of Chemical Technology Prague, Czech Republic Keywords: Acetone-butanol-ethanol fermentation; Clostridium; Flow cytometry; Gram staining 1-butanol, a possible bio-component of liquid fuels with more advantageous properties in comparison with ethanol, can be produced by various strains of the genera Clostridium at so-called acetone-butanol-ethanol (ABE) fermentation using a sacharidic substrate. A cultivation course is typically biphasic when growth of cells is connected with butyrate and acetate formations and solvents production is achieved at cells sporulation. Therefore, detailed knowledge of clostridia behaviour is necessary for fermentation control. Although, the process is nowadays studied very intensively a deep attention is devoted mainly to few solventogenic strains like C.acetobutylicum ATCC 824 or C.beijerinckii NCIMB 8052. A combination of dyes hexidium iodide and SYTO 13 together with fluorescent microscopy and flow cytometry was used for recognition of acidogenic and solventogenic metabolic phases using species C.pasteurianum NRRL B-598, C.acetobutylicum DSM 1731 and C.beijerinckii CCM 6218. Cells in acidogenic state, showing G+ response if stained according to Gram, fluoresced bright red while solventogenic cells, having G− response at traditional Gram staining, were of green-yellow colour. The method was used for cells labelling during cultivation using glucose cultivation medium. Labelling patterns were significantly diverse in case of different strains and corresponded to determined concentrations of liquid (acids and solvents) and gaseous (H2 and CO2 ) metabolites. doi:10.1016/j.jbiotec.2010.08.416