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Investigating the transcriptional differences of amylolytic Saccharomyces cerevisiae strains developed for bioethanol production via a systems based approach
New Biotechnology · Volume 29S · September 2012
Poster 1.1.30 Improved pretreatment of deoiled rice-bran for enhanced yield and productivity of acetone, butanol and ethanol Olujimi Dada 1,∗ , M. Sahaid Kalil 1 , Wan Mohtar Wan Yusoff 2 1
Dept. of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, Bangi, Malaysia 2 School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia A 3-stage novel pretreatment procedure was tested to enhance the liberation of simple sugars from deoiled rice-bran (DRB) in order to increase the amount of fermentable sugar available for fermentation. Different samples of DRB were subjected to physical, acidic (HCl, H2 SO4 and trichloroacetic acid-TCA) and enzymatic treatments to facilitate the cleavaging of the glycosidic bonds within the cellulosic structure as well as the degradation of the carbohydrate polymers previously held together by strong crystalline structure of hydrogen bonds into fermentable monomers. These substrates were subjected to anaerobic fermentation in a batch process using Clostridium saccharoperbutylacetonicum N14 as the inoculum at initial pH of 6 ± 0.2 and temperature of 30◦ C at known initial cell density and substrate concentration. The highest concentration of total fermentable sugar, 33.07 g L−1 was obtained from the TCA-treated DRB hydrolysate compared to 27.14 g L−1 and 31.93 g L−1 for HCl and H2 SO4 -treated DRB hydrolysates respectively. In addition, the highest concentration of solvents, that is acetone, butanol and ethanol (ABE); 11.64 g L−1 ,was obtained from the TCA-treated hydrolysate compared to 10.51 g L−1 and 6.56 g L−1 for H2 SO4 and HCl-treated hydrolysates respectively. The ABE yield and productivity of the TCA treated hydrolysates were 0.47 g/g and 0.097 g L−1 h−1 respectively. These values were higher than 0.2 g/g, 0.05 g L−1 h−1 and 0.32 g/g, 0.08 g L−1 h−1 for HCl and H2 SO4 respectively. In conclusion, the use of TCA for pre-treatment of de-oiled rice bran enhanced the yield and productivity of ABE. http://dx.doi.org/10.1016/j.nbt.2012.08.129 Poster 1.1.31 Investigating the transcriptional differences of amylolytic Saccharomyces cerevisiae strains developed for bioethanol production via a systems based approach Ceyda Kasavi 1,∗ , Ebru Toksoy Oner 2 , Steve G. Oliver 3 , Betul Kirdar 1 1
Department of Chemical Engineering, Bogazici University, Istanbul, Turkey 2 Department of Bioengineering, Marmara University, Istanbul, Turkey 3 Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, UK Limited oil reserves, increasing energy demand, fast growing population, and concerns about climate changes have promoted global interest in renewable energy sources, especially biofuels. Among biofuels, bioethanol plays a significant role in expanding generation of biofuels due to its relatively low production
cost. Conventional bioethanol production from starchy substrates requires an energy-intensive, high-temperature cooking process followed by enzymatic hydrolysis of starch to fermentable sugars. Use of amylolytic yeasts for one-step conversion of starch into ethanol via a simultaneous saccharification–fermentation process is an alternative to the conventional multi-stage process which is economically inviable in long term. Hence, our research group is mainly focused on the development of non-cooking fermentation systems utilizing amylolytic yeast strains. In our previous studies, plasmid-bearing and integrant Saccharomyces cerevisiae strains that can stably maintain and express multiple copies of DNA sequences encoding Bacillus subtilis ␣-amylase and Aspergillus awamori glucoamylase enzymes, were constructed. In addition to soluble starch, these recombinant amylolytic integrant strains were also found to utilize raw starch substrates like wheat and corn starch. High starch utilization rate and stable amylolytic activity of these strains made them worthy of further investigation to elucidate their true potential in hydrolyzing agro-industrial starchy residues as well as in other bioconversion processes. In this study, constructed amylolytic strains were cultivated in fully controlled fermenters, their fermentation performances were compared and genome-wide transcriptional differences underlying strain performances were investigated by using a system based approach at transcriptional level. This research has been financially supported by Bogazici University Research Fund Project 6530. http://dx.doi.org/10.1016/j.nbt.2012.08.130 Poster 1.1.32 Production of Pure Microbial Oil (PMO) with oleaginous yeasts N.A. van Biezen 1,∗ , C.R. Ruprecht 1 , A. Walravens 1 , T. Goosen 1 , D.E. Martens 2 , R.H. Wijffels 2 , B.C. Lokman 1 1
HAN BioCentre, Nijmegen, The Netherlands Wageningen University, Bioprocess Engineering, Wageningen, The Netherlands
2
A potentially promising alternative for the production of triacylglycerides (TAG) could be the use of oleaginous yeasts that produce Pure Microbial Oil (PMO). These micro-organisms have substantial advantages compared to other production systems; - High volumetric production rates (doubling time of two hours, >60 g biomass/kg broth). - More efficient use of rural area (vertical versus horizontal cultivation). - No competition (yet) with the food market. These second generation bio fuel producing micro-organisms have a high potential for future energy solutions. After initial shake flask screening, seven promising strains were identified and screened further on their robustness with respect to fermentation parameters (pH and T). Strain HBC025 came out of this screening as a good strain and was further evaluated in shake flasks and bioreactors. Altering the medium composition to higher C/N ratios results in nitrogen starvation and forces the cells to produce lipids. This gave rise to higher biomass-yields (Yx/s ), which is an unexpected phenomena under nitrogen starvation. Growth www.elsevier.com/locate/nbt S47
Poster 1.1.30 Improved pretreatment of deoiled rice-bran for enhanced yield and productivity of acetone, butanol and ethanol Olujimi Dada 1,∗ , M. Sahaid Kalil 1 , Wan Mohtar Wan Yusoff 2 1
Dept. of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, Bangi, Malaysia 2 School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia A 3-stage novel pretreatment procedure was tested to enhance the liberation of simple sugars from deoiled rice-bran (DRB) in order to increase the amount of fermentable sugar available for fermentation. Different samples of DRB were subjected to physical, acidic (HCl, H2 SO4 and trichloroacetic acid-TCA) and enzymatic treatments to facilitate the cleavaging of the glycosidic bonds within the cellulosic structure as well as the degradation of the carbohydrate polymers previously held together by strong crystalline structure of hydrogen bonds into fermentable monomers. These substrates were subjected to anaerobic fermentation in a batch process using Clostridium saccharoperbutylacetonicum N14 as the inoculum at initial pH of 6 ± 0.2 and temperature of 30◦ C at known initial cell density and substrate concentration. The highest concentration of total fermentable sugar, 33.07 g L−1 was obtained from the TCA-treated DRB hydrolysate compared to 27.14 g L−1 and 31.93 g L−1 for HCl and H2 SO4 -treated DRB hydrolysates respectively. In addition, the highest concentration of solvents, that is acetone, butanol and ethanol (ABE); 11.64 g L−1 ,was obtained from the TCA-treated hydrolysate compared to 10.51 g L−1 and 6.56 g L−1 for H2 SO4 and HCl-treated hydrolysates respectively. The ABE yield and productivity of the TCA treated hydrolysates were 0.47 g/g and 0.097 g L−1 h−1 respectively. These values were higher than 0.2 g/g, 0.05 g L−1 h−1 and 0.32 g/g, 0.08 g L−1 h−1 for HCl and H2 SO4 respectively. In conclusion, the use of TCA for pre-treatment of de-oiled rice bran enhanced the yield and productivity of ABE. http://dx.doi.org/10.1016/j.nbt.2012.08.129 Poster 1.1.31 Investigating the transcriptional differences of amylolytic Saccharomyces cerevisiae strains developed for bioethanol production via a systems based approach Ceyda Kasavi 1,∗ , Ebru Toksoy Oner 2 , Steve G. Oliver 3 , Betul Kirdar 1 1
Department of Chemical Engineering, Bogazici University, Istanbul, Turkey 2 Department of Bioengineering, Marmara University, Istanbul, Turkey 3 Cambridge Systems Biology Centre & Department of Biochemistry, University of Cambridge, UK Limited oil reserves, increasing energy demand, fast growing population, and concerns about climate changes have promoted global interest in renewable energy sources, especially biofuels. Among biofuels, bioethanol plays a significant role in expanding generation of biofuels due to its relatively low production
cost. Conventional bioethanol production from starchy substrates requires an energy-intensive, high-temperature cooking process followed by enzymatic hydrolysis of starch to fermentable sugars. Use of amylolytic yeasts for one-step conversion of starch into ethanol via a simultaneous saccharification–fermentation process is an alternative to the conventional multi-stage process which is economically inviable in long term. Hence, our research group is mainly focused on the development of non-cooking fermentation systems utilizing amylolytic yeast strains. In our previous studies, plasmid-bearing and integrant Saccharomyces cerevisiae strains that can stably maintain and express multiple copies of DNA sequences encoding Bacillus subtilis ␣-amylase and Aspergillus awamori glucoamylase enzymes, were constructed. In addition to soluble starch, these recombinant amylolytic integrant strains were also found to utilize raw starch substrates like wheat and corn starch. High starch utilization rate and stable amylolytic activity of these strains made them worthy of further investigation to elucidate their true potential in hydrolyzing agro-industrial starchy residues as well as in other bioconversion processes. In this study, constructed amylolytic strains were cultivated in fully controlled fermenters, their fermentation performances were compared and genome-wide transcriptional differences underlying strain performances were investigated by using a system based approach at transcriptional level. This research has been financially supported by Bogazici University Research Fund Project 6530. http://dx.doi.org/10.1016/j.nbt.2012.08.130 Poster 1.1.32 Production of Pure Microbial Oil (PMO) with oleaginous yeasts N.A. van Biezen 1,∗ , C.R. Ruprecht 1 , A. Walravens 1 , T. Goosen 1 , D.E. Martens 2 , R.H. Wijffels 2 , B.C. Lokman 1 1
HAN BioCentre, Nijmegen, The Netherlands Wageningen University, Bioprocess Engineering, Wageningen, The Netherlands
2
A potentially promising alternative for the production of triacylglycerides (TAG) could be the use of oleaginous yeasts that produce Pure Microbial Oil (PMO). These micro-organisms have substantial advantages compared to other production systems; - High volumetric production rates (doubling time of two hours, >60 g biomass/kg broth). - More efficient use of rural area (vertical versus horizontal cultivation). - No competition (yet) with the food market. These second generation bio fuel producing micro-organisms have a high potential for future energy solutions. After initial shake flask screening, seven promising strains were identified and screened further on their robustness with respect to fermentation parameters (pH and T). Strain HBC025 came out of this screening as a good strain and was further evaluated in shake flasks and bioreactors. Altering the medium composition to higher C/N ratios results in nitrogen starvation and forces the cells to produce lipids. This gave rise to higher biomass-yields (Yx/s ), which is an unexpected phenomena under nitrogen starvation. Growth www.elsevier.com/locate/nbt S47