Presentation Information
[P02-203]Scale-up cultivation of Shewanella livingstonensis by using Jar-fermenter for improvement of conversion and enzyme expression
○Taku Matsumoto1, Naofumi Yamada1, Akiko Hida1, Takahisa Tajima1 (1. Program of Biotechnology, Graduate School of Integrated Sciences for Life, Hiroshima University, (Japan))
Keywords:
Scale-up cultivation,Psychrophilic bacterium,Bioprocess engineering,Biocatalysis
Bioprocess that utilizing microorganisms as biocatalysts is attracting attention for their low energy and environmental impact as it involves few reaction steps under moderate temperature and pressure. However, when the conventional biocatalysis that is used the mesophilic bacteria, such as Escherichia coli, as host cells was used for the conversion process, substrates were sometimes incorporated into its cellular metabolism, resulting in the formation of undesirable products as by-products. To produce target products efficiently, we developed innovative biocatalysis called Psychrophile-based-Simple bioCatlysis (PSCat). In PSCat, mesophilic enzyme was expressed in the psychrophilic bacterium that are heat-treated at moderate temperature to inactivate intrinsic bacterial enzymes of them, while expressed enzymes retained their catalytic activity. Therefore, substrates could be consumed by expressed enzymes efficiently. In our previous studies, we could convert over 90% of substrates to target products without producing by-product.
However, it takes a long time to prepare PSCat because the psychrophilic bacteria have lower growth rates. In addition, the practical use of PSCat requires scale-up cultivation, such as at a pilot or industrial scale level. In this study, we investigated the scale-up cultivation of the psychrophilic bacterium to improve the growth rate and enzyme activity. Then, we applied them for the aspartate production as a model of enzyme conversion. Shewanella livingstonensis Ac10 that expresses E. coli aspartase was inoculated into 500 mL flask or 2 L Jar-fermenter with Triptic soy broth medium and incubated at 18℃ for 48 hour. To investigate the optimal culture condition, various environmental factors, such as agitation rate and pH, were altered during the cultivation period. The cultured cells were treated at a moderate temperature to inactivate intrinsic bacterial metabolic enzymes. The resulting cell suspension was used as PSCat.Using the jar-fermenter improved the overall oxygen transfer coefficient (kLa) and enhanced the growth rate by approximately 1.6 times at an agitation rate of 400 rpm compared to flask scale. Furthermore, the expression level of aspartase remained consistent throughout the scaling up of the cultivation process. As the concentrations of cells and produced enzymes in the medium increased, the aspartase activity was approximately 7.0 times compared to the flask scale. Therefore, by preparing PSCat derived from cells cultivated by jar-fermenter yielded approximately 1.5 times more aspartic acid than the flask scale. Additionally, as the growth rate increased with scale-up cultivation enabling 24-hour reduction in PSCat preparation time.
However, it takes a long time to prepare PSCat because the psychrophilic bacteria have lower growth rates. In addition, the practical use of PSCat requires scale-up cultivation, such as at a pilot or industrial scale level. In this study, we investigated the scale-up cultivation of the psychrophilic bacterium to improve the growth rate and enzyme activity. Then, we applied them for the aspartate production as a model of enzyme conversion. Shewanella livingstonensis Ac10 that expresses E. coli aspartase was inoculated into 500 mL flask or 2 L Jar-fermenter with Triptic soy broth medium and incubated at 18℃ for 48 hour. To investigate the optimal culture condition, various environmental factors, such as agitation rate and pH, were altered during the cultivation period. The cultured cells were treated at a moderate temperature to inactivate intrinsic bacterial metabolic enzymes. The resulting cell suspension was used as PSCat.Using the jar-fermenter improved the overall oxygen transfer coefficient (kLa) and enhanced the growth rate by approximately 1.6 times at an agitation rate of 400 rpm compared to flask scale. Furthermore, the expression level of aspartase remained consistent throughout the scaling up of the cultivation process. As the concentrations of cells and produced enzymes in the medium increased, the aspartase activity was approximately 7.0 times compared to the flask scale. Therefore, by preparing PSCat derived from cells cultivated by jar-fermenter yielded approximately 1.5 times more aspartic acid than the flask scale. Additionally, as the growth rate increased with scale-up cultivation enabling 24-hour reduction in PSCat preparation time.
Comment
To browse or post comments, you must log in.Log in