High density cell culture of recombinant bacillus megaterium pCK108 producing cellulase

Abstract

For the production of extracellular carboxymethylcellulase(CMCase), fed-batch culture was carried out using a Bacillus megaterium transformed with a plasmid, pCK 108 harboring CMCase gene. It was observed that specific cellulase productivity of the cells was decreased significantly when high cell density($> 50$ g/l) was achieved with continuous feeding of glucose or lactose as a carbon source. This phenomenon was more significant when glucose was used as a carbon source. As glucose feeding rate increased above 4g/hr, cell density was increased up to 77.1 g-DCW/l but yield of CMCase based on cell mass was lowed sharply. To find out the main reason, inhibitory metabolites accumulated in the culture broth were analyzed and identified using gas chromatography and HPLC. More than 10 kinds of organic acids were detected, including intermediates of TCA cycle, acetate, propionate, and isobutyrate etc. Among them propionic acid and isobutyric acid showed strong inhibitiory effect on both cell growth and CMCase production. Especially, isobutyric acid was found to inhibit CMCase production by 70\% even at 10mM concentration. By reducing the inhibitory metabolites accumulation under slow feeding of lactose as a carbon source, cell growth and CMCase production could increase up to 55 g/l and 15.5 U/ml which were increased by 10 fold and 7 fold, respectively, comparing to the result obtained in batch culture. Lactose as a carbon source was found superior to glucose for CMCase production