Investigation of bioreactor operation conditions for serine alkaline protease production by Bacillus subtilis

Özdemir, Elif
In this study, complex medium was designed for increasing serine alkaline protease (SAP) production by recombinant Bacillus sp. In this context, the effects of glucose, sucrose, inorganic nitrogen source, casein, defatted soybean, and molasses and their concerted effects were investigated in agitation (N=200 min"1) and heating rate (T=37°C) controlled laboratory scale bioreactors. For this purpose, experiments were started by microorganism selection. It was found that, most suitable microorganism for complex medium was r-B.subtilis carrying pHV1431. ;:subC gene. After considering soybean as a nitrogen source, the effects of (NH4)2HP04 concentration were investigated. As maximum SAP activity was obtained in the medium which did not contain (NH4)2HP04, soybean was selected as the sole nitrogen source. Then, binary effects of defatted soybean and glucose; defatted soybean and sucrose; defatted soybean and molasses; and casein and sucrose were investigated. Among these, maximum activity was obtained as 3852 U cm"3 at t=46 h with CSOybean0 =20 kg m"3 and CSUcrose°=15 kg m"3. Thereafter, effects of inorganic compounds were investigated and it was found that except phosphate, inorganic compounds found in soybean source were sufficient for SAP production, and the activity increased to 5347 U cm"3 with the optimization of the phosphate concentration. Finally, by using the medium designed the variations in the cell growth, protease production, oxygen uptake rate, and oxygen transfer coefficient with the cultivation time were determined in 2.0 dm3 bioreactor systems having temperature, pH, foam, air inlet and stirring rate controls. The maximum SAP activity reached a maximum at t=27 h with a value of 4737 U cm"3 and mass transfer coefficient varied between 0.0096 -0.0435 s"1; whereas, volumetric oxygen uptake rate varied between 0.001-0.006 mol m"3 s'1; and specific oxygen uptake rate varied between 0.0001-0.0022 mol kg"1 s1 throughout the bioprocess.
Citation Formats
E. Özdemir, “Investigation of bioreactor operation conditions for serine alkaline protease production by Bacillus subtilis,” M.S. - Master of Science, Middle East Technical University, 2003.