Fujio Mukumoto

Comparison of biotin production by recombinant Sphingomonas sp. under various agitation conditions

Biotin production by fermentation of recombinant Sphingomonas sp./pSP304 was investigated. A complex medium containing 60g/l of glycerol and 30g/l of yeast extract was suitable for biotin production. Biotin was produced in the late logarithmic or stationary phase after glycerol starvation. The optimum pH value for biotin production was 7.0. When the dissolved oxygen concentration (DO) was controlled at a constant level, the biotin concentration produced after 120h was significantly lower than that obtained in a test tube culture. Therefore, a batchwise jar-fermentor culture with a constant agitation speed and without DO control was conducted for investigating the effect of agitation conditions on biotin production. Six types of impeller were tested: turbine-blade type, turbo-lift type, rotating mesh type (EGSTAR((R))), screw with draft tube type, Maxblend((R))type, and anchor type. With some impellers, agitation speed was also changed. Both the maximum cell concentration and biotin production varied depending on agitation conditions. Relatively high cell concentrations were attained with four of the impeller types, turbine-blade type, rotating mesh type, Maxblend((R)) type, and anchor type. Among these impellers, the turbine-blade impeller with sintered sparger was suitable for biotin production. After 120h, the cell concentration reached an OD(660) of 43 and a biotin concentration of 66mg/l was obtained, which was comparable with the results from the test tube culture. Morphological variation was also observed depending on the agitation conditions: oval-shaped, rod-shaped, and elongated-shaped cells. Biotin production was relatively high in slightly long rod-shape cells but low in elongated cells. The difference in morphology appeared to depend on the shear stress. It was found that biotin production was strongly correlated with cell length and the oxygen transfer coefficient (k(L)a); cell lengths in the range 4-7µm and k(L)a values in the range 1.5-2.0/min were found to be suitable for biotin production in jar-fermentor culture.