High Cell And Spore Density Culture Of L.sporogenes (Bacillus Coagulans TQ33)

Applying cell engineering technology, we studied on the high cell density culture of Bacillus coagulans TQ33.The methods and operation parameters for fed-batch cultivation and filtration cultivation were determined. The main contents and concludes were as follow:During the fed-batch culture, the feeding method was determined about which the ratio of glucose to yeast extract in feeding fluid was 6:1(w/w), and the glucose concentration in the broth was ranging from 3.0g/L to 6.0g/L. In 5L auto-fermentor, the final cell concentration was 4.5 × 109cfu/mL, and the spore concentration was 1.2× 109cfu/mL, which was as much as 5.9 times and 3.8 times of batch culture respectively. But in the fed-batch culture, the spore-formating percentage was low,. Feeding the glucose or yeast extract in the end of the logarithmic phase results in increasing the energy change level, and the sporulation was repressed. The main reason of the low spore-formating percentage in fed-batch culture was glucose concentration's fluctuation result from adding the feeding fluid.In the filtration culture, biomass recycle was started after 18h when the lactic acid concentration was 15g/L. The filtering rate as well as the feed rate of fresh medium was around 1L/h, and the dilution rate was 0.4h-1, operation pressure <0.1Mpa.In 5L auto-fermentor, the cell concentration was 1.5 × 1010cfu/mL, and the spore concentration was 0.9 × 1010cfu/mL, about four-fold and nine-fold greater than that obtained in fed-batch fermentation respectively, which was as much as 20 times and 31.2 times of the batch culture respectively. The reason of the high cell density in the filter process was the special growth rate at high level of 0.3h-1.Sporulation of Bacillus Coagulans TQ33 was density-dependent. In the filter process, the cell concentration was 10 10cfu/niL,and the the spore-formating percentage has been increased to 60%. In the flask condition, adding the condition medium from the Bacillus Coagulans TQ33 will promote the spore formation, and the spore-formating percentage was above 62.5%, which agree to the theory that an extracellular factor was require for efficient sporulation.