Optimization of Conditions for High Cell Density Cultivation of Bacillus Subtilis
Lew, Kok Cheong (2006) Optimization of Conditions for High Cell Density Cultivation of Bacillus Subtilis. Masters thesis, Universiti Putra Malaysia.
The objectives of this study are (i) To investigate the effect of using different types of carbon source and their concentration on the growth of B. subtilis, (ii) To optimize the pH condition for improvement of growth of B. subtilis through the different pH control strategies and (iii) To develop high cell density cultivation methods for B. subtilis based on exponential fed-batch fermentation. Preliminary, the effects of different carbon sources such as glucose, sucrose, starch hydrolysate and sago starch on growth performance of B. subtilis was investigated using batch fermentation technique. Subsequently, the effects of different initial glucose concentrations, which is the preferred carbon source for growth of this bacterium, was studied in batch fermentation. The experiment was designed to find the repression effect of glucose on growth of B. subtilis. The culture pH condition for growth of B. subtilis was also optimized using different pH control strategies. The exponential fed-batch fermentation aimed at production of high cell density of B. subtilis was developed using kinetic parameter values and information generated from the preliminary batch fermentation data. Fed-batch fermentations were carried out at different required specific growth rates and their effects on final cell concentration attained, yield and productivity were analyzed. The performance of fed-batch fermentation with controlled glucose concentration at certain level in the culture for improvement of B. subtilis cultivation was also investigated. Among the different carbon sources investigated, the highest growth was obtained when glucose was used as the carbon source. B. subtilis was also capable to grow in sucrose and sago starch, indicating that B. subtilis was able to secrete invertase and amylolytic enzymes. Final cell concentration obtained in cultivation using glucose, starch hydrolysate, sucrose and sago starch all at a concentration of 20 g/L was 15.66 g/L, 9.71 g/L, 9.16 g/L and 5.72 g/L, respectively. This gave the cell yield (Yx/s) of 1.08, 0.85, 0.50 and 0.32 g/g based on glucose, starch hydrolysate, sucrose and sago starch consumed, respectively. Initial glucose concentration was found to have inhibitory effect on growth of B. subtilis. Growth increased with increasing initial glucose concentration from 15 to 20 g/L. A slight reduction in growth was observed at 35 g/L glucose and a drastic reduction on growth was observed at high glucose concentration (50 g/L). Growth of B. subtilis was also greatly influenced by the culture pH. The highest growth, in term of viable cell, was obtained in cultivation where the culture pH was controlled at 7 throughout the process. Reduction in growth was observed when the culture pH was controlled at lower and higher than pH 7. In exponential fed-batch fermentation with controlled specific growth rate, higher final viable cell (1.93 x 1010 cfu/mL) was obtained in fermentation where was controlled at 0.30 h-1 as compared to cultivation when was controlled at 0.20 h-1, where final viable cell obtained was only 3.52 x 109 cfu/mL. Further improvement of the cultivation performance was obtained in fed-batch fermentation with controlled glucose level in the culture throughout the process. The highest final viable cell at 3.50 x 1010 cfu/mL was obtained when glucose in the culture was maintained at the very low level of 0.20 g/L. Reduction in growth was observed when glucose in the culture was maintained at high levels, above 1 g/L, suggesting that glucose has repression effect on growth of B. subtilis. As a conclusion, among the techniques tested in this study, the best fermentation technique to achieve high cell density cultivation of B. subtilis was fed-batch fermentation where glucose in the culture was maintained at very low level (0.20 g/L), while the culture pH was maintained at 7 throughout the process. Using this cultivation technique, improvement in term of final viable cell was increased up to 324% compared to batch fermentation. This method of cultivation may be used for industrial application to produce large quantity of B. subtilis for subsequent use as a probiotic in the aquaculture industry, especially in controlling the water quality of the shrimp pond.
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