Production of L-lactic acid from starch by recombinant Bacillus subtilis 1A304 (Φ105MU331)

xxxi, 252 leaves : ill. (some col.) ; 30 cm.PolyU Library Call No.: [THS] LG51 .H577M ABCT 2010 WongThe interest in the production of lactic acid has significantly increased recently. Besides the increasing demand, it is also due to the prospects of using environmental friendly, renewable resources instead of petrochemicals as feedstocks. Lactic acid is widely used in the food, cosmetic, pharmaceutical and chemical industries. One of the most promising applications of lactic acid is its use for biodegradable and biocompatible lactate polymers, such as polylactic acid. The worldwide demand of lactic acid is about 130,000 150,000 metric tones per year and it may increase to 500,000 metric tones per year by 2010. An UV-induced mutant strain of recombinant Bacillus subtilis 1A304 (Φ105MU331) was used for the production of lactic acid. Shake flask experiments were performed to characterize the growth patterns and lactic acid production by recombinant Bacillus subtilis using glucose as the carbon source. Optimization of culture medium was conducted. The effect of complex nitrogen sources on the growth and lactic acid production of recombinant Bacillus subtilis was studied. 2% yeast extract and 3% martone was chosen for further studies. The effect of cell density and the oxygen transfer rate on the growth and lactic acid production of recombinant Bacillus subtilis was studied. It was found that the lactic acid concentration increased with increasing cell density and culture volumes in shake flasks (lower oxygen transfer). The optimal pH for the production of lactic acid was at pH 7.5. Fed-Batch fermentation experiments were conducted to establish optimum operating conditions for the production of lactic acid. Two kinds of fermentation strategies were compared. One was two stage fermentation and the other one was one stage fermentation. It was shown that 40 g/L lactic acid was produced in 53.5 h in one stage fermentation. The effect of stir rate and air flow was studied. The optimum condition was achieved in 50 rpm and 2 vvm. Inorganic nitrogen was found to have some inhibitory effect on the production of lactic acid. The lactic acid production was further improved by eliminating the inorganic nitrogen content in the medium. It was found that 79 g/L and 93 g/L lactic acid was produced in 25 h and 51 h respectively. Cultivation temperature was another important factor for lactic acid production. Higher lactic acid production was obtained at 40°C. The effect of different yeast extract concentrations on lactic acid production has been investigated. When 8% yeast extract was used in the medium, the lactic acid concentration was 125.6 g/L, 158.6 g/L and 170 g/L in 24 h, 51 h and 77 h respectively. The maximal lactic acid productivity was 16 g/L/h in 6 h. The lactic acid yield was about 89%.Starch was also used as the substrate for lactic acid production. This recombinant Bacillus subtilis was able to produce alpha amylase under heat induction. There was nearly no lactic acid produced when liquefied starch was used as the carbon source for lactic acid production by recombinant Bacillus subtilis. Thus, saccharification of liquefied starch to glucose was needed. Saccharification process was optimized by using different temperatures and forms of Aspergillus niger (mycelium, pellet and supernatant of the culture). The effect of starch concentration on saccharification was also studied. Simultaneous saccharification and fermentation (SSF) of starch to lactic acid was conducted using the recombinant Bacillus subtilis and Aspergillus niger. The effect of temperature and starch concentration on SSF was studied. The result shown that 84.8 g/L and 136.9 g/L lactic acid was produced in 24 h and 65 h respectively. Lactic acid productivity was 4.38 g/L in 13 h. The optical purity of L-lactic acid was greater than 96%. The results suggested that starch can be used as the carbon source to produce lactic acid by a mix culture of the recombinant Bacillus subtilis and Aspergillus niger.Department of Applied Biology and Chemical TechnologyM.Phil., Dept. of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, 201