| Bacitracin is a new type of ideal broad-spectrum antibiotics, and is produced by Bacillus licheniformis and some strains of Bacillus subtilis. It has the qualities of wide antimicrobial spectrum, discharge faster, less livestock absorption, good safety assessment, no resistance, and so on. Bacitracin is one of the world’s biggest selling forage antibiotics. This paper studies the metabolic regulation of the Bacillus licheniformis producing bacitracin. The result is as follows:(1) Optimization additions of ornithine (Orn) and KNO3 for bacitracin production by Bacillus licheniformis were investigated in this work. Addition of Orn (0.04 g/L), an insufficient precursor, at 16 h and KNO3 (0.5 g/L), a substrate of nitrate-reducing system, in production medium was conducted, respectively. The yield was enhanced by 5.8% and 3.7% compared with the control, respectively, in shake-flasks cultivation. The comprehensive effects of KNO3 and Orn were conducted further by a two-level central composite design, and a maximum yield (821.81 U/mL) with 514.39 mg/L and 45.35 mg/L additions corresponding to KNO3 and Orn, respectively, was predicted. This predicated yield was then verified experimentally in a 10 L fermentor, achieving a 24.1% improvement of cell growth and 10.8% increased bacitracin production (825 U/mL), compared to those of the control.(2) In the research of batch cultures of Bacillus licheniformis for bacitracin production, the phenomena that a low level of dissolved oxygen (DO) at the early phage of cultivation, cell lysis and secondary growth were observed. H2O2, a kind of carrying oxygen agent, can enhance the DO in batch culture and not influence the product purification process, therefore, the effects of H2O2 addition on the bacitracin production by Bacillus licheniformis in a 10 L bioreactor were investigated. H2O2 addition with a rate of 3 mmol/(L-h) from 24 h to 34 h facilitated the cell growth, and the biomass reached the max value (73.5×109 CFU/mL) at 34 h, which was 6.5% higher than that of the control. The time of cell lysis was delayed 8 h, and the lowest cell biomass after cell lysis was 62×109 CFU/mL at 42 h, which was enhanced by 77% compared with the control. The volatile fatty acid (VFA) content reached the max value (12.8 g/L) during 24 h-34 h, which was 11% lower than the control. The glucose consumption rate was 0.36 g/(L-h) that was 3% higher than the control. The bacitarcin production rate was 44.9 U/(mL-h) that was 12% higher than the control (40.1 U/(mL-h)) during 24 h-34 h. The bacitracin production was 1021 U/mL that was 9.9% higher than the control. The results suggested the H2O2 addition promoted cell growth and increased bacitracin biosynthesis efficiency.(3) In the research of batch cultures of Bacillus licheniformis for bacitracin production, the phenomena that a low level of dissolved oxygen (DO) at 3 h of cultivation, cell lysis at 26 h of cultivation and the degree reached above 60% were observed. Time course of the extracellular metabolites of propionic acid, butyrate, acetoin and 2,3-butanediol were dectected. Under the restricted condition of oxygen, the primary cause of acid stress and cell lysis were the pressure of oxidation of NADH. After added MnCl2 of 0.03 g/L, obviously, the carbon flux from pyruvate to propionic acid and butyrate reduced, and that to acetoin and 2,3-butanediol for coenzyme of NADH enhanced. Meanwhile, the contradiction of NADH/NAD+ imbalance and acid stress were alleviated. The results suggested that the carbon metabolic flux shife was realized, which delayed the time of cell lysis and lessen the degree of cell lysis. The bacitracin production was enhanced by 11.6% in the end. |
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