Comparison Of Cephalosporin C Fermentation Performance Under Different Substrates Feeding Strategies

Cephalosporin C is one kind of semi-synthetic cephalosporin antibiotic intermediates which can be synthesized by Cephalosporium mold fermentation. Cephalosprins are widely used in clinical and the market demands are increasing because of its advantages of high efficiency, low toxicity, broad antimicrobial spectrum and high stability onβ-lactamase. In this dissertation, various substrates feeding strategies for cephalosporin C (CPC) efficient production by Cephalosporins acremonium were studied and conducted in a 7 L fermentor. The effects of different C and N sources feeding manners on CPC fermentation performance were explored and compared, while metabolic analysis under different soybean feeding strategies during the main CPC synthesis phase was conducted to find out the reasons of enhanced CPC fermentation performance achieved by the optimal feeding method. The main content and conclusions of the dissertation are summarized as follows:(1) A novel ammonium sulfate feeding strategy in couple with soybean oil addition was proposed. With this strategy, NH4+ concentration in fermentation broth could be stably maintained in a range of 3?6 g/L throughout fermentation period, simultaneously satisfying the demands on nitrogen and sulfur sources for cells growth and CPC synthesis, promoting Cephalosporins acremonium hyphal split, and creating the prerequisite for the subsequent CPC efficient production.(2) On the top of the proposed ammonium sulfate feeding strategy, performance of the subsequent CPC fermentations with different soybean oil feeding strategies, namely intermittent, constant rate and automatic DO-Stat feeding was compared. Feeding ammonium sulfate in couple with the soybean oil feeding in a manner of DO-Stat+oxygen-enriched air aeration at late fermentation phase could control carbon source concentration and DO at adequate levels, ensuring efficient CPC synthesis in a way of high CPC concentration and low by-product accumulation. With this combinational feeding strategy, final CPC concentration and CPC yield could reach 35.77 g/L and 13.3%, respectively, the major by-product, de-acetoxycephalosporin (DAOC) and DAOC/CPC were only 0.178 g/L and 0.5%.(3) Metabolic analysis of CPC fermentation under different soybean feeding strategies during the main CPC synthesis phase was conducted. The results indicated that under the operation conditions of limiting soybean oil feeding by DO-Stat, more carbon fluxes were directed into the formation of CPC synthesis precursors and extracellular methionine (methionine in the medium) could be directly utilized to run the entire CPC biosynthesis in a more efficient way. More importantly, DO-Stat based soybean oil feeding strategies could adequately weakened TCA fluxes, leading to higher yields of both cells and CPC because of the reduced CO2 release in TCA cycle.(4) The optimal substrates feeding strategies were applied and tested in a 50 L fermentor on site of a pharmaceutical plant. The automatic substrates feedings were successfully activated in late stage of the fermentation and the resulting maximal CPC titer was equivalent with that using the existing experience-based feeding method. The feasibility and advantages of the proposed automatic feeding strategy were verified as expectation.