Optimization Of DHA Production By Schizochytrium Sp. And The Studies On Feeding Strategy In Fermentation

Docosahexaenoic acid has the biological functions in anti-hyperlipidemia, anti-leukocyte,anti-tumor etc. DHA production by microorganism fermentation has lots of advantages.Schizochytrium sp. is a promising microorganism in producing DHA as it has high content ofDHA, stable product quality, less peculiar taste and odor. Study the production of DHA bySchizochytrium sp. fermentation has great theoretical significance and potential applicationvalue.In this paper, the extracting methods of intracellular oil in S. limacinum JN168werestudied. We compared the oil extraction capacity of Soxhlet extraction, acid-heatingextraction, and ultrasonic assisted extraction. We found that the best method is acid-heatingextraction, and the extraction solvent was chloroform: methanol=1:1. Then we studied theprocess of separating DHA from microbial lipids. DHA was separated and purified frommicrobial lipids by the combined method of urea inclusion reaction and reversed-phase highperformance liquid chromatography. At first, DHA was concentrated from free fatty acids by aurea inclusion reaction. The DHA content was increased from42.27%to80.44%, and thenDHA was purified by HPLC, The purity of DHA after two steps purification reached95.55%.In order to increase the yield of DHA, the medium components and culture condition forDHA production by S. limacinum JN168were optimized with single factor experiment,orthogonal design and uniform design. The optimum fermentation culture was glucose120g·L-1, yeast extract11g·L-1, ammonium acetate1.5g·L-1, MgSO4·7H2O4.6g·L-1, KCl0.5g·L-1, NaCl8g·L-1, CaCl2·2H2O3.5g·L-1. Appropriate culture conditions was liquid volume50mL/250mL, culture initial pH6.0, culture temperature25℃, rotate speed200r·min-1,fermentation period96h, and the DHA production reached11.12g·L-1.At last, we did a small scale experiment test in5L fermenter. The effects of intermissionfed-batch fermentation, constant velocity fed-batch at low speed, constant velocity fed-batchat middle speed, variable velocity fed-batch on DHA production were studied. In all of thefour fed-batch ways, variable velocity fed-batch achieved the highest DHA yield and DHAproductivity, the highest DHA yield and DHA productivity reached15.60g·L-1and130.00mg·L-1·h-1respectivily, which were39.04%and11.23%over the best results obtained bybatch fermentation. This way also can achieve the highest Yx/s and Yp/s, so the variablevelocity fed-batch was the best way for producing DHA.