| DHA (docosahexaenoic acid) is a kind of polyunsaturated fatty acids (PUFAs) which has important physiological functions and high economic value. It is essential in promoting the development of brain and eyes of infants and it can also decrease blood lipid, choke back thrombosis, reject self-immune reactions and inhibit tumour for the old. Therefore DHA has been widely used in producing infants' food, health food and assistant therapeutic dose, etc. Thus more and more attention has been payed on the preparation process of DHA. Nowadays, DHA is mainly extracted from deep sea fish oil. Due to the lack of fish oil, the low producion of DHA from fish oil and the high cost, DHA can not be widely applied in the food industry and the pharmaceutical industry. What is worse, the polution of the sea is deteriorating day after day, DHA extracted from fish oil is being doubted of safety problems. As a result, more and more people are focusing their eyes on how to find a new source for DHA, which should not only be stable and secure but also with low cost.DHA production by microorganism fermentation has lots of advantages compared to fish oil. Schizochytrium limacinum SR21 is a promising microorganism in producing DHA as it has high content of DHA and the composement of PUFAs in SR21 is simple. In this dissertation, the extraction process of lipid, the transmethylation method of DHA and the culture conditions of Schizochytrium limacinum SR21 in shake flask were studied and optimized and then the original strain was mutagenized under UV and a DHA high-yield strain was found.Firstly, the extraction process of lipid and transmethylation method of DHA were studied. After 5 days-cultivation, cells were dried under the temperature of 120 ℃ for 3 hours after they were centrifuged. Chloroform :methaol=2:1 were used to extract lipids from the dry cells, after they were disposed by hydrochloric acid for 40min at the temperature of 65℃. This method is not only fast but also efficient. Before DHA was detected by gas chromatography (GC), it needed to be transmethylated. The dosage of KOH-MeOH (0.8M) is quite important in the transmethylation process. The most suitable amount would be 0.0707ml per mg lipid. The parameters of GCwere determined finally.Secondly, the cultural conditions of producing DHA with Schizochytrium limacinum SR21 in shake flask were investigated. First of all, the medium components such as carbon source, nitrogen source and metallic ions were studied. The concentrations of glucose, yeast extract and peptone have positive effect on dry cell weight.Dry cell weight increased with K+ in the medium, but lipid content decreased. After that, other factors including inoculum age, inoculum size, liquid volume, initial pH value and temperature were ulteriorly optimized and determined. Enventually, Uniform Design was used to optimize DHA yield. According to the results of the designed experiments, the recursive equation gave us an opitimized medium:Glucose 145g·L-1,Yeast extract 2.6g·L-1,Peptone 5.2g·L-1, K+ 10mM·L-1 in half the concentration of sea wa-ter,phosphate buffer.The optimum culture conditions would be: initial pH 6.8,inoculum age 24h, inoculum size 4%, culture time 5days, incubated at the temperature of 25℃ in the first three days, 20℃ in the remaining two days. Under this condition, dry cell weight achieved 55.64g/L, DHA yield 12.61g/L. DHA yield was increased by more than two times compared to pre-optimization conditions.Finally, the original strain was mutagenized under UV in order to get a strain which could grow at a normal temperature and also with higher DHA content. The mutagenized strains were screened with the designed screening model. A high-yield strain was screened out, whose DHA yield increased by 26.73%.
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