| Docosahexaenoic Acid(DHA)is one ofω-3-polyunsaturated fatty acids,plays an important role in the development of brain and eyes for infants,and it is beneficial for cardiovascular disease,and it also can improve body immunity.Conventional fish oils may not be suitable to meet the increasing demand for DHA owing to their limited supply,lower content of DHA and peculiar taste and odor.Thus the search for oleagiuous microorganisms rich in DHA as commercial sources is being more urgent.In this study,Strain WZ-3 was isolated from a mangrove,Wenzhou,China,and identified as Schizochytrium sp.by 18S rDNA analysis.DHA is accumulated in the cell lipid of Schizochytrium sp.,and EPA is almost not existed in it,so it is a good source for infant and pregnant women.The variables with the biggest effect on the growth,lipid and DHA accumulation are medium compositions and culture conditions.Through one-factor-at-a-time experiments,glucose and yeast extract were found to be most suitable carbon and nitrogen source for DHA production by Schizochytrium sp.,respectively.The optimized operational culture conditions by shaken flasks were temperature 25℃,culture volume 100 ml/500 ml,initial pH 6.0,inoculating age 48h, inoculating quantity 4%-6%and in seawater at a half salt concentration.Statistical analysis of the effect of carbon and mix-nitrogen source on DHA production by employing statistical optimization methods such as Box-Behnken design and surface response methodology indicated that the optimized medium should contain(g·L-1)glucose 126,yeast extract 10,corn steep liquor 2,soy peptone 5 and the seawater at a half salt concentration.By using the optimized medium,dry cell mass of 42.9 g·L-1,total fatty acid of 34.1 g·L-1and DHA accumulation of 13.8 g·L-1could be achieved after 120 h of incubation,respectively.The effect of Vitamin B Complex on the oil accumulation of Schizochytrium sp.were considered in synthetical medium,when vitamin B2 was added at the beginning of fermentation, lipid accumulation was decreased,incading vitamin B2 adverse to lipid accumulation.Vitamin B1, Vitamin B12,biotin,lipoic acid and folic acid were favorable for growth,and these helpful vitamin were optimized consist of Vitamin B15 mg·L-1,Vitamin B1214 mg·L-1,biotin 6 mg·L-1, lipoic acid 30 mg·L-1and folic acid 40 mg·L-1by orthogonal experimental design.Amino acids act as nitrogen source in synthetical medium.Using single factor absence design,10 kinds of amino acids were selected out of 20 elementary amino acids as indispensable amino source for Schizochytrium sp.produce DHA.Then each necessary amino acid was analyzed by HPLC and calculated wastage during fermentation course.Uniform Design and Multiple stepwise linear regression analysis were employed to optimize these amino acids.The optimal necessary amino acids were contained:alanine 2.0 g·L-1,methionine 1.2 g·L-1,cysteine 0.2 g·L-1,lysine 0.3 g·L-1,histidine 1.3 g·L-1,glutamic acid 1.4 g·L-1,glutamine 1.4 g·L-1, isoleucine 1.3 g·L-1,threonine 1.0 g·L-1and tryptophan 1.6 g·L-1,respectively.
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