Breeding Of Schizochytrium Sp. For Docosahexenoic Acid Production And Optimization Of Its Fermentation Conditions

Docosahexenoic acid (DHA) is a neutraceutical ω-3polyunsaturated fatty acid withsignificant biological functions, such as, enhancing the growth of infant brains, maintainingproper visual sight and precaution of heart diseases, hypertension, arteriosclerosis andthrombosis, etc. Based on its various functions, DHA has been adopted for a long time in thepharmaceutical and neutraceutical products and in infant formula. The decreasing traditionalsource limits the further application of DHA, which could be overcomed by microbiologytechniques. The strain Schizochytrium sp. SR21is cultured in our laboratory to produceunsaturated fatty acid. This starin has a potential to be biologically restructioned to producehigh content of DHA. According to the above reasons, the strain breeding of SR21andoptimization of fermentation process are carried out in this research. The main works arecarried out as follows:First, optimizing the culture conditions of Schizochytrium sp. SR21was studied at250mL shake flask level. Many factors influencing the cell growth and DHA synthsis in thisstrain were investgated, including carbon and nitrogen sources, inorganic salt, incubationtemperature, vitamin, inculum size and age. The optimal conditions for producing DHA werelisted below (w/v): glucose9%, corn steep0.6%and yeast extract0.9%, artificial sea water50%, incubation temperature25℃, pH6.0, culture volume60mL, inculum age30h andincubation size5%(v/v). At the end of fermentation, Schizochytrium sp. SR21canaccumulate64.0%of its weight as fatty acids and has DHA content up to30.3%of total fattyacids.Second, an efficient screening method based on metabolism pathway analysis andstatistics was established. Iodoacetic acid, malonic acid and low temperature were chosen asgrowing inhibitors in the method. Diethyl sulfate (DES) treatment, ultraviolet radiation (UV)and DES-UV complex mutagnesis were applied to induce mutation resulting in enhancing theactivities of key enzymes for DHA production. Primary screening on selective medium platesbased on the colony size was carried out and then the biggest single colony was inoculatedinto the test tubes containing culture medium. After72h cultivation, the total amount of fattyacid and their compositions were analyzed by gas chromatography. Finally, three promisingstrains which have the ability to accumulate high ratio of DHA in total fatty acids wereobtained. The productivity of these strains was thereafter checked out by shake-flaskcultivation. It was shown that mutant strain Schizochytrium sp. D-U-1accumulated43.7%(w/w) of DHA in total lipid, which showed45.3%improvement than the parent strain. Theproductivity of another mutant strain Schizochytrium sp. D-U-3was125mg/L/h, which was61.9%higher than that of the wild strain. The results indicate that this combined mutationmethod is effective for increasing yield and productivity of the DHA in Schizochytriumsp.SR21.Finally, the selected three strains mentioned above were cultured in a7L fermentor toproduce DHA in two different modes, batch and fed batch, respectively. In batch fermentation,the biomass of Schizochytrium sp. D-U-1reached35.4g/L, the lipid conent was64.9%and the DHA content was42.7%of total fatty acids. Schizochytrium sp. D-U-3showed the bestDHA productivity. In fed batch fermentation, the initial culture with9%glucose was added300mL saturated glucose solution at48h. When the cultivation stopped, the biomass ofSchizochytrium sp. D-U-1reached61.5g/L, and the DHA yield was16.8g/L, which was63.3%higher than that of the parent strain.