Screening And Breeding Of A High-yield Docosahexaenoic Acid Strain Of Crypthecodinium Cohnii And Study On Fermentative Character

The fermentation of docosahexaenoic acid by Crypthecodinium cohnii hassuch advantages as short fermentation period and high content of product, but thedisadvantages of low output also exist. Therefore, it is meaningful to screeneminent strain. In this dissertation, the best method for the storage ofCrypthecodinium cohnii, mutation breeding of high-yield DHA strains,investigation and optimization of the culture conditions of the screened mutationsand fed-batch research in bio-raector were carried out.Crypthecodinium cohnii was preserved with low-temperature refrigeration andlow temperature dark, respectively. Using algae survival rate, re-culture ability, thefermentation ability as evaluation index, the best method for the storage ofCrypthecodinium cohnii was low temperature dark. After90days,82.4%algae wassurvival with strong re-culture ability and stable genetic traits.Using Crypthecodinium cohnii ATCC30556as original strain, the mutagenesisby nitrosoguanidine was carried on in order to enhance the docosahexaenoic acidyield. The mutant LS1057was selected through high temperature（30℃） asinhibitors with the result of the docosahexaenoic acid content in total fatty acidsincreasing from25.78%to32.02%.The fermentation conditions for LS1057were optimized via single-factor test.The optimal fermentation conditions for LS1057were to grow the strain in media atpH6.5at26℃with a rotate speed at150r/min for216hours. Under this optimizedconditions, LS1057could achieve biomass at14.06g/L, total lipid at3.14g/L anddocosahexaenoic acid at1.057g/L, which were21.31%,69.73%,121.59%higher,respectively, than its parent strain.Plackett-Burman design was used to evaluate the effects of mediumcomponents on the yield of docosahexaenoic acid by Crypthecodinium cohniiLS1057. The result showed that, glucose, yeast extract and KH₂PO₄were the mostimportant factors that influence the yield of docosahexaenoic acid. Based on theexperimental results, the path of steepest ascent was undertaken to approach theoptimal region of these factors, central composite design and response surfaceanalysis were subsequently employed for further optimization. The optimal mediumfor Crypthecodinium cohnii LS1057was obtained, which was composed of glucose79.76g/L, yeast extract14g/L, KH₂PO₄0.5g/L, sea salt2.0g/L, MgSO₄7H₂O 5.0g/L, KNO₃8.0g/L, FeSO₄7H₂O0.2g/L and solution M(VB10.6g/L,VB₁₂0.1mg/L)1%（v/v）. Under the optimum conditions, the maximum yield ofdocosahexaenoic acid of1.833g/L in theory and1.811g/L in the experiment, withan increase of71.33%compared to the original medium components.The fed-batch fermentation was studied in fermentation tank, to investigate thefermentation ability of LS1057. In the end, the dry cell weight increased to28.13g/L, the content of total lipid was23.49%, and DHA yield2.112g/L, whichenhanced by16.63%than in shake flask. And the content of docosahexenoic acidwas no significant difference compared with fermentation in shake flask. Theexperimental results show that mutations LS1057has good fermentationcharacteristics, and the effect of nitrosoguanidine on Crypthecodinium cohnii andfeasibility of screening method were confirmed furtherly.