Thriving For Higher Yield Of DHA Through Fermentation Technology In Thraustochytriidae Sp. PKU#Mn16

DHA is one of the indispensable nutrients for human health.Thraustochytrids are known by their ability to accumulate high PUFAs,which makes them a promising biological resource for the production of DHA.Thraustochytriidae sp.PKU#Mn16,isolated independently in our laboratory,was used in this study.Different carbon sources operated with fed-batch fermentation strategies were evaluated.Meanwhile,the mechanism of various carbon sources on DHA synthesis,as well as the influence of carbon source feeding on PKU#Mn16,were revealed through transcriptome analysis.Additionally,the environmental conditions of fermentation(temperature and dissolved oxygen)were analyzed,and the contents of three essential substances(Acetyl-Co A,NADPH,and ATP)for fatty acid synthesis under different environmental conditions were determined.Then a staged fermentation was designed,which significantly increased the yield of DHA,and then was successfully magnified in 50 L fermenter.The main results are as follows:(1)The biomass,TFA,and DHA yield of PKU#Mn16 were analyzed within batch and fed-batch fermentation with seven different strategies.The results revealed that the optimal condition was fed-batch fermentation with mixed carbon sources,where higher biomass of 52.22 g/L,higher TFA,and DHA yields of 23.70 g/L,and 9.67 g/L were respectively achieved when PKU#Mn16 was cultured for 96 h.(2)The mechanism of DHA synthesis was revealed by the transcriptome analysis.The results showed that the fermentation group with glucose as a carbon source upregulated some genes of the FAS pathway,while the fermentation group with glycerol as a carbon source upregulated the genes of the PKS pathway.The difference between mixed carbon source fermentation regulated the genes of these two pathways and significantly increased the DHA yield.In addition,some genes related to cell division,DNA replication,phagosome,carbon metabolism,fatty acid synthesis,and ?-oxidation changed significantly in the process of mixed carbon source feeding.(3)The environmental conditions of fermentation were discussed in detail.The results showed that environmental conditions had noticeable effects on biomass,TFA,and DHA accumulation.Under higher temperature and DO,biomass and TFA accumulation were faster,but with lower DHA content.In contrast,when low temperature and DO were applied,the content of DHA increased significantly,although the biomass level decreased,which indicated that there were significant differences in the environmental conditions for biomass growth and DHA accumulation.Based on the detailed analysis of the three major substances related to fatty acid synthesis,it was found that the contents of Acetyl-Co A,NADPH and ATP were significantly higher as compared to other environmental conditions,more likely under low temperature and low DO,indicating that the environmental conditions might influence the contents of Acetyl-Co A,NADPH and ATP,and then affect the accumulation of fatty acids and DHA.(4)After clarifying the nutritional and environmental conditions of the fermentation,a staged fermentation according to the idea of promoting the rapid proliferation of cells in the early stage,and the synthesis of DHA in the later stage was designed.The fermentation was carried out by applying a high temperature,and high DO in the early stage,then a low temperature and low DO in the later stage.The results showed that under this fermentation condition,biomass was 66.92 g/L,while TFA and DHA yields achieved 30.30 g/L and 14.69 g/L respectively,at 96 h,in which the DHA yield increased by 56% compared with the results obtained with the standard fed batch fermentation.(5)The optimal culture medium,carbon source feeding process,and staged fermentation process were applied to produce DHA in the 50 L fermenter.The results showed that under these conditions,biomass and DHA of PKU#Mn16 were 67.43 g/L and 14.31 g/L respectively,which had no significant difference from the results obtained in 5 L fermenter.The results indicated that the strain isolated in our laboratory had the potential industrial production of DHA.To sum up,the results revealed that the adapted carbon source feeding strategy could significantly increase the DHA yield,and its metabolic mechanism was discussed deeply through the transcriptome analysis.The nutritional as well as the environmental conditions(temperature and DO)were discussed in detail.Finally,the staged fermentation method was designed,which significantly increased the DHA yield,and was then successfully applied in 50 L fermenter.This method laid an essential foundation for the industrialization development of Thraustochytrids and provided some new ideas for the development of the microbial oil industry.