The Influence Mechanism Of Oxygen On DHA Production By Schizochytrium Sp. With High Cell Density Cultivation On Glycerol

DHA is an important omega-3polyunsaturated fatty acid to the health of human beings,especially important to the visual and neural development of infants and children. Since thecost of algal DHA has been very expensive, the application of which has been limited tohigh–end products such as supplementary food and infant formula. Schizochytrium sp. is thebest strain for DHA production, for which the conventional carbon source is glucose. In recentyears with the rapid development of biodiesel industry, rep lacing glucose with biodieselbyproduct-crude glycerol to reduce the DHA production cost is becoming the research focus.The understanding of DHA production by Schizochytrium sp. on glycerol were limited to theobservation of fermentation phenomena since the metabolic mechanisms of DHA biosynthesishas remained unknown, therefore the fermentation strategies could not be rationally designedbased on the molecular mechanism. Dissolved oxygen (DO) is usually the key parameter forDHA production by Schizochytrium sp., however, DO-limitation was commonly encounteredin high cell density cultivation(HCDC). Therefore it is imperative to find more suitableoxygen control technologies.During the batch culture of Schizochytrium sp. on glycerol to produce DHA, the keyenzymes involved in the primary metabolic steps of glycerol assimilation were in vitrocharacterized. The results indicated that the main pathway of glycerol assimilation inSchizochytrium sp. is suggested via phosphorylative pathway not dihydroxyaceto ne pathway.Secondly, phosphorylated glycerol is converted to dihydroxyacetone phosphate byFAD+-dependent glycerol-3-phosphate dehydrogenase(FAD+-G-3-PDH) not NAD+-dependentglycerol-3-phosphate dehydrogenase (NAD+-G-3-PDH), which was similar to that ofSaccaromyces cerevisiae but distinct from oleaginous yeast Yarrowia lipolytica.Three growth stages including cell growth, lipid accumulation and lipid turnover wereidentified during the batch culture of Schizochytrium sp. on glycerol to produce DHA, themetabolic fluxes in each stage presented obvious shifts. The lipid accumulation mode inSchizochytrium sp. did not completely depend on the nitrogen-trigger mechanism but wasclosely a growth-associated process. Nitrogen exhaustion enhanced the extent of lipidaccumulation. When the carbon concentration in the culture medium could not satisfy thedemand of cells, lipid turnover was induced. Glycerol kinase(GK), ATP citrate lyase (ACL)and NAD+-ICDH were all involved this process. In lipid turnover stage, FAS products(C14:0,C16:0) including odd fatty acids were preferentially degraded while PKS products(DHA,DPA) even could be newly biosynthesized with small amounts, which resulted in thesignificant increase of the percentage of PKS product in total fatty acids(TFA). Therefore,lipid turnover could be the―DHAenrichment‖process. Compared with lipid production, theaccumulation of DHA was more correlated with the production of lipid-free biomass(Xf).Compared with the carbon concentration, the carbon to nitrogen ratio, temperature andsalinity level, the effect of oxygen on DHA biosynthesis was more significant. The study ofmetabolic enzymes and genes transcriptional level showed that the influence of oxygen oncell growth, lipid accumulation and DHA biosynthesis presented different function mechanisms. In cell growth stage, when the volumetric oxygen transfer coefficient (kLa) wasmaintained at high level meaning high oxygen supply level which caused that the activities ofthe primary metabolic enzymes of glycerol assimilation were accordingly high and the TCAwas very active, which was beneficial to the cell growth; in lipid accumulation stage, kLaelevation reduced the activity of NAD+-ICDH responsible for the regulation of carbon fluxes,which could channel more carbon flux from TCA to lipid biosynthesis. Although the activityof ACL, malic enzyme (ME) and NAD+-ICDH had some correlation with the regulation oflipid accumulation, the activity changes of these enzymes with kLa shift could not explain thechanges of DHA content in TFA. The analysis results of the real-time quantitative PCRshowed that DHA content in TFA was transcriptionally co-regulated by FAS and PKS genes.Detailedly, when kLa was enhanced, the transcriptional level of FAS gene had moreadvantages than PKS system which decreased the DHA content in total fatty acids(TFA);onthe contrary,when kLa was declined, the transcriptional level of PKS genes showed morepredominant than FAS system which increased the DHA conent in TFA.During the high cell density cultivation of Schizochytrium sp. on glycerol, the culturesexhibited shear-shining behavior which presented good fit to Herschel-Bulkey. With theincrease of oxygen supply level, the extracellular polysaccharides (EPS) increased resulting inthe rising of the viscosity of broth, thus the transport resistance of oxygen was raised leadingto the continuous decrease of kLa, so DO-limitation commonly happened. Since oxygenuptake rate (OUR) could reflect the real oxygen uptake level for cells more accurately,whichis suggested to characterize oxygen supply level for the scale-up production ofSchizochytrium sp. with high cell density.After the optimization of culture conditions, applying constantly high oxygen supplystrategy combined with continuous feed-back control strategy to cultivate Schizochytrium sp.,biomass density at151.40g/L, DHA concentration at28.93g/L, DHA productivity at301mg/L/h were finally achieved. Compared with the optimal result as previously reported,biomass density increased10.5%（137g/L）, DHA concentration increased67.61%(17.26g/L)and DHA productivity increased109.03%(144mg/L/h).Based on the investigations of the metabolic pathway of glycerol assimilation, the keyenzymes, the genes responsible to fatty acid biosynthesis in the culture of Schizochytrium sp.on glycerol to produce DHA, the present study elucidated the metabolic mechanism of cellgrowth, lipid biosynthesis and lipid turnover with glycerol involvement, and disclosed theinfluence mechanism of oxygen on FAS system and PKS system regulating DHA content.Due to DO-limitation phenomenon presented in HCDC, using OUR to reflect the real oxygenuptake level for cells, the biomass density, DHA concentration and DHA productivity weresubsequently significantly increased. The present study provided theoretical guidance forindustrial DHA production by Schizochytrium sp. on crude glycerol, which has a significanteconomic and social value.