| The research and development of microalgae high-density culture technology is one of the important research contents in microalgae biotechnology field.Mixotrophy was considered as an ideal mode for high-density culture of microalgae and biosynthesis of high value-added metabolites(such as pigments,oils and proteins).However,the understanding of the growth mechanism of mixotrophy was not yet comprehensive.This paper chose C.vulgaris 31 as experiment material,on the basis of studying the physiological,biochemical and cellular components of C.vulgaris 31,the growth dynamics,biochemical components,cellular structure,internal metabolic network changes and related metabolic pathways of C.vulgaris 31 were analyzed by using flow cytometry,electron microscopy and metabonomics.The main research results are as follows:1.Physiological and biochemical regulation mechanism of C.vulgaris 31 under different trophic modes Compared with photoautotrophy,the addition of carbon source can effectively increase the biomass concentration,specific growth rate,and yield of algal cells,among which glucose was the best.When C.vulgaris 31 was cultured under photoautotrophy,mixotrophy and heterotrophy,the biomass concentration,specific growth rate and yield of C.vulgaris 31 were all mixotrophy > heterotrophy> photoautotrophy,and the growth kinetic parameters increased with the increase of glucose concentration.In addition,the increase of carbon source inhibited the synthesis of photosynthetic pigments and proteins,while promoted the synthesis of lipid and carbohydrates.2.Changes in C.vulgaris 31 cell cycle and cell structure under different trophic modes The results showed that the addition of glucose inhibited the photosynthesis of C.vulgaris 31,but significantly promoted the growth and the accumulation of metabolites of C.vulgaris 31.At the same time,it was found that the growth was synchronized under 12 h light and 12 h dark culture conditions.The structure of chloroplasts was intacted in all culture modes,and does not affect the photosynthesis of cells.The mitochondria appeared that the structure was fuzzy,swollen,and the ridges disappeared.In addition,lipid droplets and starch granules increased significantly during the late phase of mixotrophy and heterotrophy.3.Metabolomics study of C.vulgaris 31 under different trophic modes Using metabolomics to analyze the different metabolites in C.vulgaris 31 under different trophic modes and study the content of these metabolites and the main metabolic pathways involved.According to the PCA model,it was found that changes in trophic modes had significant effects on small molecular sugars,organic acids and amino acids.The OPLS-DA model was used to analyze the metabolic profiles of C.vulgaris 31 cultured for 3 and 7 days,62 and 65 metabolic markers were identified,respectively.The results of the pathway showed that the glycolysis and pentose phosphoric acid pathway(PPP)and carbon fixation efficiency were promoted under mixotrophy and heterotrophy in the late phase,while the TCA cycle was down-regulated,amino acid biosynthesis was inhibited due to the lack of precursor compounds.Furthermore,C.vulgaris 31 under mixotrophy and heterotrophy can significantly promoted the synthesis of unsaturated fatty acids such as C18: 3,C18:1 and inhibited the synthesis of saturated fatty acids C14: 0,C16: 0 and C18: 0.Through the analysis of growth dynamics,biochemical components,cell structure and internal metabolic network changes of C.vulgaris 31 under photoautotrophy and mixotrophy and heterotrophy,it was found that the trophic modes had obvious effects on the growth and the rapid accumulation of carbohydrates and fatty acids in algal cells.Under mixotrophy and heterotrophy,the contents of carbohydrates and fatty acids were higher than photoautotrophy,while the contents of photosynthetic pigments and proteins were lower than photoautotrophy.This was mainly because late phase in mixotrophy and heterotrophy,TCA cycle made more acetyl-CoA into fatty acid synthesis pathway,the strengthening of the PPP approach provided more NADPH used to carbon assimilation and fatty acid synthesis for cells,promoted the synthesis of carbohydrates and fatty acids.However,the down-regulation of TCA cycle led to the deficiency of precursor compounds,which inhibited the synthesis of amino acids and thus reduced the protein content.This study clarified the response mechanism of C.vulgaris 31 to photoautotrophy,mixotrophy and heterotrophy in terms of growth dynamics,cell structure and cycle,cell biochemical components and metabolic pathways,providing an important theoretical basis for further understanding the mixotrophy growth mechanism and high density culture of C.vulgaris 31. |
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