Study On The Process Control And Protein Enrichment Mechanism Of Chlorella Vulgaris Heterotrophic Culture In Broken Rice Hydrolysate

C.vulgaris contains high quality protein,pigment and polyunsaturated fatty acid,which has high application value in food,feed,medicine and energy.And C.vulgaris can be heterotrophic in fermenters and does not require any arable land,which makes it a potential source of protein.However,heterotrophic culture requires a large amount of glucose as an organic carbon source,which increases the production cost and makes it difficult to popularize it commercially.In order to reduce the cost of organic carbon sources,this study established a new resource-saving technology of heterotrophic culture of C.vulgaris with broken rice hydrolysate（BRH）instead of glucose.The effects of main components of BRH on C.vulgaris growth were studied.The synergistic effect of BRH and sodium acetate on the growth of C.vulgaris was investigated.The effects of initial C/N ratio on the growth,cell morphology and nutrient composition of C.vulgaris were clarified.The regulation mechanism of amino acid and protein anabolism by C/N ratio was revealed.The aim of high-cell-density culture of C.vulgaris with BRH was realized by continuous fed batch culture strategy,which provided a strong basis for large-scale industrial production.The main results are as follows:（1）The effects of BRH components on the heterotrophic growth of C.vulgaris were studied.The results showed that BRH contains a small amount of NH₄⁺.When NO₃^-and NH₄⁺coexist in the medium,C.vulgaris would preferentially absorb NH₄⁺,and release the same amount of H⁺,resulting in rapid acidification of the medium and inhibiting the growth of C.vulgaris.Under the condition of stable p H,the maximum biomass production of C.vulgaris grew in BRH was 33.44%higher than that in glucose.When the p H of the medium is adjusted intermittently with Na OH and Na₂CO₃,the dosage of p H stabilizer can be reduced,and the biomass productivity of C.vulgaris can reach 1.00 g/L/d and 0.95 g/L/d,respectively.（2）In order to further improve the specific growth rate of heterotrophic C.vulgaris,the promotion effect of BRH combined with sodium acetate as an organic carbon source on C.vulgaris growth was explored.The results showed that the p H of the medium could be kept stable under the combined action of CH₃COO^-,NH₄⁺and NO₃^-.Under the induction of acetate,the activity of isocitrate lyase increased by 3 times,which promoted the glyoxylic acid cycle and citric acid cycle,provided more energy and metabolic precursor substances for cell metabolism,and thus accelerated the growth rate of C.vulgaris.When BRH combined with 0.4 g/L sodium acetate as organic carbon source,the specific growth rate of C.vulgaris could reach 1.99 day^-1,and the biomass production（5.04 g/L）and biomass productivity（1.65 g/L/d）of C.vulgaris were 1.56 and 1.81 times that of glucose group,respectively.These results indicated that BRH combined with sodium acetate as an organic carbon source could effectively promote the heterotrophic metabolism of C.vulgaris and increase its specific growth rate.（3）In order to improve protein production,the effects of C/N ratio on protein accumulation in C.vulgaris cells were explored.The results showed that the initial C/N ratio was the key regulatory factor of protein anabolism in C.vulgaris.Excess nitrogen sources can promote the synthesis of glutamic acid in cells,which in turn directly or indirectly provides precursors for the synthesis of other amino acids and proteins,thus promoting the accumulation of proteins in cells.When C/N was 12:1,the biomass,protein content and protein productivity were higher,which were 2.02 g/L/day,61.56%and 0.54 g/L/day,respectively.The nutritional value of the protein of C.vulgaris was analyzed.The content of essential amino acids in C.vulgaris was as high as 41.80%,which was equivalent to soybean.The content of lysine in C.vulgaris protein is higher than that in cereal protein,so it is suitable to be used as dietary supplement to improve the deficiency of lysine intake in vegetarians.The high ratio of leucine to isoleucine in C.vulgaris protein is beneficial to human health and muscle tissue recovery after exercise.（4）The untargeted metabolomics based on LC-MS was used to further investigate the regulatory mechanism of amino acid and protein anabolism in C.vulgaris cells by C/N ratio.The results showed that HN（low C/N ratio）and LN（high C/N ratio）had significant differences in the metabolites of C.vulgaris.These differential metabolites are associated with 58 metabolic pathways,of which four of the top 10 pathways are involved in amino acid synthesis and one is involved in amino acid transport.This indicated that HN group significantly promoted amino acid and protein anabolism.Under the condition of low C/N ratio,intracellular enrichment of citric acid promoted the TCA cycle,and further promoted the anabolic metabolism of glutamate,which further promoted the synthesis of other amino acids and protein accumulation.（5）In a 3 L fermenter,the continuous culture strategy was adopted for high-density culture of C.vulgaris to explore its commercial application prospects.The results showed that the growth rate of C.vulgaris was the fastest when the temperature was35℃,the p H value was 6.5 and the feeding rate was 0.2 L/day.In the fed batch experiment with sodium nitrate as nitrogen source,the growth of C.vulgaris was inhibited by the accumulation of sodium ions in the culture medium due to the unbalanced absorption of ions.When using ammonium acetate instead of sodium nitrate as nitrogen source,the mass enrichment of anions or cations could be avoided.The biomass concentration was up to 51.89 g/L,and the protein content of C.vulgaris was up to 59.36%.