| Candida glycerinogenes, a high yield of glycerol strain screened by our lab, has been successfully applied in industrial production. Recently, further study on this strain was greatly prevented due to the lack of systematic understanding of it on the physiological and biochemical characteristics, genetic background and metabolism. In this paper, the morphology and physiological & biochemical characteristics of C. glycerinogenes were comprehensively studied. Metabolic flow under different carbon sources condition during different fermentation periods were also analyzed. The chromosome DNA ploidy of C. glycerinogenes was firstly identified. All the work laid a foundation for the molecular modification and establishment of transformation platform.Through the analysis of the physiological and biochemical characteristics, we found that some features were the same to the existing appraisal results, this part proved that the C. glycerinogenes has stronge stability of genetic. Besides, it had other new characteristics: ammonium sulfate could be well used as its nitrogen source, it couldn't produce starch compound and urease but could produce ester substance during the growth process, glucose could be transformed to some organic acid, the optimum growth temperature was 32℃. The 18 S rDNA sequence analysis showed that the relationship of C. glycerinogenes was close to Issatchenkia scutulata, the homoeology reached 99%. However, because of the difference between them in biochemical characteristic, they are different strains.The chromosome DNA ploidy of C. glycerinogenes was firstly identified by taking the haploid cell of Saccharomyces cerevisiae as reference. The results showed that the optimum medium components for sporulation were as follows: glucose 1 g/L, yeast extract 2.5 g/L, NaAc 8.2 g/L,KCl 1.8 g/L, agar 20 g/L. Then we obtained the haploid cell of S. cerevisiae which based on the difference between the haploid and the diploid cells. Through determined and analyzed the concentration of chromosome DNA of a single cell, we knew that it was 1.82 times higher than the haploid, 0.848 times higher than the diploid of S. cerevisiaea and 0.97 times higher than the diploid of Candida tropicalis respectively, We confirmed that C. glycerinogenes was a diploid cell preliminary. A UV irradiation test was performed to further verify that C. glycerinogenes was a diploid cell.The growth and metabolism of C. glycerinogenes using different carbon sources were studied. It was found that in the bacteria growth period, when the monosaccharide as carbon source, the biomass was significantly higher than that of disaccharide. The biomass were 11 g/L and 9 g/L under the condition of glucose and fructose respectively, and glycerol production were 100 g/L and 80 g/L respectively. The biomass was both only 7 g/L and almost no glycerol producted under the condition of sucrose and maltose. The content of lactic acid was coupled with the change of pH during the fermentation process.Metabolic flux under different carbon sources during different fermentation periods were analyzed. During the growth period, when monosaccharide as carbon source, about 39% carbon flux turned into the biomass and about 30% carbon flux turned into glycerol synthesis., while it was about 43% and 10% respectively when disaccharide as carbon source. During the glycerol synthesis period, about 60% and 4% carbon flux turned into glycerol synthesis when monosaccharide and disaccharide were used as carbon sources respectively. Disaccharide needed to be hydrolyzed into monosaccharide and then monosaccharide could be utilited . Hydrolyzation reaction limited the content of disaccharide as carbon source into EMP pathway, and disaccharide could only maintain bacteria growth and metabolic, thereby no extra carbon source used for glycerol production. |
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