Analysis Of Metabolic Characteristics And Optimization Of Fermentation Process For Astaxanthin Production By Phaffia Rhodozyma

Astaxanthin has been widely used in aquaculture, health care, food and other fields because of its special coloring function, antioxidation, antitumor, immunity improving and other biological function. Phaffia rhodozyma is one of the main microbes of natural astaxanthin synthesis, but astaxanthin production levels of most current strains are unable to meet the production requirements of large scale. Taking the study of Phaffia rhodozyma metabolic characteristics as focal and the application of metabolic regulation to improve astaxanthin production, this paper has conducted work of the following three aspects:First, the fermentation characteristics of four Phaffia rhodozyma strains, which were breed and preserved by our laboratory, were carried on the comparative study. The relationship between cellular anabolism and astaxanthin production was also studied. Results indicated that: Phaffia rhodozym JMU-17 W had exuberant growth, large sugar consumption and low astaxanthin production; the sepecific productivity of astaxanthin of Phaffia rhodozym JMU-VDL668 and JMU-7B12 were low(only 0.3-0.5 mg/g), and the Crabtree Effect, which could produce large amounts of by-products such as ethanol, was obviously during the two strains’ fermentation; Phaffia rhodozym JMU-MVP14, which was established through EMS mutagenesis from JMU-VDL668, grew slowly, consumed less sugar and had high yield of astaxanthin(the sepecific productivity of astaxanthin reached 6.8 mg/g). The production of astaxanthin reached 116.86 mg/L in 7 L bioreactor while batch culture Phaffia rhodozym JMU-MVP14, and lots of other composition of carotenoids were detected. In the meantime, the study also found that cellular anabolism was closely related to astaxanthin synthetic of Phaffia rhodozym strains. The cellular protein and fatty acids content changed similarly in the early phase of fermentation: increased and decreased sharply. However, when the protein content leveled off in the stationary phase of cell growth, the content of fatty acids exhibited a slight increase. Conversely, the content of astaxanthin in cells fluctuated in contrast to that of protein. JMU-MVP14, an astaxanthin-overproducting mutant P. rhodozyma strain, had the highest specific productivity of astaxanthin as 6.8 mg/g while the cellular protein and fatty acids content were the lowest.Second, several fermentation promoter were used to metabolic regulation and control the growth and astaxanthin synthetic of Phaffia rhodozym. The results showed that: adding pyruvic acid, lactic acid and citric acid into the culture medium could increase sugar consumption, biomass, astaxanthin production and the fatty acid content of Phaffia rhodozym JMU-VDL668 and JMU-MVP14 while protein synthesis was certain inhibition. To Phaffia rhodozym JMU-MVP14, the sepecific productivity of astaxanthin decreased after adding pyruvic acid, lactic acid and citric acid, and after adding glycerol and ethanol, the cellular protein, fatty acid and astaxanthin production were higher, but the biomass decreased. After optimization, adding 2 g/L of pyruvic acid at 0 h was advantageous to the production of astaxanthin, biomass and astaxanthin prodiction reached 7.65 g/L and 46.79 mg/L respectively; and adding 1 g/L ethanol at 96 h could increase the sepecific productivity of astaxanthin significantly(7.43 mg/g) which was 32% than the control group during Phaffia rhodozym JMU-MVP14’s fermentation.Third, optimized the pH condition and tried several sugar-feeding strategies, results indicated that: when controlled the pH condition of batch fermentation in 7 L bioreactor at the valule 5.0, the production of astaxanthin, astaxanthin yield to sugar and synthesis speed were all the largest, Pmax=138.38 mg/L, Yp/s=2.90 mg/g and rp =1.526 mg/(L·h) respectively. High pH condition was benefit for the synthesis of β-cryptoxanthin and β-carotene, while low pH condition was better for astaxanthin synthesis, the sepecific productivity of astaxanthin at pH 3.0 and 4.0 reached 8.25 mg/g and 7.30 mg/g respectively. When pH control in stages: the first stage(0-72 h) to control the pH 5.0, the second stage(72-144 h) to control the pH of 4.0, the production of astaxanthin, astaxanthin yield to sugar and synthesis speed reached Pmax=150.11 mg/L,Yp/s=3.11 mg/g and Yp/x=7.10 mg/g respectively which were higher than the control group. The glucose mix ethanol two-phase fed-batch strategy was the most advantageous to Phaffia rhodozym strains of high density cultivation and the mass production of astaxanthin. Biomass, total carotenoids and astaxanthin has been significantly improved, reached 35.87 g/L, 393.17 mg/L and 304.71 mg/L respectively, which was 1.6 times, 2.4 times and 2.2 times of the control group.