Screening Of Axtaxanthin-Producing Phaffia Rhodozyma And Its Disruption By Mixed Culture With Bacillus Circulans

A new screened strain, Phaffia rhodozyma. ZJB OOO10, which could produce astaxanthin, was isolated from soil. According to the physiological and biochemical characteristics and its ITS rDNA gene sequence analysis, it was identified as Phaffia rhodozyma. The sequences obtained were compiled and compared with sequences in the GenBank databases using BLAST program. Sequence analysis was performed using Phylips （version 3.572） softwares and the alignment match was then used to construct the neighbor-joining phylogenetic tree.As the low production of astaxanthin for wild type strain, low-energy ions beam implantation was performed for improving the yield of astaxanthin. The optimum energy and dose of ion beam implantation were 15 keV and 80×2.6×10¹³ ions/cm², respectively. After ion beam implantation, a best mutant, E5042, was obtained and its production of astaxanthin was 2511.9μg/g （DCW）, while the wild type strain was about 1113.9μg/g （DCW）, which increases by 125.5%. Moreover, the astaxanthin production under the optimized conditions was scaled in a 50-L fermentor. The optimal maximum weight of cell biomass could reach 0.0307 g/mL at 136 h, and the astaxanthin concentration of 2510.4μg/g （CDW） could be obtained at the same time.It could be considered that oxygen might play an important role in astaxanthin biosynthesis, we focused on KMnO4 and H2O2 as the strong oxidizing agent to discuss the relationship between oxygen and astaxanthin synthesis. When fermented at 48h, after additiving KMnO4 （0.02%） and H202 （0.01%）, the final production of astaxanthin was 2602.1μg/g （DCW）. But the effect of oxidizing agent was not as sharply as above statement when scaling up addition or deferring the addition time.Mixed culture conditions of Bacillus circulans and Phaffia rhodozyma for enzymatic removal of the cell wall were investigated. It suggested the optimal carbon and nitrogen source of Bacillus circulans were colloidal chitin and corn steep, yeast farina, respectively. The optimal conditions for cell wall disruption by mixed culture were as follows: temperature was 35℃, the initial pH value of growth medium was 5.0-6.0, dosage of inoculation was 15%, Mixed culture time was 72 h, supplementary material additive on initiation were com steep liquor 2 g/L, yeast farina 2 g/L, Mg²+ 1.0 mmol/L and Ca²+ 1.0 mmol/L, Under these conditions, over 88% of astaxanthin from Phaffia rhodozyma could be extracted after 72 h of mixed culture.