Optimization of the production of astaxanthin by Phaffia rhodozyma

Abstract

The red pigment astaxanthin is the principal carotenoid pigment responsible for the distinctive orange red pigmentation of marine invertebrates, fish and birds. Astaxanthin can be used as a colorant for the red-pink color of farmed salmon, trout and shrimp. Relatively few species can produce astaxanthin. Although the intracellular astaxanthin content is relatively low when comparing with H. pluvialis, which is another promising microorganism for industrial use, the yeast P. rhodozyma is a possible candidate for commercial production because of its high biomass yield. In order to increase astaxanthin content of P. rhodozyma to meet increasing market needs in the future, one of the fast and efficient approaches seems to be the strain improvement of P. rhodozyma by mutagensis. However, the methods for both astaxanthin and biomass production are also of prime importance. As P. rhodozyma is a Crabtree positive yeast, its cell yield and pigment production are reduced at high sugar concentrations. In order to minimize the effect of high sugar concentration, fed-batch fermentation has to be used. D.O.-stat and exponential fed-batch cultures of P. rhodozyma have been investigated previously.In this study, pH-stat cultures of P. rhodozyma were extensively investigated. To optimize biomass and carotenoid production by Phaffia rhodozyma in pH-stat cultures, two methods of feeding glucose were studied. In the first method, which is comparatively simple to operate, the glucose feeding set point (pH 5.02) was higher than the culture pH (5.00) and P. rhodozyma grew at a low specific growth rate (u = 0.055 h-1). In the second method, the glucose feeding set point (pH 4.98) was lower than the culture pH 5.00 and the yeast grew at a specific growth rate (u = 0.095 h-1). With the second method of glucose feeding, which is more complex, in order to prevent overfeeding of glucose, a 'time interval" was added to the control strategy of the glucose pump and allowed to expire before the next dose of glucose was added. The length of the "time interval" affected biomass and carotenoid production. A critical time interval (Tc) was defined. In pH-stat cultures of P. rhodozyma, it was found that if the "time interval" was set longer than the critical time interval, the yeast did not grow. Apart from investigating the cultivation method, the effect of adding ethanol to D.O.-stat cultures of P. rhodozyma was examined. The time of addition of ethanol to the system and the concentration of ethanol added are important. When ethanol was added to the system simultaneously with glucose during the log phase, a low concentration of feeding ethanol (0.01% volume of ethanol / volume of fermentation medium) increased the total carotenoid content. On the other hand, a higher concentration of feeding ethanol (0.02% - 0.04% volume of ethanol / volume of fermentation medium) increased the cell biomass. If ethanol concentrations in the culture were too high, the cells were inhibited. When ethanol was added to the system during the stationary phase, provided that the ethanol concentration was not too high (less than 4.4% volume of ethanol / volume of fermentation medium) in the medium, it increased the total carotenoid production of P. rhodozyma. Moreover the increase of the total carotenoid content might be inversely proportional to the concentration of ethanol.