2.4.1 Media compostion and cultivation mode

The production biotechnological process proceeds essentially in two stages: fermentation and product recovery. An important aspect of the fermentation process is the development of a suitable culture medium to obtain the maximum amount of desired product. In recent years, cheap raw materials and by-products of agro-industrial origin have been proposed as low-cost alternative carbohydrate sources for microbial metabolite production, with the view also of minimizing environmental and energetic problems related to residues and effluent disposal. For fermentation, seed cultures are produced from the original strain cultures and subsequently used in an aerobic submerged batch fermentation to produce a biomass rich in carotene pigment and other additional metabolites, e. g. ergosterol, metal ions etc. In the whole-cell strategy product isolation is not necessary and, moreover, complex biotechnological product in the form of slightly modified biomass could be obtained.

The traditional batch production system has the disadvantage of inducing the Crabtree effect (characterized by the synthesis of ethanol and organic acids as fermentation products), due to high concentrations of initial sugars, diminishing pigment and biomass yield. The strategy for solving this problem is the fed-batch culture. Maximum astaxanthin production (23.81 mg/l) by P. rhodozyma was achieved in fed-batch fermentation with constant pH = 6.0, 4.8 times greater that the one obtained in a batch culture and the biomass concentration (39.0 g/l) was 5.3 times higher than that in the batch culture (Ramirez et al., 2006). The maximum astaxanthin concentration by X. dendrorhous at fed-batch fermentation with pH-shift control strategy reached 39.47 mg/l, and was higher by 20.2 and 9.0% than that of the batch and fed-batch fermentation, respectively, with constant pH = 5.0. However, the maximal cell density at fed-batch fermentation with pH-shift control was 17.42 g dry cells/l, and was lower by 2.0% than that of fed-batch fermentation with constant pH = 5.0. As a result of the two stage fed-batch culture P. rhodozyma, cell and astaxanthin concentrations reached 33.6 g/l and 16.0 mg/l, respectively, which were higher when compared with batch culture. The final specific astaxanthin concentration (mg/ g dry wt of cells) in the second stage was ca. threefold higher than that in the first stage and 1.5-fold higher than that in the dissolved oxygen controlled batch culture, indicating that the astaxanthin production was enhanced mush more in the second stage than in the first stage (Hu et al., 2007).

The astaxanthin production was enhanced by a high initial C/N ratio in the medium (second stage), whereas a lower C/N ratio was suitable for cell growth (first stage). A significant increase (54.9%) in astaxanthin production by X. dendrorhous was achieved in pulse fed-batch process when compared with batch process. The astaxanthin concentration was 33.91 mg/l in pulse fed-batch when compared with 30.21 mg/l in constant glucose fed — batch and 21.89 mg/l in batch fermentation. In contrast with this strain producing high yields of biomass and astaxanthin in pulse fed-batch process, another strain of P. rhodozyma demonstrated high astaxanthin-synthesizing activity during continuous fed-batch process (Hu et al., 2005). The utilization of continuous feeding showed to be the most efficient feeding method in fed-batch processes, as it did not lead to a reduction in the cellular astaxanthin concentration, as observed in the pulsed feeding. In the pulsed and continuous fed-batch processes, a cellular astaxanthin concentration of 0.303 mg/ g biomass and 0.387 mg/ g biomass, an astaxanthin concentration of 5.69 and 7.44 mg/l, a biomass concentration of 18.7 and 19.3 g/l were obtained, respectively.

Temperature was reported to control changes in enzyme activities that regulate metabolic activity in microorganisms. For example, Rhodotorula glutinis biosynthesized |3-carotene more efficiently at lower temperature, whereas increased torulene formation was accompanied by higher temperature (Bhosale & Gadre, 2002). The reason might be found in y-carotene that acts as the branch point of carotenoid synthesis. Subsequent dehydrogenation and decarboxylation leading to torulene synthesis is known to be temperature dependent since the respective enzymes are less active at lower temperature compared to the activity of P-carotene synthase. This is probable reason for an increase in the proportion of P-carotene at lower temperature in Rhodotorula glutinis. The moderately psychrophilic yeast Xanthophyllomyces dendrorhous also displayed a 50% increase in total carotenoids at low temperatures with elevated levels of astaxanthin (Ducrey Sanpietro & Kula, 1998).

Fed-batch co-cultures R. glutinis-D. castellii gave a volumetric production of 8.2 mg total carotenoid/l, about 150% of that observed in batch co-cultures and biomass concentration of 9.8 g/l which was about two times higher when compared with batch fermentation (Buzzini, 2001). The fedbatch technique maximized the specific growth rate of R. glutinis, resulted in higher biomass and minimized substrate inhibition of pigment formation. Molasses in the fed-batch mode led to increased biomass by 4.4- and 7-fold in double — and triple-strength feed, respectively when compared with 12.2 g/l biomass in batch fermentation. R. glutinis also produced a very high carotenoid concentration for double — and triple-strength feed supplement (71.0 and 185.0 mg/l, respectively), and was higher 2- and 3.7-fold of that observed in batch fermentation (Frengova & Beshkova, 2009).