Isolation And Identification Of Rhodotorula Mucilaginosa WP3 And Optimization Of Growth Conditions For Carotenoid Production From It

Carotenoid is a kind of natural pigment with extensive application value. Being able to prevent cancer and cardiovascular diseases, carotenoid can be widely applied to medicine field, food sector and cosmetic industry. As food additives, carotenoid has dual functions of coloring and nutrition supplement, thus being widely used in food in developed countries. Synthetic pigments are prohibited in many countries due to the toxicity problems while Rhodotorula arouses great research interest both at home and abroad as carotenoid synthesized by Rhodotorula is easily industrialized with low nutrition requirements and short training cycle.By enrichment culture in YPD broth and dilution-spread on YPD agar plate, an attempt was made to isolate yeast strain from the skin of apple and the yeast strain(WP3) was screened for producing carotenoids. Based on morphological, cultural and physiological tests and phylogenetic analysis of 18 S r DNA, internal transcribed spacer(ITS) and 26 S rDNA D1/D2 region sequences,WP3 was identified as Rhodotorula mucilaginosa.By studying the use of glucose, fructose, sucrose, maltose, glycerol as different carbon source for strain WP3,the results showed Rhodotorula mucilaginosa WP3 was able to grow and produce carotenoid pigments over a wide range of pH from 4 to 8(optimum p H 7.5) in a MS3 broth. Among the various carbon sources examined, fructose was the optimal carbon source and a highest value of the total carotenoid yield was obtained only in the case of initial fructose concentration of 40g/L. Among the carbon sources investigated, glucose was the most benificial to cell growth while fructose was the most benificial to the accumulation of carotenoids.When mixed carbon source of fructose and glucose was used, the output of carotenoids reached the highest(556.841?g/L).By studying the use of ammonium chloride, potassium nitrate and ammonium nitrate as different nitrogen source for strain WP3, the results showed that the tested nitrogen sources positively influenced the growth of the yeasts cells, as well as the concentration of total carotenoids. Among the nitrogen sources investigated, potassium nitrate was the most benificial to cell growth while ammonium chloride was the most benificial to the accumulation of carotenoids. The highest value of the total carotenoids concentration was obtained only in the case of nitrogen concentration of 0.9 g/L. N rate was held constant at 0.9g/L and N form was changed, only in the case of NH4-N: NO3-N=3:1, the highest pigments concentration was obtained(645.897?g/L).The experiment of fixed ammonium nitrate content is 1 g/L, set up three different carbon to nitrogen ratios(C/N ratios)(20:1, 70:1,120:1), glucose levels were 17.5, 61.25 and 105 g/L; and fixed in 57 g/L glucose levels, setting up three different C/N ratios(20:1,70:1,120:1), ammonium nitrate content were 3.257, 0.931 and 0.542 g/L, to study impact of C/N ratios on carotenoid concentration, biomass and glucose utilization.The results showed that carotenoid concentration was the highest(399.466?g/L) when C/N ratio was 70:1(N rate was held constant), however, residual sugar of this ratio at the end of fermentation was high; cell growth was the best when C/N ratio was 120:1(glucose concentration was held constant); carotenoids yield(to initial glucose)achieved the maximum(1.8271×10-5) when C/N ratio was 20:1(N rate was held constant).By the mono-factorial experiment, the results showed that the significant external factors affecting carotenoid concentration were initial pH, fructose concentration and yeast extract concentration. Based on these results, response surface methodology(RSM)was employed in the present work and a second-order polynomial equation for carotenoid cncentration was built. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be signi?cantly in good agreement with predicted values since the adjusted determination coef?cient was 0.9992. In combination with mono-factorial experiments and response surface experiments, the optimum process parameters for Rhodotorula mucilaginosa WP3 were obtained as: initial pH, 7.61, fructose concentration, 41.70g/L, yeast extract concentration, 7.13g/L. Under the proposed optimized conditions, 678.048?g/L of carotenoid concentration was achieved. The predicted values for optimization process conditions were in good agreement with experimental data, indicating that the optimum process parameters by RSM were reliable.