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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011The cells growth was quantified by: Optical Density (O.D.) determination at Λ =600 nm, evolution of pH and dried biomass concentration. Dry matter concentrationdetermination was done after biomass separation from the culture medium bycentrifugation. The biomass drying was achieved in the oven at 105 o C until constant mass.After cells’ separation by centrifugation three freeze-thaw cycles were performed.The pigments extraction procedure was done in accordance with the dedicated literature [9],comprising acetone extraction of the total pigments mixture including water solublespecies, followed by n-hexane extraction to separate the total carotenoids content; anotherextraction with alkaline methanol allowing the torularhodin (the only pigment with acidstructure) component isolation.The total carotenoids concentration and the torularhodin concentration weredetermined based on the spectrometric recording of the extracts on the spectrophotometerUV-VIS (Jenway Spectrophotometer).To calculate the torularhodin concentration, the specific absorption coefficient E 1%1932 was applied to the difference between the absorbance of the hexane extract before andafter methanol phase extraction, at 515 nm [10].3. RESULTS AND DISCUSSION1. Influence of the initial pHSeveral experiments were realized to determine the influence of the initial cultivationpH, in the range 3 – 8, on the yeast growth and both – total carotenoids and torularhodinformation, considering the control medium MS3 with initial pH 5.miuedmL/g,µntiotracennoC80070060050040030020010003 4 5 - control 6 7 8pHTotal carotenoids concentration as ß-caroteneTorularhodin concentration98Fig. 1 Variation of total carotenoid and torularhodin concentration depending of initial pH valuesThe studied characteristics – yeast growth, pH evolution, the total carotenoids formationand the torularhodin formation recommend the initial pH range of 6-7 as being favorable,the torularhodin fraction from the total carotenoids’ content being more than 90% for the
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011pH of 7. The pH 3 is not favorable for the pigments formation and the pH 8 is limiting theyeast growth.2. Influence of the initial inoculum’s concentrationAlso the growth and the carotenoids production, mainly torularhodin with the sameyeast Rhodotorula rubra ICCF 209 were investigated for different initial inoculum’ cellulardensity on the control medium MS3. The growth evolution is done in the fig. 3 and thepigments formation results are presented in the fig. 2.miuedmL/g, µntiotracennoC100090080070060050040030020010000.5 1 2 5Inoculum's concentration, %Total carotenoids concentration as ß-caroteneTorularhodin concentrationFig. 2 Variation of total carotenoid and torularhodin concentration depending of the inoculum sizemn06,sityenaldticpO1816141210864200 20 40 60 80 100 120 140 160Time, h0.5 % 1% - control 2% 5%Fig. 3 Optical density variation depending of the inoculum sizeAn inoculum concentration of 1-2 v/v is favorable for both yeast growth and carotenoidsformation including the torularhodin accumulation.99
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