Production Of DHA By Means Of High Cell Density Cultivation Of Schizochytrium Sp.

Docosahexaenoic acid (DHA) is one of theω-3-polyunsaturated fatty acids, which has important biological and physiological fuanctions, including the development of the infant brain, the function of the eye, the prevention of cardiovascular disease, and the improvement of body immunity. Therefore, DHA has been widely used in producing infant's food, health care food and assistant therapeutic dose, etc., and more and more attention has been paid on the production and preparation process of DHA. However, conventional fish oils may not be suitable to meet the increasing demand for DHA owing to their limited supply, lower content of DHA and peculiar taste and odor. As a result, new emphasis has been placed on the development of new DHA sources, which should not only be stable and secure but also with low cost.DHA production by microorganism fermentation has lots of advantages in comparison with fish oil. Schizochytrium sp. is a promising microorganism in producing DHA as it has high content of DHA, and the composition of PUFAs in Schizochytrium sp. is simple. In this study, the process and technics for production of DHA by means of high cell density cultivation of Schizochytrium sp. were studied.Firstly, the extraction process of lipid and DHA was investigated. A fast and efficient method is determined as follows: Cells were dried under the temperature of 125℃for 3 h after they were washed and centrifuged. n-Hexane was used to extract lipids from the dry cells, after they were disposed in hydrochloric acid solution for 40-50 min at the temperature of 60-70℃.Secondly, the fermention conditions for DHA production with Schizochytrium sp. such as pH and dissolved oxygen (DO) were optimized on 5-liter fermenter. The optimized batch cultivation conditions were temperature 25℃, pH 5.5, dissolved oxygen 30%, air speed 4 L/min, and the dissolved oxygen was controlled by the varying of the stirrer frequency. At the optimized conditions, dry cell mass of 36.7 g/L, total fatty acid of 23.8 g/L and DHA accumulation of 5.4 g/L could be achieved after 96 h of cultivation, which were 10.0%, 16.7% and 38.6% higher than those obtained at unoptimizable cultivation conditions, respectively.Afterwards, enhanced production of DHA was achieved by means of fed-batch cultivation of Schizochytrium sp. Based on the results of batch fermentation, the cell density and DHA concentration were improved by feeding the whole concentrated medium as well as high concentration of glucose solution (600 g/L), respectively. The results show that varying the feed rate of glucose to maintain glucose concentration between 10-20 g/L was the most advantageous for the growth of Schizochytrium sp. and accumulation of fatty acid. The maximum biomass, total fatty acid and DHA reached 60.6 g/L, 40.2 g/L and 8.0 g/L after 5 days fermentation, respectively, and the DHA productivity r_DHA was 66.7 mg/(L.h). In the subsequent scale-up culture on 30-liter fermenter the maximum biomass reached 55.5 g/L and the content of DHA reached 13.5 g/100 g powder after membrane filtration and spray drying of fermentation broth.Finally, the fed-batch cultivation of Schizochytrium sp. was carried out on 5-liter fermenter based on a semi-synthetic medium. The effect of vitamin and trace element on the cell density and DHA yield of Schizochytrium sp. were considered to optimize the primary culture medium father. Moreover, the fed-batch cultivation was carried out based on the optimized culture medium which the cell density and DHA concentration were improved by feeding nutrient fluid (C/N ratio 36:1) during the prophase of logarithmic growth phase (24 h) and feeding high concentration of glucose solution (600 g/L) during the anaphase of logarithmic growth phase(84 h). The maximum biomass and DHA reached 73.0 g/L, and 9.8 g/L after 5 days fermentation, which was improved 1.2 times and 1.5 times compared with the unoptimizable cultivation, respectively, and the r_DHA was 81.9 mg/(L.h).