Product Docosahexaenoic Acid By Fermentation With Schizochytrium Limacinum SR21

Docosahexaenoic acid(DHA) is one of the essential polyunsaturated fatty acids,it is widely distributed in the gray matter of the brain, retina tissue, nervous system and reproductive system, it is conducive to the development of infant brain and retinal, it also plays an important role in reducing inflammation and blood lipids. With the expanding of DHA market demand, the traditional source of DHA from deep sea fish oil can’t meet people’s need. The research focused on the production of DHA from microbial source. Microbial fermentation has become a new source of DHA due to good quality, controllable process and stable yield. In this paper, Schizochytrium limacinum was used to product DHA in the purpose of continuously improving the lipids and DHA yield. The research results are as follows:1、The culture medium of Schizochytrium limacinum was mainly composed of glucose, yeast extract, corn steep liquor, sea salt, ammonium acetate. Each ingredient of culture medium was investigated with the single factor experiment. The orthogonal experiment was used to optimize the culture conditions. The investigated factors and its level intervals were determined on the basis of single factor experiments. The optimum experimental conditions were determined: glucose 70g/L, corn steep liquor 20 g/L, yeast extract 6 g/L, pH=4, sea crystal 10g/L, ammonium acetate 0.5 g/L, 25℃,shaking culture(120rpm)for 8 days. Lipids and DHA yield were 9.2 g/L and 4.28g/L respectively under the optimum experimental conditions.2 、 pH had complicated effects on the fermentation of the Schizochytrium limacinum. pH affects the permeability of cell membrane and the activity of enzyme in the cell, thus affects the synthesis of Lipids and DHA. p H in the fermentation process was measured and controlled in order to make pH fit for the growth of fungi and the synthesis of lipids and DHA. pH 5~7 was conducive to cell growth, but the suitable pH for the accumulation of lipids and DHA was 3~4. The yield of lipids and DHA can reach to 9.92 g / L and 4.14 g / L respectively when the pH of fermentation broth was controlled at 3 stably in first four days and at 5 stably in the last two days.3、The consumption of glucose was measured in the fermentation process of Schizochytrium limacinum. According to the consumption of glucose, the addition way of glucose was determined. The initial glucose concentration in the culture solution was controlled at 30g/L, then 60g/L glucose was added into the culture solution after two days. This way not only can avoid the inhibition on cell growth from high concentration glucose in fermentation prophase, but also can ensure the supply of carbon source in fermentation anaphase. Compared to one-step addition of 90g/L glucose, the yield of lipids and DHA can increased to 9.34 g/L and 3.74g/L from 8 g/L and 3.51g/L respectively.4、Crude glycerol is the primary byproduct of the biodiesel industry. The feasibility of using crude glycerol to instead of glucose was investigated in order to reduce the cost of fermentation. The results showed that glycerol is feasibile for Schizochytrium limacinum to produce lipids and DHA. Crude glycerol could be directly used for Schizochytrium limacinum fermentation, but the fermentation period was extended and the yield of lipids and DHA decreased slightly. Crude glycerol treated by active carbon adsorption could become more suitable for Schizochytrium limacinum. The yield of lipids and DHA could reach to 10.56 g/L and 4.24 g/L respectively when the concentration of the treated crude glycerol was 120g/L.5 、 Exogenous substance was added to regulate the lipids accumulation of Schizochytrium limacinum on the basis of lipids accumulation mechanism. Effects of citric acid, malic acid and sodium dihydrogen phosphate on the synthesis of lipids and fatty acid composition were studied. The yield of lipids and DHA increased to 10.66g/L and 4.44g/L from 8.44 g/L and 3.05g/L respectively when the addition of citric was 2g/L, the yield of lipids and DHA increased to 10.35 g/L and 3.76g/L from 8.32 g/L and 3.10g/L respectively when the addition of malic acid was 4g/L. The addition of sodium dihydrogen phosphate had no significant effect on the accumulation of lipids and DHA, but it increased the content of low carbon fatty acid in lipids.6、The lipids extraction of Schizochytrium limacinum by enzymatic hydrolysis with cellulase was studied. The enzymatic hydrolysis had the same effect for lipids extraction as the sulfuric acid hydrolysis, but the enzymatic hydrolysis could reduce pollution and energy consumption. The paper further studied the production of sugar in the process of enzymatic hydrolysis. The sugar had high yield of 9.9g/((kg dry power) when the enzyme dosage was 24 g/(kg dry power), time was 3 h, temperature was 54 ℃ and pH was 4.8. The enzyme solution could be reused to the fermentation of Schizochytrium limacinum. The recyle of the enzyme solution had no detrimental effect to the yield of lipids and DHA, and could increase the cell density of Schizochytrium limacinum a little.