| Docosahexaenoic acid (DHA), whose systematic name is all-cis-4,7,10,13,16, 19-docosahexaenoic acid, is a long-chain polyunsaturated fatty acid (PUFA), which belongs to the omega-3 group. DHA has been recognized for its various physiological functions in the human body. DHA is an essential component of the cellular membranes of the visual and nervous system tissues in humans, and it is important for proper development of the brain and eye in infants and young children. Schizochytrium sp., a member of the thraustochytrid group of microorganisms, is considered a good potential source of DHA. However, in the process of producing DHA oil by using Schizochytrium fermentation, there are high salt ion concentration, DHA content is low, the production cost is too high, which seriously restricts the development of the industry.In this paper, in order to achieve the high quality DHA oil for the industrialization, Schizochytrium sp. S056 with independent intellectual property rights was used as the original strain, a new clean production technology of low cost, high content of DHA oil was established, the metabolism mechanism of promote the accumulation of DHA was revealed. Furthermore, through a series of experiments, the industrialization large-scale high yield production of the high quality of DHA oil was successful. This study solved the problem of high salt in the traditional culture medium, and broke through the bottleneck of the substantial increase of DHA systhensis, which significantly improved the DHA content and product quality. The specific research contents and results are as follows:(1) A new technique of low salt medium was established, which significantly reduced the concentration of salt ions in the culture medium. Schizochytrium is a marine microalga that requires high concentrations of sea salt for growth, although problems arise with significant amounts of ions in the culture medium and corrodes the fermenters. In this work, we eValuated that cell growth and docosahexaenoic acid (DHA) production can be improved when using 2-10 g/L sodium sulfate instead of 20 g/L sea salt in the culture medium for Schizochytrium sp. S056. In practice, the use of sodium sulfate as the sodium salt led to chloride ion levels in the medium that can be completely removed, thus avoiding fermenter corrosion during Schizochytrium sp. S056 growth, reducing cost and increasing DHA production, and simplifying the disposal of fermentation wastewater.(2) A new production technology of high content of DHA oil was established, which significantly increased the DHA content, and the correlation between DHA synthesis and primary metabolism was revealed. Here, we demonstrate the efficiency of glycerol as the sole carbon source in the production of docosahexaenoic acid (DHA) by Schizochytrium sp. S056. When glycerol and glucose were compared for their effect on the fermentation of Schizochytrium sp. S056, glycerol increased the DHA concentration of the total fatty acids to 52.28%that was 31.75%higher than that produced by glucose. NADPH plays a crucial role in supplying the essential reducing power for fatty acid synthesis. The 2 carbon sources were compared for the differences in the concentration of NADPH and the key enzyme activities, malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH). The results demonstrate that when glycerol was used, the activity of G6PDH of Schizochytrium sp. S056 was significantly lower, the concentration of NADPH was reduced, and the content of DHA did not decrease with the decrease of NADPH, suggesting that the synthetic pathway of DHA was more than one in Schizochytrium sp. S056.(3) The mechanism contributing to the improvement of the DHA content in Schizochytrium sp. S056 when glycerol was the carbon source was revealed by transcriptome and DEG analysis. The results show that gluconeogenesis and pentose phosphate pathway (PPP) were inhibited when Schizochytrium sp. S056 was cultured with glycerol, whereas, glycolysis and branched-chain amino acids (Valine, Leucine and isoLeucine) metabolism, which are involved in the synthesis of acetyl-CoA, were substantially up-regulated. Moreover, the suppression of the TCA cycle resulted in more acetyl-CoA available for fatty acid biosynthesis and the up-regulation of the FAS gene, which is involved in the FAS system. Therefore, the FAS system was enhanced and then the content of DHA significantly increased.(4) A pilot culturing technology which can produce high-quality DHA oil was provided, and a easy reference scale-up rule was proposed for the first time. Firstly, a dissolved oxygen (DO) shift with constant temperature culture strategy for Schizochytrium sp. S056 growth in 100 L fermentor was obtained. Secondly, according to the dynamic consumption curve of glycerol in 100 L fermentor when Schizochytrium sp. S056 cultured, in the premise of not prolonging the fermentation time, the optimum feeding scheme was obtained. At the end of fermentation, the DHA content up to 52.61%with the yield of DHA reach to 23.40 g/L, the yield and production efficiency of DHA increased significantly. Finally, based on the rule of the stirring power equivalent, the scale-up rule of Schizochytrium sp. S056 was explored. After the DO concentration in the process of Schizochytrium sp. S056 cultured in the 1000 L fermentor was adjusted consistent with the 100 L fermentor, a good high density culture of Schizochytrium sp. S056 in the 1000 L fermentor was achieved. Furthermore, the scale-up rule was confirmed in the 10 ton fermentor, there results indicated that the average yield of DHA over 22 g/L with the content of DHA in lipid is still more higher than 50%. Thus, a high yield and high quality DHA oil production of low energy consumption, low cost and environmental friendly technology was achieved.In conclusion, in this study, a low salt medium was established, the contents of DHA increased significantly, the metabolism mechanism of promote the accumulation of DHA was revealed, furthermore, through the precise control of dissolved oxygen and temperature, the integrated application of the above technologies in the 10 ton fermentor was successfully realized. |
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