High-density Culture Of Schizochytrium Limacinum And Utilization Of Agricultural By-products Treatment Solution

Docosahexaenoic acid(DHA)has important physiological functions such as promoting visual and intellective development of infants and preventing Alzheimer’s disease.Schizochytrium limacinum SR21 has become the main strain to produce DHA due to its high DHA content,fast growth and easy culture.At present,DHA production in China still has the problem of inaccurate fermentation control and low biomass density.And the fermentation medium is mainly glucose,which is acceptable for the production of DHA-containing foods,but the cost is too high for larger-scale DHA-containing feeds.In order to solve the above problems,the conditions of different growth stages of Schizochytrium limacinum were optimized and high-density fermentation was achieved,and organic waste such as corn steep liquor and hydrolyzed potato broth were used as fermentation raw materials to provide a new possibility of reducing the fermentation cost.In order to determine the suitable conditions for the growth and accumulation of DHA of Schizochytrium limacinum,the carbon source,nitrogen source,inorganic salt and temperature were first investigated in this paper through single-factor experiments.The results showed that 20-40 g/L glucose is the ideal concentration for the growth of Schizochytrium limacinum seed.There was no significant difference in the effect of 5-20 g/L yeast extract on the growth of Schizochytrium limacinum seed.Ammonium ions as an inorganic nitrogen source have a great influence on the growth of Schizochytrium limacinum.In the range of 0.2-4 g/L,the higher the concentration,the lower the cell density.A total nitrogen concentration of 0.5 g/L forms a nitrogen limitation and cells can accumulate more than 20%(DCW,w/w)of DHA.Under the same nitrogen source concentration,the ratio of organic nitrogen source to inorganic nitrogen source was 7:3,which favored the growth and proliferation of Schizochytrium limacinum.Carbon to nitrogen ratio of 12:1 is suitable for cell proliferation and 55:1 is suitable for cell growth and accumulation of DHA.30℃ is the most suitable for the growth of Schizochytrium limacinum,although 25℃ and 35℃ can reach the same level but with a lag period.On the basis of single-factor experiments,three high-density fermentation processes were designed.The results showed that the fermentation according to the carbon-nitrogen ratio feeding obtain the dry cell weight at 104 h was 105 g/L,the highest DHA content was 14.3% of dry weight,and the highest dry cell weight at 115 h was 115 g/L,DHAcontent was 12.6% of dry cell weight.But the medium of this process was complex and no effective nitrogen limitation was formed.Although the pH feeding fermentation was simple and easy to control,the dry cell weight was only 73.5 g/L at 120 h,and the DHA content accounted for dry cell weight was 12.2% and the effective nitrogen limitation was still not formed.According to the nitrogen source consumption rate,the feed fermentation control was simple.Three nitrogen source feed rates were tried and the highest dry cell weights were 133 g/L,145 g/L and 153 g/L and the highest DHA content was 14.4%,4.3% and 21.2% respectively.The medium of this process was simple and the results were good.The third type of feeding according to nitrogen consumption rate formed effective nitrogen limitation,therefore this type of fermentation process was used in subsequent experiments.The shake flask experiment was used to explore the culture of Schizochytrium limacinum using corn steep liquor and hydrolyzed potato broth as raw materials.It was proved that these two raw materials can provide carbon source and nitrogen source for its growth.And according to the different properties of the corn steep liquor and hydrolyzed potato broth,the determined high density fermentation process was changed correspondingly.High-density fermentation using corn steep liquor had the highest dry weight of 97.6 g/L and DHA content of 12.2%.The highest cell dry weight of the hydrolyzed potato broth was 123 g/L,and the DHA content was 12.0%.These two fermentation achieved the goal of Schizochytrium limacinum high-density fermentation of using these two agricultural by-products treatment solution to reduce the cost.The fermentation process determined in this study can be used in actual production to achieve high-density fermentation.The process of high-density fermentation using agricultural by-products treatment solution is expected to further reduce the production cost of DHA and provide a new way to realize the economical efficiency of containing-DHA feed.