Research On The Breeding And Biological Characteristics Of Schizochytrium Sp.for DHA Production

Docosahexaenoic acid(DHA)is a kind of ?-3 polyunsaturated fatty acids(?-3PUFAs),it plays a great important role on the normal physiological function of the body,especially oncardio cerebrovascular,blood lipid,intelligence development and vision protection.Based on its various functions,it has a broader market.DHA synthesis in human body cannotmeet the daily needs.DHA is traditionally extracted from fish oil,but due to so many restrictive factors,the marine microalga fermentation production of DHA gradually becomes a hotspot and developmenttrend.Schizochytrium is a great potentialresource of industrial production as it has advantages of rapid growth,easy to culture,simple composition of PUFAs,high content ofDHA and easy to purification.In this study,Schizochytrium sp.ATCC20888 was used as the original strain.Firstly,through low energy N + implantation the strain was mutagenic improvement,and obtainedthe high yield of DHA producing mutant with scientific screening method.Then,the original and mutant strainwere observed and studied for their cell growth and fermentation morphology of oil synthesis.The fermentation medium was optimized according to response surface methodology,and the fermentation kinetics was established to explore a set of a reasonable way ofglucose supplement and verified in 50 L fermentor.Finally,gas chromatography-mass spectrometry(GC-MS)was applied for detection of the algal oil.The main results are as follows:1.Mutagenesis of Schizochytrium sp.mutant strainsLow energy N+ implantation was an efficient approach for Schizochytrium sp.mutagenesis.The dominant strain was preliminary screened through culture by flat plate,Sudan black B staining for microscopy and tube separation,and the final high-yield mutant strain,named 15-4-M16 was obtained according to the indicators of the biomass,yield of oil,oil content,DHA content,and yield of DHA.The biomass of mutant strain was 48.91 g/L,the yield of oil was 14.89 g/L,and the yield of DHA reached up to 6 g/L,which has been increased by nearly 57%.The genetic performance was stable after 10 generations.2.The growing development and the changes of oils synthesis process of Schizochytrium sp.From the optical microscopic observation,it was found that the cells appeared significantly cleaved along with the accumulation of intracellular oil.Compared with the original strain,the mutant strain grew more vigorously,its cell division was more consistent,and at late fermentation period,cells were more ductile and not easy to crack.Observed by ultrathin section transmission electron microscopy,it was found that the original algae was the whole with the cells encapsulated in the fermentation cycle,while the mutant had been the growth of single cell,and the longer growth time was,the larger the algal cells were.The oil droplets of original algae are mostly single without block or connecting,and at the final period they were easily cracking with oil spilling.However the intracellular oil droplets of the mutant were becoming more and more with the growth time increasing,and in the fourth day,oil droplets gradually connected into a flake,finally almost filled the whole algae cells at late fermentation period.3.Optimization of fermentation medium for Schizochytrium sp.mutantAt first,the single factor test was used to investigate the main effects of glucose,sodium glutamate,yeast extract and sea salt concentration on the yield of DHA,and then,the response of Design-Expert8.0 software was used to optimize fermentation medium.The optimal fermentation medium was that the mass concentration of glucose,sodium glutamate,yeast extract and sea salt were 83.58 g/L,11.58 g/L,7.74 g/L and 18.26 g/L,respectively.At the optimal condition,the maximum yield of DHA was 6.67 g/L,which was increased by 11.35%compared to the original(5.99 g/L)and close to the predicted value.4.Establishment of fermentation kinetics and research of glucose supplement processing for Schizochytrium sp.mutantAccording to the changes of biomass,oil yield and glucose concentration during the fermentation process,the growth kinetics model of the algae was constructed by Logistic equation;the product formation kinetics model was built by the Luedeking-Piret equation;and the substrate consumption kinetics model was established by Luedeking-Piret-Like equation.The non-linear fitting of the fermentation curve was carried out by Origin 8.5 software,and the fitting index was obtained respectively as 0.988,0.991,0.981,and the experimental test values were almost with the relative deviations less than 10%compared the simulated values,indicating that the model is basically consistent with the actual fermentation situation.Based on the kinetic model,4%glucose was added at one time after the strain fermented for 96 h,with the yield of oil 25.62 g/L and the yield of DHA 11.87 g/L.Finally under 50 L pilot-scale fermentation at the optimal conditions for three batches,the oil and DHA production are similar to the fermentation results,so glucose supplement processing has been verified.5.GC-MS detection of algae oil produced by Schizochytrium sp.mutantThe DHA algae oil extracted from the 50 L fermentor was used as the raw material.Three kinds of methyl esterification methods including sodium hydroxide-methanol,sulfuric acid-methanol and acetyl chloride-methanol were compared.The fatty acid composition was determined by GC-MS.The results showed that sodium hydroxide-methanol was the optimal methyl esterification method.Based on this method,19 kinds of fatty acid components were detected,and the content of DHA was higher,indicating that the method had high efficiency and more and more DHA was esterified in the process of methyl esterification.Finally,the esterification temperature was optimized,and the optimum temperature was 55?.