Study On The Production Of DHA From Defatted Silkworm Pupae Using Schizochytrium Limacinum SR21

At present,high cost of fermentation is still the major restrictive factor for the industrial production of high-added value microbial lipid.As the featured traditional resource of our country,more than 600,000 tons of silkworm cocoons are harvested every year.However,the bioavailability of silkworm pupae acquired from silk reeling industry is limited.Given this background,in this paper,a novel process of docosahexaenoic acid(DHA)production by Schizochytrium limacinum SR21 was established with defatted silkworm pupae hydrolysate(DSWPH)as nitrogen source under non-sterile conditions.The biotransformation of DSWPH to DHA-rich lipid by S.limacinum SR21 was investigated.The additives which can enhance lipid and DHA accumulation of Schizochytrium limacinum SR21 were screened out.The specific results were as follows:Pretreated low-cost DSWPH by high pressure hydrothermal method was used as nitrogen source for DHA production using the S.limacinum SR21 cultivated in bioreactor.The impact of the condition of culture on biomass,lipid production,and DHA production of S.limacinum SR21 was investigated.The results indicated that the optimum fermentation conditions were as follows:amount of DSWPH 20 g/L,temperature 26°C,initial pH value6.0,concentration of artificial seawater salt 40 g/L,and dissolved oxygen 20%.The fermentation was elapsed after 120 hours,and biomass,lipid yield and DHA productivity were 39.27 g/L,22.44 g/L and 62.63 mg/L/h,respectively.Under the optimal conditions,the growth curves of S.limacinum SR21 were measured using DSWPH as nitrogen source.A simple model was proposed to use respectively the Logistic equation and Luedeking-Pire equation for cell growth,lipid production and glucose consumption of S.limacinum SR21 in the fermentation process(R~2>0.99),and the model can provide a good basis for Fed-batch fermentation.Compared with commercial yeast extract as nitrogen source,there was no statistical difference for the change of fatty acid profiles and lipid yield in S.limacinum SR21 at the end of fermentation(P>0.05).Hence,DSWPH can serve as organic nitrogen source to culture microalgae such as S.limacinum SR21 for the production of DHA.In order to reduce costs of fermentation,the strategy of using DSWPH as nitrogen source under non-sterile conditions for the growth of S.limacinum SR21 was adopted.Glucose consumption,cell growth,lipid production and fatty acid profiles of batch fermentation under sterile conditions and non-sterile conditions were comparatively analyzed,and the feasibility of S.limacinum SR21 under non-sterile culture conditions was demonstrated.The non-sterile culture conditions were as follows:initial pH 5.5,concentration of artificial seawater salt 50 g/L,inoculum dose 15%,and after culturing for120 hours,biomass,lipid and DHA yield was 32.05 g/L 16.53 g/L and 6.08 g/L,respectively.In addition,by comparing results obtained from three fed-batch fermentation modes,we found that the fed-batch fermentation with continuous feed was superior to the disposable fed-batch and intermittent fed-batch.In fed-batch fermentation with continuous feed,biomass reached 47.63 g/L,and compared with batch culture,the yield and productivity of DHA was increased by 63.25%and 18.72%,respectively.However,novel ideas were promoted for DHA production using fed-batch fermentation with continuous feed under non-sterile conditions.In order to further reduce costs of fermentation,the enhancer of DHA production by S.limacinum SR21 using DSWPH as nitrogen source was screened out.For the first time,inositol has been found to enhance DHA and lipid production of S.limacinum SR21.It was found that with 120 mg/L of inositol added to the medium at 48 h,the yield of lipid and DHA were 22.86 g/L and 8.53 g/L,which were 13.90%and 20.82%higher than the control group,respectively.In addition,the content of unsaturated fatty acids in lipid increased significantly,and UFAs/SFAs increased by 23.38%.It was found that the supplement of inositol enhanced activities of PC,G6PDH,NADP-ME,and ACL during the lipid accumulation stage.In particular,the effect of inositol added at 48 h was more obvious.Therefore,inositol can enhance the lipid accumulation of S.limacinum SR21 and changes of fatty acid composition.As well as,inositol can be used as an enhancer of S.limacinum SR21 fermentation.In summary,the traditional nitrogen source can be replaced with DSWP for producing DHA-rich lipid by S.limacinum SR21 under non-sterile conditions.It can give a basis for producing microbial DHA with low cost.This biotechnological process has good application prospects.