Study On The Application Of Microalgae-yeast Co-culture System In Shrimp Aquaculture Wastewater Treatment

In the past 30 years,China's aquaculture industry has developed rapidly,and the aquaculture model has achieved a transition from extensive one to high-density and industrialized one.However,with the rapid development of aquaculture,the emission of aquaculture wastewater has also increased dramatically.The decomposition products of the bait in the aquaculture wastewater,the metabolites of the cultured species,and the addition of antibiotics during the culture make the aquaculture wastewater rich in nitrogen and phosphorus nutrients and organic pollutants.Marine aquaculture wastewater is also characterized by large water volume and high salinity.If a large amount of aquaculture wastewater is discharged into the adjacent sea area without treatment,the concentrations of nitrogen,phosphorus,and organic pollutants in these water bodies will be increased,which will cause eutrophication of water bodies and seriously damage the ecological environment.The current study used the microalgae-yeast co-culture system to purify aquaculture wastewater,and biomass of microalgae and yeast were produced simultaneously,which can be used as a raw material for the production of high-value products.It is a recyclable and efficient wastewater bio-remediation method.Furthermore,aquaculture was applied as a biological treatment approach in China's alkali-saline areas.By culturing shrimp in alkali-saline land,the salinity and alkalinity of alkali-saline water can be reduced,and large areas of alkali-saline land in China can be reused.However,the immunity and yield of shrimp cultured in alkali-salinei areas have decreased.Therefore,the current study uses molecular biology methods to examine the key regulatory genes and responding mechanisms of shrimp in high-alkali environments,and provides theoretical guidance for molecular breeding of high-alkali-resistant shrimp and for better development of aquaculture in alkali-saline land.The main research contents of this article are as follows:?1?Suspended co-culture of Rhodosporidium sp.with three different microalgae?marine Chlorella vulgaris,Isochrysis galbana and Platymonas helgolandica?was studied,respectively,and the co-culture system of Rhodosporidium sp.with C.vulgaris was found best in pollutants removal performances,in which 84.50%of NH₄⁺and 100%of NO₂^-were removed.Based on this system,the effects of different symbiotic forms on pollutants removal performances were studied.Among three different microalgae-yeast symbiotic forms?co-culture of suspended Rhodosporidium sp.and immobilized C.vulgaris,suspended co-culture of Rhodosporidium sp.and C.vulgaris and co-immobilized of Rhodosporidium sp.and C.vulgaris?,it was found that the suspended Rhodosporidium sp.and immobilized C.vulgaris co-culture system had highest removal efficiency and the removal rate of NH₄⁺,NO₂^-,NO₃^-and PO₄^3-was 85.47%,100%,96.09%and 93.38%,respectively.?2?Based on the suspended Rhodosporidium sp.and immobilized C.vulgaris co-culture system,the effect of different inoculation ratios was studied,four different inoculum ratio was established,including 5:1,10:1,1:1 and 1:5?C.vulgaris/Rhodosporidium sp.?,and it was found that pollutants removal was highest when the inoculum ratio was 5:1.The removal rates of NH₄⁺,NO₂^-,NO₃^-,PO₄^3-and COD were 86.20%,100%,100%,100%and 100%,respectively.In a semi-continuous experiment,the optimized microalgae-yeast system showed stable and efficient removal efficiency.In three repeated batches,the removal rate of NO₃^-was 100%,the removal rate of NO₂^-remained above 95%,and the removal rate of NH₄⁺was74.57-86.19%.PO₄^3-and COD removal rates are 90.68-100%and 92-100%,respectively.?3?The high-pH tolerance gene POLR2H cDNA of Litopenaeus vannamei was cloned by772 base pair?bp?,containing an open reading frame?ORF?of 456 bp,encoding 151 amino acids;quantitative real-time PCR?qPCR?experiments found that the tissue with the highest expression of POLR2H was gills;The results of in situ hybridization showed that POLR2H was mainly distributed in the second gill filaments of shrimp;under high pH stress,low salinity stress,high concentration of NH₄⁺and NO₂^-stress,the relative expression of POLR2H was significantly up-regulated.Under various biological stimuli?injection of LPS and poly?I:C??,the relative expression of POLR2H did not change significantly;after siRNA injection was conducted to silence the POLR2H gene,in high pH stimulation experiments,the survival rate of shrimp injected with siRNA was only 33.33%,and the survival rate of all shrimp in DEPC water?blank control group?and NC siRNA?negative control group?under high pH stimulation?pH=9.0?and control group?pH=8.2?was higher than 83.33%,which indicated that Litopenaeus vannamei POLR2H was of significance in the response to high-pH environment.