Nitrogen Removal Mechanism In FeS_x-based Denitrification Enhanced Recirculating Aquaculture System

Recirculating aquaculture system（RAS）is a kind of aquaculture mode with the advantages of high efficiency,energy saving,high quality of aquatic products and environmental protection.RAS usually includes a series of units such as aquaculture pond,physical filtration,biological treatment and disinfection,biological treatment set nitrification tank,the toxicity of ammonia nitrogen to aquatic organisms is larger into less toxic nitrate,nitrate,however,with the recycling of water system in the accumulation,and harm the health of cultured fish.In this study,the iron-sulfur-based autotrophic denitrification process was used to remove nitrate from RAS,to select the appropriate electron donor for RAS denitrification,to build a new type of RAS,and to add a FeS_x-based denitrification reactor to explore the denitrification performance of RAS and identify the growth status of aquatic organisms.This study was aimed at promoting the iron-sulfur-based denitrification process from basic research to engineering practice.The main research and conclusions are as follows:（1）The acute and chronic toxicities of sulfate,a by-product of thioautotrophic denitrification,to zebrafish were investigated in acute and chronic toxicity experiments involving aquatic organisms.The results showed that zebrafish had a high tolerance to sulfate,and no acute toxicity was observed in the range of 200-3200 mg/L.The chronic toxicity experiment showed that when the sulfate concentration was less than 1500 mg/L,the mortality of zebrafish was reduced with the increase of sulfate concentration,indicating that low concentration of sulfate did not pose a threat to the health of zebrafish.In addition,the salinity of aquaculture water was increased by lower concentration of sulfate,which increased the survival rate of zebrafish.However,the mortality of zebrafish increased with the increase in sulfate concentration from 1500 mg/L to 3000 mg/L,suggesting that high sulfate concentrations had serious adverse effects on aquatic growth.At the same time,it was found during the experiment that after long-term exposure to sulfate at a concentration higher than 250 mg/L,zebrafish showed different degrees of behavioral abnormalities.Finally,histopathological analysis revealed the histopathological effects of sulfate on zebra fish gills.The results showed that when zebrafish was exposed to sulfate concentration higher than 250 mg/L for a long time,gill filaments would swell and fall off,gill epithelium would proliferate and necrosis,and gill tissue lesions would get worse with the increase of sulfate concentration.（2）Through batch experiments,the denitrification performance of elemental sulfur（S⁰）,natural pyrite（FeS₂）,ferrous sulfide（FeS）and fired iron-sulfur mixture（FeS_x）was compared.S⁰ has a stable denitrification rate,but it produces a lot of sulfate,and the sulfate output is 7.54 mg SO₄^2-/mg N.In contrast,when FeS_x is an electron donor,the nitrate removal rate can reach 100%,and the sulfate yield is less than mg SO₄^2-/mg N.Then,FeSx with different S/Feratios was prepared.When the molar ratio of Fe:S is 1:1,the denitrification performance of the sintered FeS_x is stable,and the accumulation of nitrite is not obvious,and no ammonia nitrogen is produced.The sulfate yield is 0.72 mg SO₄^2-/mg N.The effect of coexisting sulfate on the denitrification performance of FeS_x was investigated under the optimal molar ratio of Fe:S（1:1）.When the concentration of coexisting sulfate is50 mg/L,the nitrate removal rate is 8.33 mg N/L/d;when the concentration is 250 mg/L and500 mg/L,the nitrate removal rate is 5.56 mg N/L/d,and the coexisting sulfate slightly inhibits the nitrate removal rate.However,coexisting sulfate has a significant inhibitory effect on nitrite reduction,and the greater the concentration of coexisting sulfate,the slower the nitrite reduction rate.BET,XPS and XRD analysis show that FeS_x has larger specific surface area and porous structure,and its main components are iron and sulfur compounds,including FeS,FeS₂ and Fe₇S₈.High-throughput sequencing technology revealed that the microorganisms involved in nitrate removal in the process of FeS_x-based denitrification were mainly sulfur autotrophic microorganisms Thiobacillus.（3）A RAS was built,and FeS_x-based denitrification reactor was added to explore the denitrification performance of iron-sulfur autotrophic denitrification technology in the system.Combined with high-throughput sequencing,the microbial community changes with and without RAS denitrification column were compared.In the stage of simulating aquaculture wastewater operation and not running denitrification reactor,nitrite and ammonia nitrogen in RAS were lower than 0.20 mg/L and 0.50 mg/L respectively,and nitrate gradually accumulated.After adding cultured fish and adding FeS_x-based denitrification reactor,nitrite and ammonia nitrogen were lower than 0.01 mg/L and lower detection limit,respectively,and the nitrate removal rate was 79.04%.After 24 days of operation,the sulfate concentration was lower than 200 mg/L,and the p H was stable at around 7.During the whole experiment,the survival rate of carp was 95%,and at the end of the experiment,the fish increased by 6-7 cm.The average weight gain of fish was 5.31 g,and the culture density increased from the initial 10 kg/m³ to 26.54 kg/m³.The results of microbial diversity analysis showed that the microbial diversity of denitrification column（C1）connected to the system were higher than that of denitrification column（C2）not connected to the system,and the abundance of Proteobacteria in C1 was 44.82%,which was34.07%higher than that of C2.At the genus level,there are more heterotrophic denitrifying bacteria such as Thauera and Arenimonas in C1 than in C2.In this study,the effects of sulfate on the health of aquatic organisms were revealed through acute and chronic experiments,which provided a reference for the application of sulfur-based denitrification process in water treatment.At the same time,the denitrification performance of different electron donors was compared,and a suitable electron donor was selected for the denitrification of RAS.It was also clear that the FeS_x-based denitrification reactor could effectively remove nitrate from RAS,which provided a new idea for the development of green ecological aquaculture.