Removal Of Nitrogen From Aquaculture Wastewater By HN-AD Compound Bacteria Enhanced MBBR

Compared with traditional fishery,Recirculation Aquaculture System(RAS)is widely used in aquaculture because of its high breeding density,high water cycle rate,effective removal of toxic and harmful substances in water by physical and biological technologies,and environmentally friendly.As the core unit of RAS water treatment,biological filtration system mainly depends on the full contact between biofilm attached to the biocarrier and water to increase the biomass and biological species in the reactor,so as to improve the treatment efficiency of the reactor and provide a healthy aquatic environment for aquaculture products.In this study,the heterotrophic nitrification-aerobic denitrification(HN-AD)compound bacteria with obvious denitrification effects were selected,and the optimal carbon source and Carbon/Nitrogen(C/N)suitable for growth of the bacteria were determined,which was applied to strengthen the start-up process of the moving bed biofilm reactor(MBBR).The nitrogen removal performance of MBBR under different substrate concentrations,hydraulic retention time(HRT)and Dissoved Oxygen(DO)conditions was further investigated,the structural changes of microbial communities in the long-term operation of MBBR,as well as the composition structure and correlation analysis of microbial communities related to nitrogen removal was explored,so as to provide theoretical support for the study of nitrogen removal effect of MBBR in RAS.The main research results are as follows:(1)HN-AD compound bacteria BW-2 with the best growth and nitrogen removal performance was selected.The removal rates of ammonia(NH₄⁺-N)and nitrate(NO₃^--N)were 54%and 100%,and and there was no accumulation of nitrite(NO₂^--N).BW-2 can use glucose,sodium acetate and brown sugar as carbon sources.When brown sugar as the external carbon source,BW-2 can remove 64%and 100%of NH₄⁺-N and NO₃^--N,and and there was no accumulation of NO₂^--N,showing the best treatment effect.The optimal C/N was 20.When C/N was lower than 20,the growth performance and NH₄⁺-N removal capacity of BW-2 increased with the increase of C/N;when C/N was higher than20,the carbon source provided could fully meet the growth and metabolism of bacteria,resulting in little effect of C/N on the growth and denitrification performance of bacteria.(2)MBBR starts quickly within 11 days and starts less than most reported MBBRs.MBBR has a wide range of adaptation to substrate concentration.When the influent Total Nitrogen(TN)(NH₄⁺-N+NO₃^--N)concentration is 70(30+40)mg/L-190(90+100)mg/L,the removal rates of NH₄⁺-N and NO₃^--N are not less than 96%and 99%,and the effluent NO₂^--N concentration is not more than 0.3 mg/L.MBBR had the most obvious removal effect when HRT was 5 h,and the average removal rates of NH₄⁺-N and NO₃^--N were 98%and 100%,respectively,with little accumulation of NO₂^--N.When HRT was too high(10 h),the growth of dominant bacteria was inhibited due to the untimely supplement of organic carbon source.When HRT is too low(2 h),the contact time between microorganisms and pollutants in the biocarrier is short,and MBBR needs to take a long time to achieve effluent stability.MBBR had the best treatment effect when DO was 6 mg/L.After stabilization,the average effluent concentrations of NH₄⁺-N and NO₃^--N were 0.59 mg/L and 0 mg/L,respectively,and no NO₂^--N accumulation was detected.DO had significant effect on NH₄⁺-N treatment,followed by NO₂^--N,and had little effect on NO₃^--N.The long-term operation efficiency of MBBR was investigated under the condition of close to the maximum substrate concentration of RAS(NH₄⁺-N=15 mg/L,NO₃^--N=20 mg/L).The results showed that MBBR entered the stable effluent stage after 20d of adaptation.The average effluent concentrations of NH₄⁺-N,NO₃^--N,NO₂^--N and Chemical Oxygen Demand(COD)in the stable stage(21–60 d)were 0.47,0.10,0.18 and 34.71 mg/L,respectively.The effluent could meet the first-level emission standard of(GB 8978-l996)and meet the suitable growth conditions of fish.During the 80 d operation of MBBR,the protein(PN)content of extracellular polymeric substances(EPS)was the highest in the stable period,with an average of182.40 mg/(g/VSS),while the polysaccharide(PS)content increased slowly to 61.94 mg/(g/VSS)during the reaction period.The results of Three Dimensional fluorescence spectrum(3D-EEM)analysis showed that humic substances appeared in the later stage of the reaction,indicating the weakening of the stability of the biofilm and the lysis and apoptosis of the bacteria.Based on the sequencing results,although there are some differences in the community structure at the phylum level,the species with higher abundance have certain similarity.The relative abundance of Proteobacteria,Acidobacteriota and Bacteroidota were relatively high in each period.Chloroflexi was a rich species in the stable period.Desulfobacterota and Firmicutes suddenly increased in the reaction recession period,which was the main reason for the formation of a small amount of biological flocs and biocarrier anaerobic zone in the reactor.The changes of biological community structure were observed at the genus level,and there were great differences among different samples.Paenarthrobacter,Kouleothrix and Nakamurella are the dominant bacteria in stable period.According to the heatmap of genus level species abundance clustering,the four samples in stable period showed strong affinity,which could be clustered into a large branch,and the adaptation period and recession period were clustered into one branch,which was very consistent with the division of MBBR reaction stage in the early stage.The correlation analysis between bacteria at genus level and pollutant removal efficiency showed that Kouleothrix and Chloroflexi species had positive effects on the removal of NH₄⁺-N,NO₃^--N,COD and the reduction of NO₂^--N.Moreover,according to the network diagram of bacteria at genus level and pollutant removal,the complex relationship between microorganisms is conducive to promoting the efficient removal of nitrogen.