Water Purification Effect And Bacterial Community Structure Analysis Of Internal Circlation Culture Systems Based On Nitrification Biofilm

Aquaculture is an important source of animal protein worldwide and China has the highest aquaculture production in the world,accounting for 60%of the global aquaculture production.Pond culture that is a Chinese traditional aquaculture mode mainly relies on exchanging water to maintain the health of the aquaculture water environment resulting in a large amount of aquaculture tail water discharge and serious water resource consumption.Therefore,the most important task of protecting and improving the quality of water environment and measures for the sustainable development of aquaculture are to strengthen the control of water pollutant discharge in aquaculture,develop ecological intensive aquaculture mode and reduce environmental pollution and dependence on water resources.The main toxic substances in aquaculture waters are ammonia and nitrite that are removed through nitrification.During nitrification,ammonia oxidizing bacteria（AOB）can oxidize ammonia to nitrite,and nitrite oxidizing bacteria（NOB）can oxidize nitrite to nitrate with low biological toxicity.In this study,polyurethane fillers were selected as carriers to preculture nitrifying biofilm,and internal circulation culture systems for in-situ water purification based on nitrifying biofilm were established.The water purification effects of nitrifying biofilms on the internal circulation culture system of Penaeus monodon（P.monodon）,Litopenaeus vannamei（L.vannamei）with different salinities,and Hippocampus abdominalis（H.abdominalis）were compared and studied.The high throughput sequencing was used to analyse the bacterial community structure of biofilms in internal circulation culture systems under different operating conditions.Redundancy analysis was used to explore the factors effecting the composition of bacterial community structure,and the function of bacterial community was predicted by FAPROTAX.The main conclusions obtained through the study are as follows:（1）Nitrifying biofilms can maintain the concentrations of NH₄⁺-N and NO₂^--N in the internal circulation culture systems for P.monodon,L.vannamei with moderate salinity（15‰）,L.vannamei with low salinity（3‰）,and H.abdominalis within the recommended range of the aquatic organisms.The peak concentration of NH₄⁺-N in the H.abdominalis culture system was the highest（0.82±0.04 mg/L）,and the mean concentration of NH₄⁺-N in the P.monodon culture system was the highest（0.34±0.03 mg/L）.The peak（0.04±0.01 mg/L）and mean（0.02±0.01 mg/L）concentrations of NH₄⁺-N in the L.vannamei culture system under moderate salinity was the lowest.The peak concentration（1.16±0.08 mg/L）and mean concentration（0.70±0.04 mg/L）of NO₂^--N concentration in the L.vannamei culture system under low salinity were the highest,while the peak concentration（0.20±0.04 mg/L）and mean concentration（0.10±0.02 mg/L）of NO₂^--N concentration in the H.abdominalis culture system were the lowest.（2）The changes of salinity and temperature affected the activity of nitrifying biofilms.There was no significant difference in the concentration of NH₄⁺-N between the moderate and low salinity culture systems of L.vannamei,and the concentration of NO₂^--N in the medium salinity culture system of L.vannamei was significantly higher than that in the low salinity culture system of L.vannamei,indicating that the decrease of salinity inhibited the activity of nitrite oxidizing microorganisms.The concentration of NH₄⁺-N in the P.monodon culture system was significantly higher than that in the medium and low salinity culture systems of L.vannamei,indicating that increase of salinity inhibited the activity of ammonia oxidizing microorganisms in nitrifying biofilms.The peak concentration of NH₄⁺-N in the H.abdominalis culture system was significantly higher than that in the P.monodon culture system.However,the mean concentration of NH₄⁺-N in the H.abdominalis culture system was significantly lower than that in the P.monodon culture system,indicating that the biofilm still had the ability to control the concentration of NH₄⁺-N at a lower level under low temperature conditions but the low temperature environment at the early stage of culture would inhibit the activity of ammonia oxidizing microorganisms in the biofilm.（3）The survival and average daily growth（ADG）of the four internal circulation culture systems were all at a high level.The survival of aquatic organisms in the P.monodon,L.vannamei with moderate salinity,L.vannamei with low salinity,and H.abdominalis culture systems were 80.5±0.9%,79.7±0.8%,74.1±1.2%,71.2±1.9%,respectively.The ADG were 0.066±0.003 g/d,0.132±0.005 g/d,0.138±0.008 g/d and 0.010±0.001g/d,respectively.（4）The analysis results of biofilm bacterial community structure and diversity showed that the richness and diversity of the initial biofilm bacterial community were the lowest.The richness and diversity indexes of the biofilm bacterial community in the internal circulation culture system under different operating conditions were in the order of L.vannamei with moderate salinity>L.vannamei with low salinity>P.monodon>H.abdominalis.The bacterial community difference of biofilm between systems under different operating conditions was greater than that of bacterial community within systems.And the number of OTUs（23）shared by all samples was small,while the shared OTUs（342）number of three samples in H.abdominalis culture system was large.The clustering differences between samples from different culture systems and initial biofilm samples were greater than those from the same culture system.The bacterial communities of L.vannamei culture systems under different salinity were relatively similar,while the bacterial communities of P.monodon and H.abdominalis culture systems were relatively similar,which was related to the operating conditions of the culture systems.（5）The eight biofilm samples were classified into 35 phyla and 86 classes.The dominant phyla included Proteobacteria,Bacteroidota,Desulfobacterota,Planctomycetota,Actinobacteriota,etc.The relative abundance of Proteobacteria in the initial biofilm was the highest（71.59%）,while that of Bacteroidota was the lowest（12.33%）.The relative abundance of Proteobacteria and Bacteroidota in other samples were 42.90～54.48%,13.72～47.99%,respectively.The dominant classes include Gammaproteobacteria,Bacteroidia,Alphaproteobacteria,Desulfuromonadia,Phycisphaerae,Actinobacteria,etc.The relative abundances of Alphaproteobacteria（33.51%）and Gammaproteobacteria（38.06%）in the initial biofilm were the highest,while the relative abundances of Bacteroidia（35.40%～47.85%）in the samples from the H.abdominalis culture system were the highest.（6）There were significant differences in the dominant genuses of biofilms in internal circulation culture systems under different operating conditions.Nitrococcus,Nitrosococcus,and Halomonas were the dominant genuses in the initial biofilm,in which the relative abundance of nitrifying bacteria（10.08%）was the highest.The relative abundance of nitrifying bacteria in the used sample biofilm was between 0.05%and 2.97%.The relative abundances of Nitrococcus and Nitrosococcus in the used biofilm significantly decreased,while the relative abundance of Nitrospira significantly increased.There was no significant change in the relative abundance of Nitrosomonas.The shared dominant genuses of the L.vannamei culture systems with different salinity included SM1A02 and Pseudohongiella,while the unique dominant genuses of the L.vannamei culture system with moderate salinity included Ruegeria and norank＿f＿＿A4b,Phaeodactylibacter,and the unique dominant genuses in the L.vannamei culture system with low salinity include Paracoccus,SWB02,Rheinheimera,Gemnobacter,and Pseudomonas.The dominant genus in the P.monodon and H.abdominalis culture systems was Ulvibacter,which can degrade proteins in high salinity environments.The halophilic and pathogenic Vibrio had been found both in the P.monodon and H.abdominalis culture systems with a relative abundance of 0.15%to 3.01%.The unique dominant genuses in the P.monodon culture systems included Mycobacterium and Marinicella.The unique dominant genuses in the H.abdominalis culture system included Colwellia and Psychroserpens.（7）The results of redundancy analysis（RDA）and correlation heatmap analysis showed that the composition of bacterial community of biofilm at the level of phylum,class and genus was mainly effected by salinity,temperature and nitrite,while there were no significant correlation between temperature,salinity,ammonia,nitrite,nitrate,and TP of Nitrospirota,Nitrosporia,Nitrosococcus and Nitrospira.These indicated that nitrifying bacteria in biofilms hae strong environmental adaptability.FAPROTAX function prediction showed that there were 54 functions of biofilm bacteria,among which the relative abundance of chemoheterotrophy and aerobic＿chemoheterotrophy was the highest（37.76～75.94%）.And there were 15 functional types related to nitrogen cycle,including nitrate＿reduction,nitrogen＿respiration,nitrate＿respiration,nitrite＿respiration,nitrate＿denitrification,nitrite＿denitrification,denitrification,nitrification,aerobic＿nitrte＿oxidation,aerobic＿ammonia＿oxidation,etc.