Study Of Salt And Cold Tolerant Heterotrophic Nitrificationdenitrifying Bacteria And Enhancement Of Nitroge Removal In Constructed Wetlands

Planktonic cells,a vital part of biogeochemical nutrient cycling,affect the biogeochemical cycling of nutrients.In this study,samples from three seasons（spring,summer and winter）were collected 10 km along the Bailang River estuary,and the relationship between environmental factors and bacterial community structure was studied by high-throughput sequencing.The correlation of physicochemical properties including p H value,salinity,inorganic nitrogen（NH4+,NO3-,NO₂-）was analyzed.Non-metric multidimensional scaling analysis（NMDS）and principal coordinate analysis（PCo A）showed that the samples formed three clusters according to seasons,clearly seasonal distribution and no significant difference in spatial composition.The results of redundancy analysis（RDA）showed that p H,salinity and inorganic nitrogen（NH4+,NO3-,NO₂-）were the key factors in shaping the composition of bacterial communities.In different seasons,the core taxa of bacterial communities were the same,including Actinobacteria,Cyanobacteria and Proteobacteria.According to the prediction of nitrogen metabolism function of bacterial communities by PICRUSt,the nitrogen cycle related processes include four main processes: nitrogen mineralization（NM）,nitrogen fixation（NF）,dissimilar nitrate reduction（DNRA）and denitrification（ND）.These results indicate that the bacterial community structure in the estuary of Bailang River is affected by seasonal changes rather than spatial changes,and these findings provide new evidence for the study of seasonal changes in the estuarine bacterial community.A strain WD0301 that salt and cold tolerance of efficient heterotrophic nitrification-aerobic denitrifies bacteria was isolated from the surface water of the Bailang River estuary in winter.Morphological observation and 16 s r DNA sequence analysis were performed.The effects of initial p H,C/N,rotational speed,temperature and salinity on the heterotrophic nitrification and aerobic denitrification characteristics of WD0301 were studied,and the enhancement effect of microbial agent of WD0301 on biological nitrogen removal in laboratory constructed wetland system was further research.The results showed that WD0301 belong to Acinetobacter sp.The optimal conditions for heterotrophic nitrification and aerobic denitrification performance are as follows: p H of 7-8,C/N ratio of 15²0,a rotating speed of 180 r·min-1 and temperature of 30℃.WD0301 could grow in the medium with NH4 Cl or KNO3 as the only nitrogen sources,and the removal rates of NH4+-N and NO3--N all reached 95% within 36 h,respectively.The strain could grow at a salinity of 40 g·L-1（Na Cl）and the removal rates of NH4+-N and NO3--N were 99.3% and 95.4%,respectively,within 36 h.The removal rates of NH4+-N and NO3--N within 36 h at 15℃ were 65.2% and 68.3%,respectively.When microbial inoculum was added to the constructed wetland to treating the tail water of mariculture,the NH4+-N degradation efficiency of the constructed wetland was increased by 16.9% compared with that of the unenhanced wetland.The results indicated that strain WD0301 had good heterotrophic nitrification performance and good treatment effect in the test of bioenhanced constructed wetland for the treatment of mariculture tail water,which provided theoretical support for the application of theoretical support and bioenhanced nitrogen removal technology.High-throughput sequencing results of the bio-enhanced constructed wetland and unenhanced constructed wetland indicated that the addition of microbial inoculum to the constructed wetlands changed the structure and function of microbial communities and structure.Bio-enhanced constructed wetland nitrogen metabolism-related microorganisms such as Nitrosomonas,Nitrospira,Bacillus,Aliiroseovarius,Bacteroidetes_BD2-2,Phaeodactylibacter,Sedimenticola,Candidatus_Thiobios obviously higher than unenhanced constructed wetland.Bio-enhanced constructed wetland nitrogen metabolism related enzymes genes that [EC:1.7.2.2],[EC:1.7.7.1],[EC:1.7.99.4],[EC:1.18.6.1],[EC:4.2.1.104],[EC:3.5.1.49],[EC:1.4.1.4] were higher than unenhanced constructed wetland.These results indicate that salt-tolerant microbial inoculum can be used to improve the nitrogen removal capacity of constructed wetlands under low temperature and salt stress.