The Ecological Distribution Of Nitrifying Microorganisms In The Estuary-underground Estuary And The Ecological Restoration Of Ammonia Nitrogen Pollution

Nitrification plays an important role in the removal of ammonia-nitrogen in natural environments such as land,river and ocean,and is an indispensable part of microbial nitrogen cycle.However,the community structure and ecological distribution of microorganism for nitrification(including ammonia-oxidation bacteria-AOB,ammoniaoxidation archaea-AOA,and nitrite-oxidation bacteria-NOB)in estuaries and subterranean estuaries,as well as the removal of ammonia-nitrogen,are not well understood.Based on the high-throughput sequencing analysis of functional genes of amoA,nxrB and nxrA,this paper systematically studied the nitrification rate,the abundance,diversity and the distribution characteristics of nitrifying microorganisms' communities in the Pearl River Estuary and the Houmen Subterranean Estuary.Results of this study deep insight into the understanding ecological functions of nitrification processes in estuaries and subterranean estuaries,and will help to reveal the role of nitrification processes in the removal of ammonia-nitrogen pollution.The main conclusions of this paper are as follows:1.A method of analyzing AOB amoA gene diversity and community structure was built and optimized on Mothur software.In this study,a reference database of AOB amoA functional gene(including sequence files and taxonomic files)was constructed.Furthermore,87% sequence similarity was selected as the similarity threshold of AOB amoA gene to classify OTU at species level.Based on the reference database and similarity threshold,a high-throughput sequencing method for AOB amoA amplicon analysis based on Mothur software was established and optimized.2.Gradient change of environmental conditions in the Pearl River Estuary(PRE)affected the activity of nitrification process and the distribution of nitrifying microorganisms in the PRE.Nitrifying microorganisms are mainly distributed in surface sediments and upstream water in the PRE.The nitrification rate and abundance in the upstream were higher than those in the downstream.Nitrification plays an important role in the removal of ammonia-nitrogen in the PRE.AOB and Nitrospira NOB were dominant in the upstream water where ammonia-nitrogen was converted to nitratenitrogen,while AOA and Nitrospina NOB are dominant in the downstream water.After fractional filtration of water in the PRE,we found that the abundance of AOA was higher than AOB in the suspended matter with particle size less than 1.2 ?m in the upstream water,and the proportion of total AOM was increased in the suspended matter with particle size less than 5 ?m.There was a significant positive correlation between nitrification rate and relative gene abundance(p < 0.01).High-throughput sequencing results showed that the dominant AOB is Nitrosomonas in upstream and shifted to Nitrosospira in downstream.OTU1,OTU2,OTU3 and OTU4 of Nitrospina NOB are widely distributed in the PRE,and their relative abundance is relatively high in the upstream sites.Other OTUs of Nitrospina NOB had higher relative abundance at downstream stations.In addition,statistical analysis showed that salinity and substrate concentration were the main environmental factors affecting the nitrification process in the PRE(p < 0.05).In brief,under the influence of gradient distribution of salinity and substrate,nitrification mainly occurred in upstre am water and surface sediments in which ammonia-nitrogen was quickly converted to nitrate-nitrogen by nitrifying microorganisms under the condition of sufficient oxygen.Nitrifying microorganisms play an important role in the ecological restoration of ammonia-nitrogen pollution in the PRE.AOB and Nitrospira NOB palyed dominant funtions in the upstream,while nitrification process is relatived lower in the downstream,and AOA and Nitrospina NOB were the main drivers.3.The unique nitrification process was formed by the environmental factors in the Houmen Subterranean Estuary(HSE).The distribution of nitrification rate were matched to the abundance of nitrification genes in the HSE.The nitrification rate and abundance can be detected in the sediment cores of the offshore-end(depth < 1 m),while almost can not be detected in the sediment cores of the nearshore-end lower depth of 60 cm.This distribution partern was possible reaulted from available of dissovole oxygen.In the deep zone,because dissovole oxygen difficultly reached,aerobic nitrifying microorganisms would stop grow or play an active role,resulting in the accumulation of ammonia-nitrogen.High-throughput sequencing analysis showed that the structure of AOA community in the offshore-end of the HSE was obviously different from that of the nearshore-end.The AOA community of the offshore-end was dominated by the marine cluster AOA,while the proportion of soil cluster AOA was increased at nearshore-end.The community structure of AOB and Ntrospira NOB was relatively invariable,and most of them were unidentified species.Statistic analysis showed that the substrate concentration,p H and salinity were the main environmental factors affecting the nitrification process of the HSE(p < 0.05).In brief,under the influence of salinity,p H,substrate and tide,the AOA in HSE showed niche differentiation,while community composition of AOB and Ntrospira NOB did not change.Nitrification mainly occurred in the nearsurface sediments(depths l ess than 60 cm)at the offshoreend and the nearshore-end,while the nitrification rate and abundance could not be detected in the bottom sediments of the nearshore-end.In the low oxygen concentration,the activity of the nitrifying microorganisms in the HSE was low,resulting the low transformation rate of ammonia-nitrogen which was accumulated in the HSE.In the PRE and HSE,nitrifying microorganisms transformed excessive ammonianitrogen into nitrate-nitrogen,then be transformed into nitrogen gas by denitrifying microorganisms to remove nitrogen from the estuarine area.Nitrifying microorganisms play an important role in purifying estuarine water quality and removing excess nitrogen.This study systematically studied the ecological distribution of nitrification process in the PRE and HSE,which provided a research basis for microbial ecological restoration of nitrogen pollution in estuarine area.