| The estuary wetland is located in the land-sea interactive zone harboring active geobiochemical process.As a result of economic development and increasing human activities,large amount of reactive nitrogen has been imported into the estuary environment,causing a series of ecological and environmental problems.Understanding the microbial processes would help us interact with these environmental issues.The nitrification process plays a vital role in the nitrogen cycle.The recently discovered complete ammonia oxidizers(comammox),which catalyze ammonia to nitrate in a single organism,has changed the understanding of the nitrification which was thought as a process mediated by two different groups of microorganisms for more than 100 years.Comammox related studies have become a research hotspot in the nitrification area.The nitrification process is affected by various environmental factors,among which salinity is an important one.Estuary wetlands are located at the confluence of land and sea,harbor strong tidal process in the coastal area.The coastal sediments form a natural salinity gradient,which is a hot area for studying the nitrification process.At present,the mechanism of the influence of salinity on nitrifying microorganisms(especially comammox bacteria)is not clear.Therefore,we here investigated the community distribution patterns of comammox Nitrospira,and canonical nitrifiers including ammonia-oxidizing archaea(AOA),ammonia-oxidizing bacteria(AOB),and Nitrospira(including strict nitrite-oxidizing bacteria(NOB)and comammox Nitrospira)along a salinity gradient in Nanliu River estuary and also delineated the responses of the comammox bacterium Nitrospira inopinata in pure culture to a wide range of simulated salinity levels.The main research results are as follows:(1)Phylogenetic analysis of functional genes based on AOA,AOB and Nitrospira showed that the main AOA communities were group?.1b and goup?.1a in low and high salinity samples,respectively.Nitrosomonas-AOB was detected in all the samples,and Nitrosospira-AOB mainly existed in the high-salt areas.Among Nitrospira-NOB,Nitrospira lineage II and IV were the main groups in most samples,while Nitrospira lineage I was only detected in the low-salt area.(2)Based on sequences retrieved with four sets of functional gene primers,it was confirmed that comammox was mainly composed of clade A Nitrospira,with clade A1 subgroup existing in all and clade A2 subgroup in low salinity samples.(3)The gene abundances of comammox Nitrospira-amoA,AOA-amoA,AOB-amoA,and Nitrospira-nxr B in the sediments of the Nanliu Estuary were1.30?6.70×106,2.29×106?1.69×107,5.47×105?2.75×106??1.36×108?2.06×1010copies g-1 The gene abundance of Nitrospira-nxr B was at the highest level in each sample;the gene abundance of AOA-amoA was always greater than the abundance of AOB-amoA under different salinity gradients;the gene abundance of AOB-amoA increased with salinity;The gene abundance of Nitrospira-nxr B in low salinity samples was less than that of high salinity;the gene abundance of AOA-amoA and comammox Nitrospira-amoA did not change significantly along with the salinity gradient.(4)According to the co-occurrence network analysis,the nitrifying microbial species in the study were mainly positively correlated(94.31%).CCA analysis and Partial mental test analysis of microflora structure and environmental factors in the study area showed that the microflora structure distribution of comammox Nitrospira,AOA,AOB and Nitrospira had a significant positive correlation with salinity(P<0.05).(5)The laboratory experiments of pure comammox strain Nitrospira inopinata under different salinity gradient showed that optimal salinity for N.inopinata was0.5‰.N.inopinata ceased to oxidize ammonia,while was still active for nitrite oxidation at 12.8‰.In addition,as a clade A1 comammox Nitrospira and a lineage II Nitrospira,N.inopinata showed consistent response to various salinity levels with community dynamics of comammox and NOB Nitrospira along a salinity gradient. |
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