The Microbial Communities Along The Dongjiang River And Their Functions In Ammonia-nitrogen Transformation

The Dongjiang River is the eastern tributary of the Pearl River in Guangdong provinceand provides vital drinking water to Hong Kong and several other cities in the Pearl RiverDelta (PRD). The booming economic growth and urban development along the river basinhave led to excessive release of nitrogen pollutants into the river region. However, the overallwater quality of the river is a generally quality source of drinking water, which suggest thatefficient natural nitrogen removal occurs in the river and microorganism may play animportant role in this process. It is still limited known that microbial distribution in the riverecosystem and its role in the conversion of ammonia. In this study, water and sediments weresampled from different sections of164.51km along the Dongjiang River, andhigh-throughput pyrosequencing, quantitative PCR, clone libraries and multivariate statisticalanalysis were used to exploit the composition and stucture of microbial communities and theircharacteristics in transformation of ammonia-nitrogen. The following main results wereachieved.一. The Dongjiang River between Huizhou and Qiaotou have the nitrogen removalcapacity.The water qulity of the Dongjiang River during2011-2012showed that water qualitydecreased from upstream to downstream and the gradients of nitrogen content graduallyincreased from Xinfeng to Huizhou. However, ammonia nitrogen decreased about half fromHuizhou to Qiaotou and the potential nitrification rates in downstream sampling sites(Huizhou and Qiaotou) was significantly higher than upstream sampling sites (Xinfeng,Heyuan and Guzhu). These results suggest that Dongjiang shows strong nitrogen removalcapacity with gradually increasing ammonia pollutants.二. Bacterioplankton community from upstream to downstream of Dongjiang water areaffected by the composition of the nitrogen gradient, which show significant seasonal andspatial variation characteristics.Here we investigated intra-annual bacterial community structure in the Dongjiang Riverby the application of high-throughput pyrosequencing during the dry and wet seasons in2011.Our observations demonstrate that Proteobacteria with dominant class of Betaproteobacteria,Actinobacteria, and Bacteroidetes were the dominant members of the bacterioplanktoncommunity in the Dongjiang River. The bacterioplankton varied in compositions anddistributions between dry and wet seasons and exhibited profound seasonal changes, although the diversities of bacterioplankton communites between the two seasons were similar.Hierarchical clustering analysis of microbiological communities showed the communities inthe upstream sampling sites Heyuan and Guzhu were well grouped together and separatedwith those in the downtream sites Huizhou and Qiaotou in both seasons, suggesting that thediscrepancy of community composition between different sampling sites may be affected bythe contents of nitrogen from upstream to downstream. Temperature and nitrient contents(especially NO-3-N concentration) represent important factors contributing to the observedvariations based on a series of statistical analyses.三. The main groups of ammonia oxidizing archaea (AOA) and ammonia-oxidizingbacteria (AOB) in the water column of the Dongjiang River are Group1.1a ofThaumarchaeota and Nitrosospira, and their distributions were significantly correlated withthe content of NO-3-N and total suspended solids (TSS), respectively. Group1.1b andGroup1.1b-associated of Thaumarchaeota were dominant in sediments with high carbon andnitrogen contents and AOB in sediments was dominant with Nitrosomonas. The distributionof AOA in sediments was significantly associated with pH and the content of TC.In order to elucidate that ammonia-oxidizing archaea (AOA) and bacteria (AOB) mayplay important roles in the ammonia-nitrogen transformation in the Dongjiang River. Thisstudy examined the community structures, distributions and abundance of AOA and AOB inthe water column and sediments of the Dongjiang River and their responses to changes inenvironmental parameters through quantitative polymerase chain reaction, cloning, andsequencing of ammonia monooxygenase (amoA). The abundance ratios of AOB to AOAvaried from0.07to9.4along the river and were positively correlated with the concentrationof NH4+-N. Phylogenetic analyses showed that most sequences of AOA communities in thewater column were belonged to Group1.1a of Thaumarchaeota, while Group1.1b andGroup1.1b-associated sequences of AOA predominated in the sediments with comparativelyhigh carbon and nitrogen contents (e.g. total carbon content (TC)>13g kg-1sediment,NH4+-N>144mg kg-1sediment), and Group1.1a and Group1.1a-associated sequences weredominant in the sediments with opposite conditions (e.g. TC <4g kg-1sediment, NH4+-N <93mg kg-1sediment). The majority of AOB sequences in the water column were affiliated withNitrosospira species. Although Nitrosomonas-and Nitrosospira-related sequences of AOBwere detected in the sediments, nearly70%of the sequences fell into Nitrosomonas-like Bcluster. Multivariate statistical analyses indicated that the community distributions of AOAand AOB in the water column were significantly correlated with the concentrations of NO3--N sediments in the Dongjiang River, while significant correlations were only detected betweenthe distribution of AOA and the sediment pH and TC, which suggested that AOA in sedimentsresponded more sensitively than AOB to the variations of environment factors.四. Candidatus Jettenia related anammox bacteria was the dominant community in theDongjiang River. The abundance and diversity of anammox bacteria in the water column weresignificantly related with the content of NH4+-N, and NO-2-N content was the key factorinfluencing the abundance of anammox bacteria in sediments.Anaerobic ammonium-oxidizing (anammox) process has recently been recognized as animportant pathway for removing fixed nitrogen (N) from aquatic ecosystems. Anammoxorganisms are widely distributed in freshwater environments. However, little is known abouttheir presence in the water column of riverine ecosystems. Here, the existence of a diverseanammox community was revealed in the water column and sediments of the DongjiangRiver by analyzing16S rRNA and hydrazine oxidation (hzo) genes of anammox bacteria.Candidatus Brocadia and two new anammox bacterial clusters were detected based onphylogenetic analysis of16S rRNA genes in the water column and sediments of theDongjiang River. Phylogenetic analyses of hydrazine oxidation enzyme (HZO) proteinsequences showed that Candidatus Jettenia related clades of anammox bacteria were bothdominant in the river water column and sediments. The abundance of anammox bacteriavaried significantly among the water and sediment samples based on qPCR. The abundanceand diversity of anammox bacteria in the water column were both significantly correlatedwith concentrations of NH4+-N based on Pearson and partial correlation analyses.Redundancy analyses showed the contents of NH4+-N, NO-3-N and the ratio of NH4+-N toNO-2-N significantly influenced the spatial distributions of anammox bacteria in the watercolumn. In contrast, Pearson correlations analysis indicated that NO-2-N concentration wasthe key factor influencing the abundance of anammox bacteria in sediments. The redundanceanalysis (RDA) confirmed that the combination of the contents of NO-2-N, NO-3-N, and theratio of total nitrogen versus total carbon (TN/TC) had significant impact on the anammoxbacterial community structure in sediments.五. Dynamic model of nitrogen removal in the Dongjiang RiverThe overall water quality were good in upstream of the Dongjiang River (Xinfeng,Heyuan and Guzhu) and the relative abundance of water column AOA were dominant. Withthe injection of nitrogen pollutants, the water quality gradually deteriorated at Huizhou and Qiaotou in downstream, and ammonia oxidizing communities also changed accordingly. Theobvious decrease of the relative abundance of AOA and the dominance of AOB wereobserved in water column and sediments, and the relative abundance of anammox bacteriaincreased. These suggest that AOA plays an important role in the transformation of ammoniaat low ammonia content in the water column, while AOB plays an important role in thetransformation of ammonia with increasing levels of ammonia contents, and collaborates withanammox bacteria to complete nitrogen removal process. Correspondingly, the potentialnitrification rate of Huizhou and Qiaotou in downstream sites were higher than upstream sites,suggesting that the dynamic changes of ammonia oxidizing communities enhanced theself-purification capacity of water column.Overall, bacterioplankton and benthic microbial have an importantly ecological functionsin the biogeochemical processes, which were related with the strong nitrogen removalcapacity of the Dongjiang River. Diverse aerobic ammonia oxidation and anammoxcommunites were broadly distributed in the oligotrophic water columns and sediments of theriver. Their diversity and distritution changes were closedly related with environmentalconditions. These communities play an important role in maintaining self-purificationcapacity of the Dongjiang River through ammonia oxidation and anammox processes andimproving the conversion efficiency of ammonia.