Anaerobic ammonium oxidation (anammox) as an important process of nitrogen cycle has been studied in estuarine environments. However, knowledge about the dynamics of anammox bacteria and their interactions with associated activity remains scarce in these environments. Here we report the anammox bacterial diversity, abundance, and activity in the Yangtze Estuary, using molecular and isotope‐tracing techniques. The phylogenetic analysis of 16S rRNA indicated that high anammox bacterial diversity occurred in this estuary, including Scalindua, Brocadia, Kuenenia, and two novel clusters. The patterns of community composition and diversity of anammox bacteria differed across the estuary. Salinity was a key environmental factor defining the geographical distribution and diversity of the anammox bacterial community at the estuarine ecosystem. Temperature and organic carbon also had significant influences on anammox bacterial biodiversity. The abundance of anammox bacteria ranged from 2.63 × 106 and 1.56 × 107 gene copies g−1, and its spatiotemporal variations were related significantly to salinity, temperature, and nitrite content. The anammox activity was related to temperature, nitrite, and anammox bacterial abundance, with values of 0.94–6.61 nmol N g−1 h−1. The tight link between the anammox and denitrification processes implied that denitrifying bacteria may be a primary source of nitrite for the anammox bacteria in the estuarine marshes. On the basis of the 15N tracing experiments, the anammox process was estimated to contribute 6.6%–12.9% to the total nitrogen loss whereas the remainder was attributed to denitrification.

d Diversity, abundance, and activity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using the ammonia monooxygenase ␣ subunit (amoA) in the intertidal sediments of the Yangtze Estuary. Generally, AOB had a lower diversity of amoA genes than did AOA in this study. Clone library analysis revealed great spatial variations in both AOB and AOA communities along the estuary. The UniFrac distance matrix showed that all the AOB communities and 6 out of 7 AOA communities in the Yangtze Estuary were statistically indistinguishable between summer and winter. The studied AOB and AOA community structures were observed to correlate with environmental parameters, of which salinity, pH, ammonium, total phosphorus, and organic carbon had significant correlations with the composition and distribution of both communities. Also, the AOA communities were significantly correlated with sediment clay content. Quantitative PCR (qPCR) results indicated that the abundance of AOB amoA genes was greater than that of AOA amoA genes in 10 of the 14 samples analyzed in this study. Potential nitrification rates were significantly greater in summer than in winter and had a significant negative correlation with salinity. In addition, potential nitrification rates were correlated strongly only with archaeal amoA gene abundance and not with bacterial amoA gene abundance. However, no significant differences were observed between rates measured with and without ampicillin (AOB inhibitor). These results implied that archaea might play a more important role in mediating the oxidation of ammonia to nitrite in the Yangtze estuarine sediments.

Dissimilatory nitrate reduction processes, including denitrification, anaerobic ammonium oxidation (ANAMMOX), and dissimilatory nitrate reduction to ammonium (DNRA), play an important role in controlling the nitrate dynamics and fate in estuarine and coastal environments. We investigated potential rates of denitrification, ANAMMOX, and DNRA in the sediments of the Yangtze Estuary via slurry incubation experiments combined with isotope-tracing techniques to reveal their respective contributions to total nitrate reduction in this hypereutrophic estuarine ecosystem. Measured rates of denitrification, ANAMMOX, and DNRA ranged from 0.06 to 4.51 μmol N kg À1 h À1, 0.01 to 0.52 μmol N kg À1 h À1, and 0.03 to 0.89 μmol N kg À1 h À1, respectively. These potential dissimilatory nitrate reduction process rates correlated significantly with salinity, sulfide, organic carbon, and nitrogen. Denitrification contributed 38-96% total nitrate reduction in the Yangtze Estuary, as compared to 3-45% for DNRA and 1-36% for ANAMMOX. In total, the denitrification and ANAMMOX processes removed approximately 25% of the external inorganic nitrogen transported annually into the estuary. In contrast, most external inorganic nitrogen was retained in the estuary and contributes substantially to the severe eutrophication of the Yangtze Estuary.

Ammonia oxidation plays a pivotal role in the cycling and removal of nitrogen in aquatic sediments. Certain bacterial groups and a novel group of archaea, which is affiliated with the novel phylum Thaumarchaeota, can perform this initial nitrification step. We examined the diversity and abundance of ammonia-oxidizing β-Proteobacteria (β-AOB) and ammonia-oxidizing archaea (AOA) in the sediments of Chongming eastern tidal flat using the ammonia monooxygenase-α subunit (amoA) gene as functional markers. Clone library analysis showed that AOA had a higher diversity of amoA gene than β-AOB. The β-Proteobacterial amoA community composition correlated significantly with water soluble salts in the sediments, whereas the archaeal amoA community composition was correlated more with nitrate concentrations. Quantitative PCR (qPCR) results indicated that the abundance of β-AOB amoA gene (9.11 × 10(4)-6.47 × 10(5) copies g(-1) sediment) was always greater than that of AOA amoA gene (7.98 × 10(3)-3.51 × 10(5) copies g(-1) sediment) in all the samples analyzed in this study. The β-Proteobacterial amoA gene abundance was closely related to organic carbon, while no significant correlations were observed between archaeal amoA gene abundance and the environmental factors. Potential nitrification rates were significantly greater in summer than in winter and correlated strongly with the abundance of amoA genes. Additionally, a greater contribution of single amoA gene to potential nitrification occurred in summer (1.03-5.39 pmol N copy(-1) day(-1)) compared with winter (0.16-0.38 pmol N copy(-1) day(-1)), suggesting a higher activity of ammonia-oxidizing prokaryotes in warm seasons.

Silver nanoparticles inhibit nitrification but enhance nitrous oxide production by nitrifying organisms.

Complete ammonia oxidizers (comammox), as novel microbial communities, are predicted to play an important role in the nitrogen cycle. Here we reported the presence of complete nitrification in tidal sediments and examined the diversity and abundance of comammox in natural ecosystems. Metagenome and metatranscriptome of the enrichment culture from tidal sediments harbored the genes of comammox. Near-complete comammox AmoA/B/C- and Hao-like sequences showed close relationships to the known comammox (with sequence identity from 79 to 99%) rather than classical betaproteobacterial ammonia-oxidizing bacteria (β-AOB) (57 to 66%) and ammonia-oxidizing archaea (AOA) (24 to 38%). To analyze the diversity of comammox in natural environments, a new primer set targeting clade A comammox Nitrospira (COM-A) amoA genes was designed based on sequences obtained in this study and sequences from published database. In silico evaluation of the primers showed the high coverage of 89 and 100% in the COM-A amoA database. Application of the primers in six different ecosystems proved their strong availability. Community composition of COM-A suggested a relatively higher diversity than β-AOB in similar environments. Quantification results showed that COM-A amoA genes accounted for about 0.4-5.6% in total amoA genes. These results provide novel insight into our perception of the enigmatic comammox and have significant implications for profound understanding of complex nitrification process.

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Denitrification plays a critical role in nitrogen removal in estuarine and coastal ecosystems. In this study, the community composition, diversity, abundance, and distribution of cytochrome cd1-type nitrite reductase gene (nirS)-harboring denitrifiers in intertidal sediments of the Yangtze Estuary were analyzed using polymerase chain reaction (PCR)-based clone libraries and quantitative PCR techniques. Clone library analysis showed that the nirS-encoding bacterial biodiversity was significantly higher at the lower salinity sites than at the higher salinity sites. However, there was no significant seasonal difference in the nirS gene diversity between summer and winter. Phylogenetic analysis revealed that the nirS-harboring denitrifier communities at the study area had distinctive spatial heterogeneity along the estuary. At the lower salinity sites, the nirS-harboring bacterial community was co-dominated by clusters III and VII; while at the higher salinity sites, it was dominated by cluster I. Canonical correspondence analysis indicated that the community compositions of nirS-type denitrifiers were significantly correlated with salinity, ammonium, and nitrate. Quantitative PCR results showed that the nirS gene abundance was in the range of 1.01 × 10(6) to 9.00 × 10(7) copies per gram dry sediment, without significant seasonal variation. Among all the environmental factors, the nirS gene abundance was only significantly related to the change of salinity. These results can extend our current knowledge about the composition and dynamics of denitrification microbial community in the estuarine ecosystem.