| Over the past few decades, global nitrogen overload in aquatic ecosystems is an important environmental issue, due mainly to the excessive input of anthropogenic nitrogen. The anthropogenic perturbation of nitrogen cycle continues to cause serious environmental problems, ranging from severe eutrophication, frequent occurrences of harmful algal blooms to hypoxia. Thus, the biogeochemical cycle of nitrogen in estuarine and coastal regions has been paid more attentions recently.Aerobic and anaerobic ammonium oxidations, which are the key processes of the whole nitrogen cycle, play an important role in the biogeochemical cycle of nitrogen. Aerobic ammonium oxidation, oxidizes ammonium into nitrite, is the first and rate-limiting step of nitrification. Anaerobic ammonium oxidation, oxidizes ammonium via reducing nitrite into dinitrogen under anaerobic conditions, is a newly discovered and important nitrogen removal pathway. However, knowledge about aerobic/anaerobic ammonium oxidation processes and their associated microbial dynamics remain scarce in estuarine and coastal ecosystems. Thus, the Yangtze Estuarine intertidal wetland was selected as a typical study area to study the aerobic and anaerobic ammonium oxidation microbial diversity, abundance, and activity. This work may provide novel insights into the biogeochemical cycle of nitrogen in estuarine and coastal areas, and it also can help to enhance the environmental protection and nitrogen-pollution control of the Yangtze Estuary. The main findings are as following.(1) Based on amoA gene, the diversity, abundance and activity of ammonium oxidizing bacteria (AOB) and ammonium oxidizing archaea (AOA) were investigated in the surface intertidal sediments of the Yangtze Estuary. Clone library analysis revealed that AOB communities were dominated by Nitrosomonas, while AOA communities were dominated by the Marine group 1.1a. 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. Generally, AOB had a lower diversity of amoA gene than AOA in this study. Quantitative PCR results indicated that the abundance of AOB amoA genes (7.36 × 104～5.20×106 copies g-1) was greater than that of AOA amoA genes (5.70 ×104～6.84 × 105 copies g-1) in 10 of the 14 samples analyzed in this study. Potential nitrification rates were significantly greater in summer (4.2～186.3 nmol N g-1 h-1) than in winter (2.9-30.8 nmol N g-1 h-1) and had a significant negative correlation with salinity. In addition, potential nitrification rates were only correlated strongly with archaeal amoA gene abundance but not with bacterial amoA gene abundance. The ampicillin inhibition experiment showed that AOA contributed 73.4%～86.9% to the aerobic oxidation of ammonium to nitrite in the surface sediments of the Yangtze Estuary.(2) Using molecular and isotope-tracing techniques, we studied the anammox bacterial diversity, abundance and activity in the intertidal sediments of the Yangtze Estuary. Phylogenetic analysis 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. The abundance of anammox bacteria in the surface sediments ranged from 2.63 × 10 and 1.56 × 10 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. On the basis of the 15N tracing experiments, the anammox process was estimated to contribute 6.6%～12.9% to the total nitrogen loss.(3) The intertidal flat can be divided seaward into three different habitats:the high, middle, and low tidal flats. The tide has different effects on different tidal habitats, which may affect the ammonium oxidation rate and the associated functional microorganisms. Here, we analyzed the aerobic/anaerobic ammonium oxidation processes at different habitats. Clone library analysis showed that the diversity of AOB increased from the high tidal flat to the low tidal flat, while the diversity of AOA decreased from the high tidal flat to the low tidal flat. Quantitative PCR results indicated that the abundance of amoA gene increased from the high tidal flat to the low tidal flat in summer, while decreased from the high tidal flat to the low tidal flat in winter. Potential nitrification rate showed the same spatial distribution pattern as the amoA gene abundance. There were different anammox bacterial genera in different tidal flats:Scalindua was the predominant group in the low tidal flat, while Kuenenia was the major population in the high tidal flat and the middle tidal flat. In comparison with the high and low tidal flats, the community structure of anammox bacteria in the middle tidal flat was the most complicated. The abundance of anammox bacterial 16S rRNA gene increased from the high tidal flat to the low tidal flat, which was the same as the anammox rate.(4) The tidal effect can help regulate the transport and transformation of nutrients near the sediment-tidal water interface (SWI). Also, tidal dynamics can induce groundwater table fluctuation, which may contribute to the pulse input of groundwater-transported nutrients into deeper sediment depths in the marshes. Thus, tidal effect may serve as an important driver of anaerobic ammonium oxidation in intertidal zones. The depth distribution of anammox activity indicated that the highest potential rates (0.61 nmol N g-1 in April and 0.66 nmol N g-1 in October) appeared in the surface sediments. Below the surface, anammox decreased with depth to the lowest values (0.16 nmolN g-1h-1 in April and 0.28 nmol N g-1h-1 in October) at 60-70 cm. However, anammox activity was stimulated again in the groundwater fluctuating layer. In addition, the vertical distribution pattern of anammox bacterial abundance was similar to that of the anammox rate, and the elevated activity and abundance at depth may be attributed to the tidal effect. Although anammox bacterial biodiversity was not significantly affected, our results confirmed that tidal effect may be a newly-discovered driving mechanism of anammox process in intertidal marshes. The tidal effect was further supported by nutrient profiles, tidal-induced groundwater fluctuation, and a tidal simulation experiment. |
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