Yanling Zheng

Community Dynamics and Activity of Ammonia-Oxidizing Prokaryotes in Intertidal Sediments of the Yangtze Estuary

ABSTRACT Diversity, abundance, and activity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using the ammonia monooxygenase α 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.

Effects of sulfamethazine on denitrification and the associated N2O release in estuarine and coastal sediments.

Denitrification is an important pathway of nitrogen removal and nitrous oxide (N2O) production in estuarine and coastal ecosystems, and plays a significant role in counteracting aquatic eutrophication induced by excessive nitrogen loads. Estuarine and coastal environments also suffer from increasing antibiotic contamination because of the growing production and usage of antibiotics. In this study, sediment slurry incubation experiments were conducted to determine the influence of sulfamethazine (SMT, a sulphonamide antibiotic) on denitrification and the associated N2O production. Genes important for denitrification and antibiotic resistance were quantified to investigate the microbial physiological mechanisms underlying SMTs effects on denitrification. SMT was observed to significantly inhibit denitrification rates, but increasing concentrations of SMT enhanced N2O release rates. The negative exponential relationships between denitrifying gene abundances and SMT concentrations showed that SMT reduced denitrification rates by restricting the growth of denitrifying bacteria, although the presence of the antibiotic resistance gene was detected during the incubation period. These results imply that the wide occurrence of residual antibiotics in estuarine and coastal ecosystems may influence eutrophication control, greenhouse effects, and atmospheric ozone depletion by inhibiting denitrification and stimulating the release of N2O.

Dissimilatory nitrate reduction processes and associated contribution to nitrogen removal in sediments of the Yangtze Estuary

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−1 h−1, 0.01 to 0.52 µmol N kg−1 h−1, and 0.03 to 0.89 µmol N kg−1 h−1, 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.

Anaerobic ammonium oxidation and its contribution to nitrogen removal in China's coastal wetlands.

Over the past several decades, human activities have caused substantial enrichment of reactive nitrogen in China’s coastal wetlands. Although anaerobic ammonium oxidation (anammox), the process of oxidizing ammonium into dinitrogen gas through the reduction of nitrite, is identified as an important process for removing reactive nitrogen, little is known about the dynamics of anammox and its contribution to nitrogen removal in nitrogen-enriched environments. Here, we examine potential rates of anammox and associate them with bacterial diversity and abundance across the coastal wetlands of China using molecular and isotope tracing techniques. High anammox bacterial diversity was detected in China’s coastal wetlands and included Candidatus Scalindua, Kuenenia, Brocadia, and Jettenia. Potential anammox rates were more closely associated with the abundance of anammox bacteria than to their diversity. Among all measured environmental variables, temperature was a key environmental factor, causing a latitudinal distribution of the anammox bacterial community composition, biodiversity and activity along the coastal wetlands of China. Based on nitrogen isotope tracing experiments, anammox was estimated to account for approximately 3.8–10.7% of the total reactive nitrogen removal in the study area. Combined with denitrification, anammox can remove 20.7% of the total external terrigenous inorganic nitrogen annually transported into China’s coastal wetland ecosystems.

Evidence of Nitrogen Loss from Anaerobic Ammonium Oxidation Coupled with Ferric Iron Reduction in an Intertidal Wetland

Anaerobic ammonium oxidation coupled with nitrite reduction is an important microbial pathway of nitrogen removal in intertidal wetlands. However, little is known about the role of anaerobic ammonium oxidation coupled with ferric iron reduction (termed Feammox) in intertidal nitrogen cycling. In this study, sediment slurry incubation experiments were combined with an isotope-tracing technique to examine the dynamics of Feammox and its association with tidal fluctuations in the intertidal wetland of the Yangtze Estuary. Feammox was detected in the intertidal wetland sediments, with potential rates of 0.24-0.36 mg N kg(-1) d(-1). The Feammox rates in the sediments were generally higher during spring tides than during neap tides. The tidal fluctuations affected the growth of iron-reducing bacteria and reduction of ferric iron, which mediated Feammox activity and the associated nitrogen loss from intertidal wetlands to the atmosphere. An estimated loss of 11.5-18 t N km(-2) year(-1) was linked to Feammox, accounting for approximately 3.1-4.9% of the total external inorganic nitrogen transported into the Yangtze Estuary wetland each year. Overall, the co-occurrence of ferric iron reduction and ammonium oxidation suggests that Feammox can act as an ammonium removal mechanism in intertidal wetlands.

Tidal pumping facilitates dissimilatory nitrate reduction in intertidal marshes

Intertidal marshes are alternately exposed and submerged due to periodic ebb and flood tides. The tidal cycle is important in controlling the biogeochemical processes of these ecosystems. Intertidal sediments are important hotspots of dissimilatory nitrate reduction and interacting nitrogen cycling microorganisms, but the effect of tides on dissimilatory nitrate reduction, including denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium, remains unexplored in these habitats. Here, we use isotope-tracing and molecular approaches simultaneously to show that both nitrate-reduction activities and associated functional bacterial abundances are enhanced at the sediment-tidal water interface and at the tide-induced groundwater fluctuating layer. This pattern suggests that tidal pumping may sustain dissimilatory nitrate reduction in intertidal zones. The tidal effect is supported further by nutrient profiles, fluctuations in nitrogen components over flood-ebb tidal cycles, and tidal simulation experiments. This study demonstrates the importance of tides in regulating the dynamics of dissimilatory nitrate-reducing pathways and thus provides new insights into the biogeochemical cycles of nitrogen and other elements in intertidal marshes.

Diversity, Abundance, and Distribution of nirS-Harboring Denitrifiers in Intertidal Sediments of the Yangtze Estuary

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 × 106 to 9.00 × 107 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.

Net anthropogenic nitrogen inputs (NANI) into the Yangtze River basin and the relationship with riverine nitrogen export

This study investigated net anthropogenic nitrogen inputs (NANI, including atmospheric nitrogen deposition, nitrogenous fertilizer use, net nitrogen import in food and feed, and agricultural nitrogen fixation) and the associated relationship with riverine dissolved inorganic nitrogen (DIN) export in the Yangtze River basin during the 1980–2012 period. The total NANI in the Yangtze River basin has increased by more than twofold over the past three decades (3537.0 ± 615.3 to 8176.6 ± 1442.1 kg N km−2 yr−1). The application of chemical fertilizer was the largest component of NANI in the basin (51.1%), followed by net nitrogen import in food and feed (26.0%), atmospheric nitrogen deposition (13.2%), and agricultural nitrogen fixation (9.7%). A regression analysis showed that the riverine DIN export was strongly correlated with NANI and the annual water discharge (R2 = 0.90, p < 0.01). NANI in the Yangtze River basin was estimated to contribute 37–66% to the riverine DIN export. We also forecasted future variations in NANI and riverine DIN export for the years 2013 to 2030, based on possible future changes in human activities and the climate. This work provides a quantitative understanding of NANI in the Yangtze River basin and its effects on riverine DIN export and helps to develop integrated watershed nitrogen management strategies.

Nitrogen mineralization and immobilization in sediments of the East China Sea: Spatiotemporal variations and environmental implications

Nitrogen (N) mineralization and immobilization are important processes of N biogeochemical cycle in marine sediments. This study investigated gross N mineralization (GNM) and NH4+ immobilization (GAI) in the sediments from the East China Sea (ESC), using 15N stable isotope dilution technique. Results show that measured rates of GNM and GAI ranged from 0.04 to 6.1 µg N g−1 d−1 and from undetectable to 9.82 µg N g−1 d−1, respectively. In general, both GNM and GAI rates were significantly greater in summer as compared to winter, and the high rates occurred mainly in the muddy area and increased gradually from the Yangtze Estuary to Zhe-Min Coastal muddy areas. The GNM and GAI processes were related closely to sediment temperature, pH, ammonium (NH4+), nitrate (NO3−), total organic carbon (TOC), and total nitrogen (TN) contents in the muddy area, while they were associated tightly with sediment temperature, pH, NH4+, TOC, TN, sulfide, and Fe(III) concentrations in the sandy area. In addition, the total mineralized and immobilized N in the East China Sea (ECS) were estimated to be approximately 2.1 × 106 t N yr−1 and 2.7 × 106 t N yr−1, respectively. Overall, these results highlight the importance of N mineralization and immobilization in controlling the N budget in the ECS and improve the understanding of both processes and associated controlling mechanisms in the coastal marine ecosystem.

Effects of thiamphenicol on nitrate reduction and N2O release in estuarine and coastal sediments.

Nitrate overload is an important driver of water pollution in most estuarine and coastal ecosystems, and thus nitrate reduction processes have attracted considerable attention. Antibiotics contamination is also an emerging environmental problem in estuarine and coastal regions as a result of growing production and usage of antibiotics. However, the effects of antibiotics on nitrate reduction remain unclear in these aquatic ecosystems. In this study, continuous-flow experiments were conducted to examine the effects of thiamphenicol (TAP, a common chloramphenicol antibiotic) on nitrate reduction and greenhouse gas N2O release. Functional genes involved in nitrogen transformation were also quantified to explore the microbial mechanisms of the TAP influence. Production of N2 were observed to be inhibited by TAP treatment, which implied the inhibition effect of TAP on nitrate reduction processes. As intermediate products of nitrogen transformation processes, nitrite and N2O were observed to accumulate during the incubation. Different TAP inhibition effects on related functional genes may be the microbial mechanism for the changes of nutrient fluxes, N2 fluxes and N2O release rates. These results indicate that the antibiotics residues in estuarine and coastal ecosystems may contribute to nitrate retention and N2O release, which could be a major factor responsible for eutrophication and greenhouse effects.