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Denitrification is an important pathway for reactive nitrogen removal from aquatic ecosystems. In this study, the biodiversity, abundance, and activity of cytochrome cd 1 -type nitrate reductase gene (nirS)-harboring denitrifiers in the sediments of the Indus River Estuary were examined by molecular and isotope-tracing techniques. Results showed that the nirS -harboring denitrifier communities showed significant geographical variations along the estuarine salinity gradient. Real-time quantitative PCR showed that the abundance of nirS -harboring denitrifiers ranged from 5.3 × 106 to 2.5 × 108 copies g−1, without significant spatiotemporal variation. The potential rates of denitrification varied from 0.01 to 6.27 μmol N kg−1 h−1 and correlated significantly to TOC and Fe(II) (P < 0.05). On the basis of 15N isotope-tracing experiments, the denitrification process contributed 18.4–99.4% to the total nitrogen loss in the sediments of the Indus River Estuary. This study provides novel insights into the microbial mechanism of nitrogen removal process in estuarine ecosystems. Unlabelled Image • nirS -type denitrifier community had significant spatial variation along the estuary. • Abundance of nirS -type denitrifier ranged from 5.3 × 106 to 2.5 × 108 copies g−1. • The potential rates of denitrification varied from 0.01 to 6.27 μmol N kg−1 h−1. • Denitrification contributed up to 99% to total N loss in the Indus River Estuary. [ABSTRACT FROM AUTHOR]

Denitrification is a dominant reactive nitrogen removal pathway in most estuarine and coastal ecosystems, and plays a significant role in regulating N2O release. Although multiple antibiotics residues are widely detected in aquatic environment, combined effects of antibiotics on denitrification remain indistinct. In this work, 5 classes of antibiotics (sulfonamides, chloramphenicols, tetracyclines, macrolides, and fluoroquinolones) were selected to conduct orthogonal experiments in order to explore their combined effects on denitrification. 15N-based denitrification and N2O release rates were determined in the orthogonal experiments, while denitrifying functional genes were examined to illustrate the microbial mechanism of the combined antibiotics effect. Denitrification rates were inhibited by antibiotics treatments, and synergistic inhibition effect was observed for multiple antibiotics exposure. Different classes of antibiotics had different influence on N2O release rates, but multiple antibiotics exposure mostly led to stimulatory effect. Abundances of denitrifying functional genes were inhibited by multiple antibiotics exposure due to the antimicrobial properties, and different inhibition on denitrifiers may be the major mechanism for the variations of N2O release rates. Combined effects of antibiotics on denitrification may lead to nitrate retention and N2O release in estuarine and coastal ecosystems, and consequently cause cascading environmental problems, such as greenhouse effects and hyper-eutrophication. [ABSTRACT FROM AUTHOR]

Anaerobic ammonium oxidation (anammox) is an important bioprocess for nitrogen removal and has been studied in estuarine environments. However, knowledge on anammox bacterial community dynamics and related controlling factors remains limited in these ecosystems. In this study, the community compositions, abundance, and activity of anammox bacteria in the surface sediments from the Indus Estuary were investigated along a salinity gradient, considering the links between the anammox bacterial community dynamics and environmental variables. The potential importance of anammox was also estimated for nitrogen removal. High anammox bacterial diversity was detected in the sediments of the Indus Estuary, including Kuenenia , Brocadia , Scalindua , Jettenia , and a novel anammox-like cluster. Kuenenia was identified as the dominant anammox bacteria in most samples. Anammox bacterial diversity was significantly correlated with sediment NO 3 −, while the distribution of anammox bacterial community was significantly related to temperature and sediment sulfide (P < 0.05). The anammox bacterial abundance based on the 16S rRNA gene varied between 1.64 × 106 copies g−1 and 8.21 × 108 copies g−1, and was significantly correlated with sediment Fe(II). Based on an 15N isotope-tracing technique, potential anammox rates were found in the range 0.01–0.32 μ mol N kg−1 h−1, and were controlled mainly by salinity, Fe(II), and TOC. It was estimated that the anammox bacteria contributed about 21.9% to the total nitrogen loss, on average. These results show the importance of anammox bacteria for nitrogen transformation and removal in estuarine and coastal environments. Image 1 • High diversity of anammox bacteria was detected in the estuarine environments. • Anammox bacterial diversity was associated with sediment NO 3 − • Distribution of anammox bacteria was related to temperature and sediment sulfide. • Anammox bacteria played an important role in nitrogen removal. [ABSTRACT FROM AUTHOR]

Global warming and acidification, induced by a substantial increase in anthropogenic CO 2 emissions, are expected to have profound impacts on biogeochemical cycles. However, underlying mechanisms of nitrous oxide (N 2 O) production in estuarine and coastal sediments remain rarely constrained under warming and acidification. Here, the responses of sediment N 2 O production pathways to warming and acidification were examined using a series of anoxic incubation experiments. Denitrification and N 2 O production were largely stimulated by the warming, while N 2 O production decreased under the acidification as well as the denitrification rate and electron transfer efficiency. Compared to warming alone, the combination of warming and acidification decreased N 2 O production by 26 ± 4%, which was mainly attributed to the decline of the N 2 O yield by fungal denitrification. Fungal denitrification was mainly responsible for N 2 O production under the warming condition, while bacterial denitrification predominated N 2 O production under the acidification condition. The reduced site preference of N 2 O under acidification reflects that the dominant pathways of N 2 O production were likely shifted from fungal to bacterial denitrification. In addition, acidification decreased the diversity and abundance of nirS -type denitrifiers, which were the keystone taxa mediating the low N 2 O production. Collectively, acidification can decrease sediment N 2 O yield through shifting the responsible production pathways, partly counteracting the warming-induced increase in N 2 O emissions, further reducing the positive climate warming feedback loop.

Nitrous oxide (N 2 O) emissions from lakes exhibit significant spatiotemporal heterogeneity, and quantitative identification of the different N 2 O production processes is greatly limited, causing the role of nitrification to be undervalued or ignored in models of a lake's N 2 O emissions. Here, the contributions of nitrification and denitrification to N 2 O production were quantitatively assessed in the eutrophic Lake Taihu using molecular biology and isotope mapping techniques. The N 2 O fluxes ranged from -41.48 to 28.84 μmol m -2 d -1 in the lake, with lower N 2 O concentrations being observed in spring and summer and significantly higher N 2 O emissions being observed in autumn and winter. The 15 N site preference and relevant isotopic evidence demonstrated that denitrification contributed approximately 90% of the lake's gross N 2 O production during summer and autumn, 27-83% of which was simultaneously eliminated via N 2 O reduction. Surprisingly, nitrification seemed to act as a key process promoting N 2 O production and contributing to the lake as a source of N 2 O emissions. A combination of N 2 O isotopocule-based approaches and molecular techniques can be used to determine the precise characteristics of microbial N 2 O production and consumption in eutrophic lakes. The results of this study provide a basis for accurately assessing N 2 O emissions from lakes at the regional and global scales.

Estuarine and coastal wetland ecosystems are important sources of atmospheric nitrous oxide (N 2 O). However, the underlying driver of emitted N 2 O from estuarine and coastal wetlands remains poorly understood. Here, natural-abundance isotope technique was applied to characterize the processes responsible for N 2 O emission from the intertidal soils of the Yangtze Estuary. Measured N 2 O emission rates ranged from 0.70 to 2.15 μmol m - 2  h - 1 , with relatively high values at the upper estuarine sites. The δ 15 N, δ 18 O and SP (intramolecular 15 N site preference) of emitted N 2 O varied from -4.5 to 6.7‰, 42.4 to 53.2‰, and 6.7 to 15.4‰, respectively. Gross N 2 O production and consumption rates were within the ranges of 3.16-14.34 μmol m - 2  h - 1 and 2.22-12.54 μmol m - 2  h - 1 , respectively, showing a similar spatial pattern to N 2 O emission. N 2 O consumption proportion varied from 69.56 to 90.31%, which was generally lower at the upper estuarine sites. The gross production rates and consumption degree of N 2 O simultaneously controlled the variations in N 2 O emission. Bacterial denitrification was the dominant production pathway (78.22-97.36%), while hydroxylamine (NH 2 OH) oxidation contributed 2.64-21.78% to N 2 O production. Soil pH, Fe 2+ /Fe 3+ , sulfide and substrate availability were probably the main factors governing the N 2 O emission dynamics. Overall, these results highlight the substantial role of NH 2 OH oxidation and N 2 O consumption in N 2 O release in redox-dynamic soils of estuarine intertidal wetlands., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 SMT's 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.

Nitrogen (N) pollution in aquatic ecosystems has attracted much attention over the past decades, but the dynamics of this bioreactive element are difficult to measure in aquatic oxygen-transition environments. Nitrogen-transformation experiments often require measurement of (15)N-ammonium ((15)NH4(+)) ratios in small-volume (15)N-enriched samples. Published methods to determine N isotope ratios of dissolved ammonium require large samples and/or costly equipment and effort. We present a novel ("OX/MIMS") method to determine N isotope ratios for (15)NH4(+) in experimental waters previously enriched with (15)N compounds. Dissolved reduced (15)N (dominated by (15)NH4(+)) is oxidized with hypobromite iodine to nitrogen gas ((29)N2 and/or (30)N2) and analyzed by membrane inlet mass spectrometry (MIMS) to quantify (15)NH4(+) concentrations. The N isotope ratios, obtained by comparing the (15)NH4(+) to total ammonium (via autoanalyzer) concentrations, are compared to the ratios of prepared standards. The OX/MIMS method requires only small sample volumes of water (ca. 12 mL) or sediment slurries and is rapid, convenient, accurate, and precise (R(2) = 0.9994, p < 0.0001) over a range of salinities and (15)N/(14)N ratios. It can provide data needed to quantify rates of ammonium regeneration, potential ammonium uptake, and dissimilatory nitrate reduction to ammonium (DNRA). Isotope ratio results agreed closely (R = 0.998, P = 0.001) with those determined independently by isotope ratio mass spectrometry for DNRA measurements or by ammonium isotope retention time shift liquid chromatography for water-column N-cycling experiments. Application of OX/MIMS should simplify experimental approaches and improve understanding of N-cycling rates and fate in a variety of freshwater and marine environments.