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Rivers play a significant role in the global nitrous oxide (N 2 O) budget. However, the microbial sources and sinks of N 2 O in river systems are not well understood or quantified, resulting in the prolonged neglect of nitrification. This study investigated the isotopic signatures of N 2 O, thereby quantifying the microbial source of N 2 O production and the degree of N 2 O reduction in the Yellow River. Although denitrification has long been considered to be the dominant pathway of N 2 O production in rivers, our findings indicated that denitrification only accounted for 18.3% (8.2%-43.0%) of the total contribution to N 2 O production in the Yellow River, with 50.2%-80.2% being concurrently reduced. The denitrification contribution to N 2 O production (R 2  = 0.44, p < 0.01) and N 2 O reduction degree (R 2  = 0.70, p < 0.01) were positively related to the dissolved organic carbon (DOC) content. Similar to urban rivers and eutrophic lakes, denitrification was the primary process responsible for N 2 O production (43.0%) in certain reaches with high organic content (DOC = 5.29 mg/L). Nevertheless, the denitrification activity was generally constrained by the availability of electron donors (average DOC = 2.51 mg/L) throughout the Yellow River basin. Consequently, nitrification emerged as the primary contributor in the well-oxygenated Yellow River. Additionally, our findings further distinguished the respective contribution of ammonia-oxidizing bacteria (AOB) and archaea (AOA) to N 2 O emissions. Although AOB dominated the N 2 O production in the Yellow River, the AOA specie abundance (AOA/(AOA + AOB)) contributed up to 32.6%, which resulted in 25.6% of the total nitrifier-produced N 2 O, suggesting a significant occurrence of AOA in the oligotrophic Yellow River. Overall, this study provided a non-invasive approach for quantifying the microbial sources and sinks to N 2 O emissions, and demonstrated the substantial role of nitrification in the large oligotrophic rivers., 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 © 2024 Elsevier Ltd. All rights reserved.)

Nitrate pollution in fresh water is becoming increasingly serious. In this study, the effects of temperature and graphene oxide materials on the potential functions of denitrification communities in lake sediments were investigated by metagenome. The addition of graphene oxide significantly affected the abundance of denitrification genes such as Nap, Nos, and enhanced the contribution of Pseudomonas, making low temperature and material addition conducive to the denitrification process. Module network implied that low temperature increased the centrality of denitrification in community functions. At low temperatures, graphene oxide enhanced community anabolism by stimulation organic carbon consumption and regulating the gene abundance in the citric acid cycle and the semi-phosphorylation Entner-Doudoroff, thus possibly stimulating extracellular polymeric substances (EPS) synthesis and secretion. In addition, graphene oxide may also regulate the transfer of reducing electrons from NADH to denitrifying enzymes by affecting the gene abundances of complex I and complex IV., 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 © 2024 Elsevier Ltd. All rights reserved.)

Iron-dependent denitrification is a safe and promising technology for nitrogen removal in freshwaters. However, the understanding of microbial physiology and interactions during the process was limited. Denitrifying systems inoculated with freshwater samples were operated with and without iron(II) at a low C/N ratio for 54 days. Iron addition improved nitrogen removal. Batch experiments confirmed that microbially mediated reaction rather than abiotic reaction dominated during the process. Metagenomics recovered genomes of the five most abundant microorganisms, which accounted for over 99% of the community in every triplicate of the iron-based system. Based on codon usage bias, all of them were fast-growing organisms. The total abundance of fast-growing organisms was 38% higher in the system with iron than in the system without iron. Notably, the most abundant organism Diaphorobacter did not have enzymes for asparagine and aspartate biosynthesis, whereas Rhodanobacter could not produce serine and cobalamin. Algoriphagus and Areminomonas lost synthesis enzymes for more amino acids and vitamins. However, they could always obtain these growth-required substances from another microorganism in the community. The two-partner relationship minimized the limitation on microbial reproduction and increased community stability. Our results indicated that iron addition improved nitrogen removal by supplying electron donors, promoting microbial growth, and building up syntrophic interactions among microorganisms with timely communications. The findings provided new insights into the process, with implications for freshwater remediation., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Denitrification is one of the major sources of N 2 O in freshwaters. Diverse forms of organic compounds act as the electron donors for microbial denitrification. However, the influences of carbon sources on N 2 O production, N 2 O reduction, isotope fractionation and functional microbes during denitrification were largely unknown. In this study, five forms of carbon sources (i.e. acetate, citrate, glucose, cellobiose and leucine) were used to enrich denitrifiers in freshwater sediments. N 2 O conversion in the enrichments was investigated by a combination of inhibition technique, natural stable isotope method and metagenomics. Acetylene was effective in inhibiting N 2 O reduction without influencing the isotopic characteristics during N 2 O production. Glucose led to the least N 2 O production and reduction, in accordance with the lowest abundance of both NO and N 2 O reductases in this enrichment. δ 18 O and site preference value (SP, =δ 15 N α -δ 15 N β ) of N 2 O were sensitive to discriminate the five carbon sources, except when comparing acetate and leucine. Isotopic values of N 2 O were not significantly different in these two enrichments due to the similarity of NO reductases - Pseudomonas-type cNorB. Specifically, the enrichment with cellobiose produced N 2 O with the lowest δ 18 O values (39.4‰±1.1‰), due to Alicycliphilus with both cNorB and qNorB. The enrichment with glucose led to the highest SP values (8.9‰±8.6‰), caused by Thiobacillus-type cNorB. Our results demonstrated the link between carbon sources, N 2 O production and reduction, isotopic signatures, microbial populations and enzymes during denitrification in freshwaters., 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 © 2022 Elsevier Ltd. All rights reserved.)

As an emerging material, graphene oxide (GO) has been widely used in recent years and will inevitably enter into natural water bodies, and it may have an impact on lake microbial communities owing to its potential toxicity and denitrification-enhancing ability. This study simulated the effect of 0.1 g/L GO on denitrification in lake sediments under summer (28 °C) and winter temperatures (8 °C). GO promoted carbon source metabolism and denitrification. Phylogenetic bin-based null model analysis suggested that GO significantly altered the contribution of heterogeneous selection in bacterial and archaeal community assembly. The co-occurrence network indicated that bacterial communities responded to the enhancement of heterogeneous selection by strategies of enhancing positive correlation and shared niche, whereas archaeal communities adopted strategies of enhancing negative correlation and competition. Bacterial networks also emerged with more non-hub connector species that could drive changes in community structure. Our study contributed to the understanding of different ecological strategies adopted by bacterial and archaeal communities in response to changes in ecological selection driven by GO., 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 © 2022 Elsevier B.V. All rights reserved.)

This study found that graphene oxide (GO) improved microbial denitrification at low temperatures (~12 °C), and the optimal concentration was 10 mg/L as the removal rate of NO 3 -N increased by 17%. At the optimal concentration, GO improved the electron transport system activity of the microbes and enhanced the activity of nitrate reductase and nitrite reductase while exhibited low microbial toxicity. The addition of GO increased the content of tightly bound extracellular polymeric substances (EPS). The results of fluorescence spectrometer indicated that GO accelerated the renewal of bound EPS (B-EPS). Fourier Transform infrared spectroscopy (FTIR) results showed that GO affected the secondary structure of the protein in B-EPS, making B-EPS more hydrophobic and promoting microbial aggregation. B-EPS affected by GO can promote the electron transfer process of microorganisms. However, high concentration (>25 mg/L) of GO may inhibit denitrification by competing for electrons, which was not conducive to denitrification thermodynamically., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The stable isotope technique has been used in tracking nitrogen cycling processes, but the isotopic characteristics are influenced by environmental conditions. To better understand the variability of nitrate isotopes in nature, we investigated the influence of organic carbon sources on isotope fractionation characteristics during microbial denitrification. Denitrifying cultures were inoculated with freshwater samples and enriched with five forms of organic compounds, that is, acetate, citrate, glucose, cellobiose, and leucine. Though the isotope enrichment factors of nitrogen and oxygen (15ε and 18ε) changed with carbon sources, 18ε/15ε always followed a proportionality near 1. Genome‐centred metagenomics revealed the enrichment of a few populations, such as Pseudomonas, Enterobacter, and Atlantibacter, most of which contained both NapA‐ and NarG‐type nitrate reductases. Metatranscriptome showed that both NapA and NarG were expressed but to different extents in the enrichments. Furthermore, isotopic data collected from a deep reservoir was analysed. The results showed δ18O‐ and δ15N‐nitrate did not correlate in the surface water where nitrification was active, but 18ε/15ε followed a proportionality of 1.05 ± 011 in deeper waters (≥ 12 m) where denitrification controlled the nitrate isotope. The independence of 18ε/15ε from carbon sources provides an opportunity to determine heterotrophic denitrification and helps the interpretation of nitrate isotopes in freshwaters. [ABSTRACT FROM AUTHOR]

Sulphur‐driven denitrification is a low‐cost process for the treatment of nitrate‐contaminated water. However, a comprehensive understanding of core populations and microbial interactions of a sulphur‐based denitrifying system is lacking. This study presents results from three replicated denitrifying systems amended with thiosulphate and operated under a low C/N ratio. Amplicon sequencing revealed gradual enrichments of a few abundant denitrifiers. Based on genome‐centred metagenomics and metatranscriptomics, a core set of microbes was identified in the systems, with Pseudomonas 1 and Thauera 2 being the most abundant ones. Although the replicates showed different enrichments, generalized observations were summarized. Most core populations conserved energy from denitrification coupled with sulphur. Pseudomonas 1 and Thauera 2 were able to finish complete denitrification. Surprisingly, they were also able to synthesize almost all amino acids and vitamins. In contrast, less abundant members, including Pseudomonas 2, were relatively auxotrophic and required an exogenous supply of amino acids and vitamins. The high expression of enzymes involved in biosynthesis and transport systems indicated their syntrophic relationships. The genomic findings suggested life strategies and interactions of the core thiosulphate‐based denitrifying microbiome, with implications for nitrate‐polluted water remediation. [ABSTRACT FROM AUTHOR]

Microbial nitrate reduction can be driven by organic carbon oxidation, as well as by inorganic electron donors, such as reduced forms of sulfur and iron. An apparent inverse oxygen isotope fractionation effect was observed during nitrate reduction in sediment incubations from five sampling sites of a freshwater lake, Hongze Lake, China. Incubations with organic and inorganic electron donor additions were performed. Especially, the inverse oxygen isotope effect was intensified after glucose addition, whereas the incubations with sulfide and Fe2+ showed normal fractionation factors. Nitrate reductase encoding genes, napA and narG, were analysed with metagenomics. Higher napA/narG ratios were associated with higher oxygen fractionation factors. The most abundant clade (59%) of NapA in the incubation with glucose was affiliated with Rhodocyclales. In contrast, it only accounted for 8%–9% of NapA in the incubations with sulfide and Fe2+. Differences in nitrate reductases might explain different oxygen isotope effects. Our findings also suggested that large variance of O‐nitrate isotope fractionations might have to be considered in the interpretation of natural isotope records. [ABSTRACT FROM AUTHOR]

The dominance of different nitrate-reducing pathways determines nitrogen cycling patterns. Denitrification (DNF) has been widely studied, but assimilatory nitrate reduction (ANR) and dissimilatory nitrate reduction to ammonium (DNRA) have received much less attention. Their ecological patterns and responsible microbes are poorly understood. Here, we studied the structure and function succession of the three functional groups in the middle route of the South-to-North Water Diversion Project, which is a 1230 km canal spanning 8 degrees of latitude. The results reflected a nitrogen-removing pattern dominated by DNF in the summer and a nitrogen-retaining pattern dominated by ANR and DNRA in the winter. Stenotrophomonas , a typical denitrifier, was the keystone species in the summer and contributed to N 2 O production. Clostridium , a genus able to conduct ANR and DNRA, was the keystone species in the winter. Notably, a significant zonation pattern was discovered. According to the community structure, the system could be separated into two biogeographic zones, and the Yellow River (about latitude 35°N) is an important cut-off line. This bacterial biogeography followed different water characteristics and ecological processes. ANR was found to be an important process and seasonally transformed its habitat from the northern zone to the southern zone. DNRA bacteria were acclimated to the northern zone and favored at this region in both seasons. The generation of N 2 O, a strong greenhouse gas, also exhibited this zonation pattern. This is the first study to consider assimilatory and dissimilatory nitrate reducers together at a molecular level, and provides new insights into the underlying patterns of a nitrate-reducing bacterioplankton community. [ABSTRACT FROM AUTHOR]

Microbial denitrification is a main source of nitrous oxide (N 2 O) emissions which have strong greenhouse effect and destroy stratospheric ozone. Though the importance of sulfide driven chemoautotrophic denitrification has been recognized, its contribution to N 2 O emissions in nature remains elusive. We built up long-term sulfide-added microcosms with sediments from two freshwater lakes. Chemistry analysis confirmed sulfide could drive nitrate respiration in long term. N 2 O accumulated to over 1.5% of nitrate load in both microcosms after long-term sulfide addition, which was up to 12.9 times higher than N 2 O accumulation without sulfide addition. Metagenomes were extracted and sequenced during microcosm incubations. 16 S rRNA genes of Thiobacillus and Defluviimonas were gradually enriched. The nitric oxide reductase with c-type cytochromes as electron donors (cNorB) increased in abundance, while the nitric oxide reductase receiving electrons from quinols (qNorB) decreased in abundance. cnorB genes similar to Thiobacillus were enriched in both microcosms. In parallel, enrichment was observed for enzymes involved in sulfur oxidation, which supplied electrons to nitrate respiration, and enzymes involved in Calvin Cycle, which sustained autotrophic cell growth, implying the coupling relationship between carbon, nitrogen and sulfur cycling processes. Our results suggested sulfur pollution considerably increased N 2 O emissions in natural environments. • N2O accumulation was up to 12.9 times higher after long-term sulfide input. • N2O production was associated more with c-type cytochrome nitric oxide reductases. • cnorB genes similar to Thiobacillus were enriched in both microcosms. • Sulfur oxidation supplied electrons to nitrate respiration. • Microbes sustained autotrophic growth through the Calvin cycle. [ABSTRACT FROM AUTHOR]

Partitioning between nitrate reduction pathways, denitrification and dissimilatory nitrate reduction to ammonium (DNRA) determines the fate of nitrate removal and thus it is of great ecological importance. Sulfide (S2−) is a potentially important factor that influences the role of denitrification and DNRA. However, information on the impact of microbial mechanisms for S2− on the partitioning of nitrate reduction pathways in freshwater environments is still lacking. This study investigated the effects of long-term (108 d) S2− addition on nitrate reduction pathways and microbial communities in the sediments of two different freshwater lakes. The results show that the increasing S2− addition enhanced the coupling of S2− oxidation with denitrification instead of DNRA. The sulfide-oxidizing denitrifier, Thiobacillus , was significantly enriched in the incubations of both lake samples with S2− addition, which indicates that it may be the key genus driving sulfide-oxidizing denitrification in the lake sediments. During S2− incubation of the Hongze Lake sample, which had lower inherent organic carbon (C) and sulfate (SO 4 2−), Thiobacillus was more enriched and played a dominant role in the microbial community; while during that of the Nansi Lake sample, which had higher inherent organic C and SO 4 2−, Thiobacillus was less enriched, but increasing abundances of sulfate reducing bacteria (Desulfomicrobium , Desulfatitalea and Geothermobacter) were observed. Moreover, sulfide-oxidizing denitrifiers and sulfate reducers were enriched in the Nansi Lake control treatment without external S2− input, which suggests that internal sulfate release may promote the cooperation between sulfide-oxidizing denitrifiers and sulfate reducers. This study highlights the importance of sulfide-driven denitrification and the close coupling between the N and S cycles in freshwater environments, which are factors that have often been overlooked. Image 1 • Long-term sulfide input favors chemoautotrophic denitrification instead of DNRA. • Thiobacillus was the key genus driving sulfide-oxidizing denitrification. • Inherent C and S in the samples influenced microbial interactions under S2− input. • Internal SO 4 2− release increased cooperation between N and S cycling microorganisms. Long-term sulfide input favors chemoautotrophic denitrifiers instead of DNRA bacteria in freshwater sediments. [ABSTRACT FROM AUTHOR]

Nitrate contamination in aquatic systems is a widespread problem across the world. The isotopic composition (δ15N, δ18O) of nitrate and their isotope effect (15ε, 18ε) can facilitate the identification of the source and transformation of nitrate. Although previous researches claimed the isotope fractionations may change the original δ15N/δ18O values and further bias identification of nitrate sources, isotope effect was often ignored due to its complexity. To fill the gap between the understanding and application, it is crucial to develop a deep understanding of isotopic fractionation based on available evidence. In this regard, this study summarized the available methods to determine isotope effects, thereby systematically comparing the magnitude of isotope effects (15ε and 18ε) in nitrification, denitrification and anammox. We found that the enzymatic reaction plays the key role in isotope fractionations, which is significantly affected by the difference in the affinity, substrate channel properties and redox potential of active site. Due to the overlapping of microbial processes and accumulation of uncertainties, the significant isotope effects at small scales inevitably decrease in large-scale ecosystems. However, the proportionality of N and O isotope fractionation (δ18O/δ15N; 18ε/15ε) associated with nitrate reduction generally follows enzyme-specific proportionalities (i.e., Nar, 0.95; Nap, 0.57; eukNR, 0.98) in aquatic ecosystems, providing enzyme-specific constant factors for the identification of nitrate transformation. With these results, this study finally discussed feasible source portioning methods when considering the isotope effect and aimed to improve the accuracy in nitrate source identification. [Display omitted] • Ignorance of isotope fractionations impedes the nitrate source identification. • Enzymatic reaction is the fundamental factor to drive isotope fractionations. • 18ε/15ε provides specific constant factors for nitrate source identification • Appropriate practices of isotopic effect improves the accuracy of identification. [ABSTRACT FROM AUTHOR]