The identification of nitrate (NO₃⁻) sources and biogeochemical transformations is critical for understanding the different nitrogen (N) pathways, and thus, for controlling diffuse pollution in groundwater affected by livestock and agricultural activities. This study combines chemical data, including environmental isotopes (δ²HH₂O, δ¹⁸OH₂O, δ¹⁵NNO₃, and δ¹⁸ONO₃), with land use/land cover data and a Bayesian isotope mixing model, with the aim of reducing the uncertainty when estimating the contributions of different pollution sources. Sampling was taken from 53 groundwater sites in Comarca Lagunera, northern Mexico, during 2018. The results revealed that the NO₃⁻ (as N) concentration ranged from 0.01 to 109 mg/L, with more than 32% of the sites exceeding the safe limit for drinking water quality established by the World Health Organization (10 mg/L). Moreover, according to the groundwater flow path, different biogeochemical transformations were observed throughout the study area: microbial nitrification was dominant in the groundwater recharge areas with elevated NO₃⁻ concentrations; in the transition zones a mixing of different transformations, such as nitrification, denitrification, and/or volatilization, were identified, associated to moderate NO₃⁻ concentrations; whereas in the discharge area the main process affecting NO₃⁻ concentrations was denitrification, resulting in low NO₃⁻ concentrations. The results of the MixSIAR isotope mixing model revealed that the application of manure from concentrated animal-feeding operations (∼48%) and urban sewage (∼43%) were the primary contributors of NO₃⁻ pollution, whereas synthetic fertilizers (∼5%), soil organic nitrogen (∼4%), and atmospheric deposition played a less important role. Finally, an estimation of an uncertainty index (UI90) of the isotope mixing results indicated that the uncertainties associated with atmospheric deposition and NO₃⁻−fertilizers were the lowest (0.05 and 0.07, respectively), while those associated with manure and sewage were the highest (0.24 and 0.20, respectively).

Microbial denitrification is a main source of nitrous oxide (N₂O) emissions which have strong greenhouse effect and destroy stratospheric ozone. Though the importance of sulfide driven chemoautotrophic denitrification has been recognized, its contribution to N₂O 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. N₂O 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 N₂O 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 N₂O emissions in natural environments.

Nitrous oxide (N₂O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH₃ oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N₂O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N₂O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH₃ oxidizers and denitrifiers, including the previously-overlooked taxa, in N₂O emission from a cropland, and address the biological and environmental factors controlling the N₂O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N₂O production than the long-studied ones. We also demonstrated that the N₂O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N₂O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N₂O production by overlooked denitrifying bacteria and N₂O reduction by long-studied N₂O-reducing bacteria after manure fertilization into the plowed layer; and (3) N₂O production by NH₃-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH₃ oxidizers and denitrifiers, which will help us understand the environmental context-dependent N₂O emission processes.

Enhanced efficiency nitrogen fertilizers (EENFs), including nitrification inhibitors (NIs) and slow-release fertilizer (SRF), are considered promising approaches for mitigating nitrous oxide (N₂O) emissions while improving crop yield. This study investigated the combined application of EENFs with improved water and fertilizer management in an intensively irrigated spring maize rotation over five years in Northwestern China. High-frequency measurements of N₂O fluxes were made throughout each year (both during crop growth and the fallow season) in five treatments: no N fertilizer as a control (CK), conventional N fertilization and irrigation (Con), optimum N fertilization and irrigation (Opt, 33% reduction in N fertilizer and 25% reduction of irrigation water), optimum N fertilization and irrigation with nitrification inhibitor (Opt + NI), and optimum N fertilization and irrigation with slow-release fertilizer (Opt-SRF). Annual mean cumulative N₂O emissions reached 0.31 ± 0.07, 3.66 ± 0.19, 1.87 ± 0.16, 1.23 ± 0.13, and 1.61 ± 0.16 kg N₂O–N ha⁻¹ for CK, Con, Opt, Opt + NI, and Opt-SRF, respectively, with annual mean nitrogen use efficiency (NUE) of 36, 54, 61 and 59% for Con, Opt, Opt + NI, and Opt-SRF, respectively. The Opt, Opt + NI and Opt-SRF treatments significantly reduced cumulative N₂O emissions by 49%, 66%, and 56% (P < 0.05), respectively, and increased NUE by 51%, 70%, and 66% (P < 0.05), respectively, relative to Con. However, mean above-ground N uptake (288–309 kg N ha⁻¹) and mean grain yields (12.7–12.8 Mg ha⁻¹) did not differ significantly between the Con, Opt, Opt + NI, and Opt-SRF treatments during the five-year study. High N₂O emissions mainly occurred within a few days of fertilization with irrigation, which could have been produced by microbially-mediated nitrifier or nitrifier denitrification processes. The fallow seasons had significantly lower cumulative N₂O emissions, which were mainly attributed to the low temperature, low N inputs of crop residues, and low soil moisture conditions. Our study clearly indicated that the combined application of EENFs with optimum N fertilization and irrigation management can reduce environmental impacts while maintaining high crop yields in dryland regions such as Northwest China.

In this work, two deep subsurface wastewater infiltration systems (SWISs) were constructed and fed with domestic sewage (control system, S1) and mixed wastewater consisting of old landfill leachate and domestic sewage (experimental system, S2). S1 and S2 exhibited favorable removal efficiencies, with TP (98.8%, 98.7%), COD (87.6%, 86.9%), NH₄⁺-N (99.8%, 99.9%) and TN (99.2%, 98.9%). Even when increasing the pollutant load in S2 by adding old landfill leachate, the almost complete removal performance could be maintained in terms of low effluent concentrations and even increased in terms of load removal capabilities, which included COD (19.4, 25.9 g∙m⁻²·d⁻¹), NH₄⁺-N (8.2, 19.9 g∙m⁻²·d⁻¹), TN (8.9, 20.6 g∙m⁻²·d⁻¹). To investigate the transformation of dissolved organic matter along depth, Three-Dimensional Excitation Emission Matrix fluorescence spectroscopy combined with Fluorescence Regional Integration analysis was applied. The results showed that PⅠ,ₙ and PⅡ,ₙ (the proportions of biodegradable fractions) increased gradually from 6.59% to 21.8% at S2_20 to 10.8% and 27.7% at S2_110, but PⅢ,ₙ and PⅤ,ₙ (the proportions of refractory organics) declined from 23.1% to 27.8% at S2_20 to 21.1% and 16.4% at S2_110, respectively. In addition, high-throughput sequencing technology was employed to observe the bacterial community at different depths, and the predicted functional potential of the bacterial community was analyzed by PICRUSt. The results showed that the genera Flavobacterium, Pseudomonas, Vogesella, Acinetobacter and Aquabacterium might be responsible for refractory organic degradation and that their products might serve as the carbon source for denitrifiers to achieve simultaneous nitrate and refractory organic removal. PICRUSt further demonstrated that there was a mutual response between refractory organic degradation and denitrification. Overall, the combined treatment of domestic sewage and old leachate in rural areas by SWIS is a promising approach to achieve comprehensive treatment.

Using Fe(II) salt as the precipitant in heterotrophic denitrification achieves improved TP removal, and enhancement in denitrification was often observed. This study aimed to obtain a better understanding of Fe(II)-enhanced denitrification with sufficient carbon source supply. Laboratory-scale experiments were conducted in SBRs with or without Fe(II) addition. Remarkably improved TP removal was experienced. TP removal efficiency in Fe(II) adding reactor was 85.8 ± 3.4%; whereas, that in the reactor without Fe(II) addition was 31.1 ± 2.8%. Besides improved TP removal, better TN removal efficiency (94.1 ± 1.1%) were recorded when Fe(II) was added, and that in the reactor without Fe(II) addition was 89 ± 0.8%. The specific denitrification rate were observed increase by 12.6% when Fe(II) was added. Further microbial analyses revealed increases in the abundances of typical denitrifiers (i.e. Niastella, Opitutus, Dechloromonas, Ignavibacterium, Anaeromyxobacter, Pedosphaera, and Myxococcus). Their associated denitrifying genes, narG, nirS, norB, and nosZ, were observed had 14.2%, 19.4%, 21.6%, and 9.9% elevation, respectively. Such enhancement in denitrification shall not be due to nitrate-dependent ferrous oxidation, which prevails in organic-deficient environments. In an environment with a continuous supply of Fe(II) and plenty of carbon sources, a cycle of denitrifying enzyme activity enhancement in the presence of Fe(II) facilitating nitrogen substrate utilization, stimulating denitrifier metabolism and growth, elevating denitrifying genes abundance, and increasing denitrifying enzymes expression were thought to be responsible for the Fe(II)-enhanced heterotrophic denitrification. Fe(II) salt is often a less expensive precipitant and has recently become attractive for TP removal in wastewater. The findings of this study solidify previous observation of enhancement of both TP and TN removal by adding Fe(II) in denitrification, and would be helpful for developing cost-effective pollutant removal processes.

Thiosulfate is frequently used as an energy source and electron donor in autotrophic denitrification (AD) for removing nitrate from wastewater. However, transforming pathways of S₂O₃²⁻ in this process is unclear. Herein, the aim of this study is to explore possible transforming pathways of sulfur compounds in thiosulfate-based AD process. After measuring the variation of NO₃⁻, NO₂⁻, and various sulfur compounds such as S⁰, SO₄²⁻, S₂O₃²⁻, acid volatile sulfide (AVS), and S²⁻ in the presence and absence of S₂O₃²⁻, the variation process of S₂O₃²⁻ and the contribution of various sulfur compounds were analyzed. The results indicated that S⁰, AVS, and S²⁻ were the intermediate products when S₂O₃²⁻ was applied as an electron donor. All S₂O₃²⁻, S⁰, AVS, and S²⁻ could act as electron donors in the nitrate removal process with the final products of SO₄²⁻. The utilization priority of these four sulfur sources was presumed in the following order: S²⁻ > S₂O₃²⁻ > AVS ≈ S⁰. Furthermore, sulfur transformation and balance in nitrate removal process was also investigated. This suggests the transforming pathways of sulfur compounds in denitrification process. Nitrogen removal and sulfur conversion process are dependent on the presence of microorganisms in the sludge.

The installation rate of denitrification devices is accelerating in Chinese urban boilers. Previous studies on pulverized coal-fired boilers without denitrification devices showed that combustion products containing mainly oxidized mercury (Hg) preferably enriched lighter Hg isotopes than feed coals. However, the magnitude of this enrichment becomes less pronounced if denitrification devices are installed. The underlying Hg isotope fractionation mechanisms are still unclear. In this study, three types of urban boilers (two pulverized coal-fired boilers, one circulating fluidized bed boiler and one municipal waste incinerator boiler) all installed with denitrification devices were measured for Hg isotope compositions of their feed fuels and corresponding combustion products. We observed little mass independent fractionation but very significant mass dependent fractionation (MDF) between feed fuels and combustion products. The fly ash and desulfurization products both enriched heavier Hg isotopes than feed coals in three coal-fired boilers, and the enrichment of heavy Hg isotopes increased with sequential removal of combustion products in all boilers. Different from previously suggested kinetic MDF for gaseous Hg⁰(g)→Hgᴵᴵ(g) and gaseous Hgᴵᴵ(g)→particulate Hgᴵᴵ(p) in coal combustion flue gases, we propose an equilibrium MDF for Hg⁰(g)↔Hgᴵᴵ(g) followed by a kinetic MDF for Hgᴵᴵ(g)→Hgᴵᴵ(p). This equilibrium MDF most likely occurs during Hg⁰(g) oxidation in denitrification devices, which enriches heavy Hg isotopes in oxidized products (Hgᴵᴵ(g) and Hgᴵᴵ(p)) that are then sequestrated in fly ash and desulfurization products. The paradigm shift of MDF in boilers with denitrification devices was further verified by parallel Hg isotope measurement in urban atmosphere particulates. Our study clearly demonstrates that modern coal-fired boilers with denitrification devices have a quite different MDF compared to traditional boilers without denitrification devices. This has important implications for estimating isotope signatures of urban boiler Hg emissions, and for isotope tracing of anthropogenic Hg emissions.

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 (SO₄²⁻), 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 SO₄²⁻, 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.

To simultaneously achieve biological denitrification and bio-energy recovery from sludge, the effects of nitrite on the two-phase anaerobic digestion (AD) of waste activated sludge were explored. Herein, effects of nitrite on the acidogenic phase are optimized, and the corresponding influence mechanisms are investigated. The experimental results show that the optimal nitrite treatment conditions for improving the acidogenic phase are an initial pH of 8.0, a nitrite addition concentration of 500 mg NO₂⁻-N·L⁻¹, and a fermentation time of six days. By comparing the effects of nitrite and nitrate on the acidogenic phase, it was found that it was the nitrite, not the nitrate, that significantly enhanced the sludge organic solubilization, hydrolysis, and acidification, which are primarily attributed to the redox property of nitrite. Based on an analysis of different forms of soluble nitrogen concentrations, there was no obvious accumulation of nitrite or nitrate during the acidogenic phase. An analysis of the methane production and the volatile solid (VS) degradation during the two-phase AD revealed that the nitrite improved the methane production from the methanogenic phase and enhanced the VS degradation of sludge during the entire two-phase AD process. These findings could provide references for simultaneously treating nitrite-rich wastewater and improving anaerobic sludge digestion via two-phase system.