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Light-activated, -inhibited and -independent denitrification by a denitrifying phototrophic bacterium.

Effects of illumination on denitrification by a freshly isolated denitrifying phototrophic bacterium were investigated. Denitrification activity was induced when cells were grown in either light or darkness in the presence of nitrate without oxygen. Denitrification of nitrate with malate as the electron donor by cells at a phase of exponential growth occurred independently of illumination while that by cells in a stationary phase was activated. Effects of illumination on denitrification varied with electron donors. Using malate or succinate, denitrification by cells in a stationary phase was accelerated by illumination, inhibited when glucose or lactate was used, and independent of illumination when pyruvate was used. Denitrification by cells in an exponential phase was independent of illumination when succinate, malate or pyruvate was used and inhibited by it when glucose or lactate was used. Effects of illumination on the denitrification of nitrite were similar to those involving nitrate. Effects of various inhibitors on denitrification were examined in light-succinate and dark-lactate systems. Differences between the two systems are discussed.

Aerobiosis

Deciphering the effects of sulfonamide antibiotics on denitrification from a metagenomic perspective: Inhibition of nitrite reduction and succession patterns of functional microorganisms.

Limited research has thoroughly elucidated the impact mechanisms of antibiotics on the denitrification process at the genomic and gene levels, which has hindered the optimization and development of nitrogen removal technology for antibiotic-containing swine wastewater. Lab-scale sequencing batch reactors were constructed in this study to treat synthetic wastewater containing different sulfonamides and nitrate. Investigations were carried out on denitrification performance, microbial community diversity, denitrifier succession patterns, and functional gene distribution. The stress of sulfonamides inhibited the nitrite reduction process, transforming complete denitrification into partial denitrification and causing significant nitrite accumulation. The average nitrogen removal efficiency in the treatment groups decreased from 81.0% ± 2.2-40.1% ± 6.1%. Alicycliphilus and Thauera were identified as the key taxa, accounting for 32.2% and 16.9% of all potential denitrifying bacteria, respectively. Although metagenome-assembled genomes (MAGs) from Thauera were enriched with genes encoding nitrate reductases (nap, nar) and nitrite reductases (nir), this genus preferentially utilized nitrate as an electron acceptor, resulting in the preferential nitrate reduction and subsequent nitrite accumulation. In contrast, Alicycliphilus MAGs developed tolerance to the sulfonamides stress during later stages, with concomitant enrichment of associated functional genes. They replaced Thauera to reemerge as the dominant group, thereby restoring complete denitrification. This study provides new insights into the regulatory mechanisms governing complete versus partial denitrification in nitrogen removal from antibiotic-containing wastewater.

Denitrifier succession

Building biofilms for saline hydrogenotrophic denitrification from contrasting origins: Convergent acclimation, divergent performance.

Hydrogenotrophic denitrification is promising for deep nitrogen removal from saline, low-C/N wastewaters, but rapid establishment of stable biofilms at high salinity remains challenging. Here, two saline-adapted inocula from two representative, functionally contrasting habitats-a functionally-diversified inoculum from mangrove sediment and a functionally-focused inoculum from seabed sediment-were acclimated in parallel H2-based membrane biofilm reactors at constant 3.5% salinity. The Diverse-derived biofilm required 80 d to reach steady state and achieved only partial denitrification with 61.1% nitrate removal and considerable nitrite accumulation. In contrast, the Focus-derived biofilm rapidly established complete denitrification within ∼40 d, which was maintained for >50 d, with effluent NOx- below 1 mg-N·L-1 and 98.7% nitrate removal. Microbiome analyses showed that identical operation promoted convergence in community structure and enriched similar community-level functional potentials. However, genome-resolved analysis revealed distinct source-dependent functional organization among dominant microbial populations. Complete denitrifiers co-encoding denitrifying, hydrogenotrophic, and autotrophic functions were preferentially enriched in the Focus-derived biofilm, whereas these functions remained partitioned among different dominant populations in the Diverse-derived biofilm, coinciding with less complete denitrification. These findings indicate that saline hydrogenotrophic denitrification performance depends not only on which functions are enriched at the community level, but also on how key functions become organized among microbial populations, providing a previously overlooked criterion for inoculum selection in saline biological nitrogen control.

Complete denitrification

Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD.

Heterotrophic nitrification and aerobic and anaerobic denitrification by Alcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (KI = 109 microM acetate). Denitrifying or assimilatory nitrate reductase were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized by A. faecalis strain TUD, with a maximum oxygen uptake rate of 140-170 nmol O2.min-1.mg prot-1. Comparison of the behaviour of A. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.

Aerobiosis

Boosting domestic wastewater treatment with quorum signal-augmented heterotrophic nitrification-aerobic denitrification bacterial-algal aerobic granular sludge.

The aerobic bacterial-algal granular sludge (ABGS) enhanced with heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria, as a novel symbiotic technology, exhibits fluctuating treatment efficiency and unstable performance primarily due to the unstable symbiotic relationship. This study proposes an innovative approach to strengthening the bacteria-algae symbiosis by introducing exogenous signaling molecules. Concurrently, high-throughput, correlation analysis of environmental factors and metagenomic sequencing techniques are employed to elucidate the enhancement mechanisms of the signaling molecules. The results demonstrate that signaling molecule enhancement boosted total nitrogen (TN) removal efficiency by 24.51 % in the bacteria-algae symbiotic system (X1). Scanning electron microscopy (SEM) characterization revealed that the addition of signaling molecules resulted in more compact aerobic granular sludge (AGS) and markedly improved stability. High-throughput sequencing showed signaling molecules enriched denitrifying bacteria (Hydrogenophaga, Pseudoxanthomonas, Thauera, Zoogloea) and organic-degrading Desulfomicrobium, optimizing microbial diversity and enhancing nitrogen/organic removal. Correlation analysis of environmental factors indicate that the addition of C8-HSL facilitates the enrichment and functional activation of specific genera. Metagenomic analysis revealed that signaling molecules enhanced the system's denitrification performance by modulating gene expression and associated metabolic pathways. Quantitative polymerase chain reaction (qPCR) analysis further confirmed that the signaling molecules upregulated the expression of the napA, nirK, and nirS genes. An increased abundance of the napA gene facilitated aerobic denitrification (NO₃⁻-N→NO₂⁻-N), while upregulated abundance of the nirK and nirS genes accelerated nitrite reduction (NO₂⁻-N→N₂). This study aims to provide theoretical and practical foundations for implementing advanced bacteria-algae symbiotic technologies.

Denitrification

Metabolic niche differentiation and napA evolution stabilize partial denitrification in wastewater ecosystems.

Although partial denitrification (PD) is increasingly applied as a nitrite-supplying strategy for anammox-based nitrogen removal, the ecological distribution, metabolic specialization, and genomic determinants of stable nitrite accumulation remain poorly understood at the ecosystem scale. Here, we reconstructed 516 high-quality metagenome-assembled genomes (MAGs) using high-depth metagenomic sequencing of 107 wastewater treatment plants and classified denitrifiers according to their nitrite production or consumption capacities. Of these genomes, 23% (120 MAGs) were classified as partial denitrifiers, 41% (211 MAGs) as complete denitrifiers, and 36% (185 MAGs) as nitrite-reducing denitrifiers, revealing pronounced functional partitioning rather than dominance by complete denitrification pathways. Comparative genomics showed that partial denitrifiers possess metabolic architectures favoring rapid carbon oxidation and NADH generation while exhibiting constrained NADPH production and biosynthetic investment, thereby promoting nitrate-to-nitrite conversion but limiting subsequent nitrite reduction. Nitrite accumulation does not result from incomplete denitrification pathways but from metabolic niche differentiation. These metabolic trade-offs were further associated with the evolutionary divergence of the periplasmic nitrate reductase gene, napA, which displayed distinct sequence characteristics and genomic contexts between partial and complete denitrifiers. Integration of carbohydrate-active enzyme repertoires further revealed metabolic complementarity between partial denitrifiers and anammox bacteria, supporting efficient carbon handoff without direct substrate competition. From an engineering perspective, operating conditions that impose moderate electron limitation, such as low or fluctuating C/N ratios and intermittent carbon feeding, may selectively enrich partial denitrifiers and enhance a stable nitrite supply for PD-anammox systems. Together, these findings identify PD as a predictable ecological state shaped by genome-encoded metabolic specialization and provide a mechanistic basis for designing robust, low-carbon nitrogen-removal processes.

Anammox

Abundance-transcription decoupling reveals functional partitioning in bioelectrochemical denitrification biofilms.

Bioelectrochemical denitrification (BED) is often attributed to electroactive microorganisms that access electrode-derived electrons, yet the relative functional contribution of electroactive taxa and denitrifying populations within complex BED biofilms remain unclear. Here, we integrated reactor measurements with genome-resolved metagenomics and metatranscriptomics to examine microbial community structure, functional potential, and gene transcription across contrasting BED operational regimes differing in dissolved oxygen (DO), hydraulic retention time (HRT)/loading, and poised potential. Nitrate removal exceeded 90% across all tested conditions, but nitrogen intermediate accumulation, current generation, and theoretical electron balance differed substantially. Electroactive taxa such as Geobacter dominated (>80% abundance) under longer HRT and stronger poised potential, but contributed minimally to the transcription of canonical denitrification genes. Weaker cathodic potential enriched transcriptionally active denitrifying taxa such as Stutzerimonas, Acidovorax, and MR-S7, while oxygen exposure induced redox-stress responses and reshaped nitrogen metabolism beyond being a competing electron acceptor. Together, these results reveal a decoupling between taxonomic abundance, genomic functional potential, and transcriptional contribution in BED biofilms, indicating that nitrate-removal performance cannot be inferred from current generation or electroactive-taxon abundance alone.

Bioelectrochemical system

Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans.

The metabolism of nitric oxide in Paracoccus denitrificans has been studied using a Clark-type electrode. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the SH reagent N-ethylmaleimide, both of which released nitric oxide from cells respiring nitrite, were found to be efficient inhibitors of nitric oxide reductase activity. Control experiments with another uncoupler, pentachlorophenol, showed that the inhibitory effect of CCCP was not the result of a decrease in membrane potential. The denitrification pathway in cells with partly inhibited nitric oxide reductase, or in a reconstituted system containing purified nitric reductase and membrane vesicles, exhibited marked sustained oscillations of nitric oxide concentration. The occurrence of the oscillations was strictly dependent on the initial concentration of nitrite. The observed oscillatory kinetics is considered to reflect two regulatory signals destabilizing the denitrification pathway, namely the inhibition of nitric oxide reductase by nitric oxide and/or by nitrite.

Biological Transport

Nitrate and nitrite microgradients in barley rhizosphere as detected by a highly sensitive denitrification bioassay.

A highly sensitive denitrification bioassay was developed for detection of NO3- and NO2- in rhizosphere soil samples. Denitrifying Pseudomonas aeruginosa ON12 was grown anaerobically in citrate (30 mM) minimal medium with KClO3 (10 mM) and NaNO2 (3 mM), which gave cells capable of NO2- reduction to N2O but incapable of NO3- reduction to NO2-. Growth on citrate minimal medium further resulted in the absence of N2O reduction. When added to small soil samples in O2-free vials, such cells could be used to convert the indigenous NO2- pool to N2O, which was subsequently quantified by gas chromatography. Cells grown in KClO3-free citrate medium with 10 mM NaNO3 as the electron acceptor were capable of reducing both NO3- and NO2-, and these cells could subsequently be added to the sample to convert the indigenous NO3- pool to N2O. Concentrations of both NO3- and NO2- were thus determined as N2O, with a detection limit of approximately 10 pmol of N. The bioassay could be used to determine NO3- and NO2- pools in 10-mg soil samples taken along a microgradient in the rhizosphere of field-grown barley plants. At both low (10%, wt/wt) and high (18%, wt/wt) water content, relatively high levels of NO2- were found in the rhizosphere compared with bulk soil. Under dry conditions, NO3- was also more abundant in the rhizosphere than in the bulk soil, whereas such a difference was not observed at the high water content. The roles of plant metabolism and bacterial nitrification and denitrification processes for NO3- and NO2- availability in the rhizosphere are discussed.

Biological Assay

Effect of chemicals used as nitrification inhibitors on the denitrification process.

Several chemicals used as nitrification inhibitors were tested to determine their effect on dentrification by a Pseudomonas sp. and in soil. Denitrification by the bacterium was suppressed by 2-chloro-6(-trichloromethyl)-pyridine (N-Serve) at a concentration of 50 ppm, while 2,5-dichloroaniline caused the accumulation of nitrite in the culture medium. The nitrification inhibitors had little effect on the denitrifying activity in soil under anaerobic conditions. 2-Sulfanilamidothiazole inhibited denitrification to some extent and samples supplied with potassium azide produced N2O rather than N2 as the predominant gas.

Anaerobiosis

Nitrous oxide as end product of denitrification by strains of fluorescent pseudomonads.

Growing cultures of several strains of Pseudomonas fluorescens and Pseudomonas chlororaphis produced N2O as the only detectable gaseous product of denitrification, and other strains produced N2 as the gaseous end product of denitrification. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2O-producing strains P. fluorescens PJ 185 and P. chlororaphis B-560 was recovered as N2O. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2-producing strain P. fluorescens PJ70 was converted to N2. Cell extracts of P. fluorescens PJ 70, PJ 185, and P. chlororaphis B-560 exhibited NO3- reductase activity when sodium succinate was the electron donor. Reduced nicotinamide adenine dinucleotide and flavine adenine dinucleotide were required to demonstrate NO2- reductase activity in cell extracts.

Anaerobiosis

Loss of nitrogen by denitrification.

A series of experiments showed the quantity and composition of nitrogen lost in gaseous form from fertilizers in soil is largely determined by the conditions of denitrification. Loss of nitrogen from ammonium sulfate or calcium nitrate was mainly through the release of nitrous oxide and molecular nitrogen, while nitrogen was released from sodium nitrite in the form of nitric oxide. Under anaerobic conditions and at neutral soil pH in the presence of glucose, the more reduced gaseous forms of nitrogen were released. However, the oxides of nitrogen predominated under conditions unfavorable for denitrification. The nitrogen oxides were not the terminal products of nitrogen conversion (nitrates and nitrites). By a process of dissimilation, the nitrogen oxides acted as electron acceptors for microorganisms, being converted to N2O and N2. The reduction of NO generally led to the formation of N2O as an intermediate, and depended on pH, aeration, and the presence of an energy source for the denitrifying organisms.

Aerobiosis

[Chemistry of denitrification in sporogenous soil bacteria].

Soil sporeforming denitrifying bacteria, Bacillus filaris and Bacillus polymyxa, differ by their cultural-morphological and physiological characteristics, but are similar in the chemistry of dissimilating nitrate reduction. Two processes occur simultaneously: denitrification yielding gaseous nitrogen forms, and nitrate respiration upon which nitrates are reduced to ammonia. The ratio between the two depends on physico-chemical conditions of the environment. The chemistry of dissimilating nitrate reduction by sporeforming bacteria differs therefore from nitrate transformation by other denitrifying microorganisms: Pseudomonas, Micrococcus, Rhizobium, Achromobacter, which, according to the published evidence, are capable only of denitrification.

Bacillus

Mechanism exploration of divergent partial denitrification performance under tetracycline stress: Insights from functional gene, electron transport and molecular docking.

Nitrates and antibiotics like tetracycline (TC) coexist in wastewater and inhibit nitrite (NO₂--N) accumulation during partial denitrification (PD), restricting anammox coupling. A moving bed biofilm reactor (PD-MBBR) and a sequencing batch reactor (PD-SBR) were compared under TC stress (0-8 mg/L). The PD-MBBR proved more robust, sustaining a high nitrate transformation ratio (NTR) of 95.11% and ∼53% TC removal. Metagenomic sequencing, quantitative polymerase chain reaction (qPCR), and molecular docking revealed this tolerance stemmed from physical shielding and metabolic compensation. Carrier-attached growth promoted extracellular polymeric substances (EPS) overproduction, forming a dense barrier preventing TC from binding to key denitrifying enzymes. The biofilm maintained stable nitrate reductase (NAR) activity via high narG and napA gene abundances, while nitrite reductase (NIR) was inhibited, ensuring efficient NO₂--N accumulation. This was supported by hyperactivated electron transport chain components, with complex III relative abundance increasing 15.08% and peak enzymatic activity reaching 149.02%. While IntI1-mediated horizontal gene transfer fortified community defense, concentrated antibiotic resistance genes (ARGs) within the biofilm pose a secondary dissemination risk. Thus, PD-MBBR provides an efficient pretreatment strategy for anammox, though downstream ARGs management is warranted.

Denitrification

Insights into iron-enhanced denitrification coupled with antibiotic resistant genes control in biochar-based biofiltration systems.

In biofiltration (BF) systems, biochar can enhance pollutant removal by promoting biofilm formation. Its abundant pore structure can also sequester antibiotics away from microbial cells, thereby reducing its bioavailability and accumulation of antibiotic resistance genes (ARGs). However, dense biofilms favor horizontal ARG transfer, especially among denitrifying bacteria, which are prone to stress under low influent C/N conditions. In this study, a strategy combining iron minerals was proposed to alleviate the ARG accumulation in BF systems. Compared with magnetite, goethite and siderite released Fe2+ through microbial dissimilatory iron reduction and chemical dissolution respectively, thereby driving iron‑autotrophic denitrification and enhancing the activity of electron‑transfer mediators (cytochrome c and Fe-S proteins). As a result, the level of nitrosative stress was reduced with significant downregulation of related genes (hmp, hcp, norR, and etc.), which was a driving force for conjugative transfer of ARGs. Specifically, the excessive accumulation of tryptophan and shortage of methionine were thus alleviated, which contributed to the regulation of global repressor gene expression and the mitigation of ARG conjugative transfer. With the combination of goethite or siderite in BF systems, the abundance of resistance genome in biofilm exhibited a reduction of 52.68 ± 3.80% and 41.26 ± 4.20%, respectively, which could effectively reduce the environment-ecological risk of antibiotic and ARGs.

Denitrification

Cattle manure suppresses methane consumption and enhances denitrification-associated nitrous oxide production in farm dams.

BACKGROUND: Farm dams (or agricultural ponds) are often heavily polluted freshwater systems because of nutrient-rich manure entering the water through direct deposition and runoff. Accordingly, these systems have among the highest greenhouse gas emissions per area, accounting for 41% of global freshwater methane emissions. Sustainable management actions, such as limiting livestock access through fencing, can significantly reduce nutrient concentrations and greenhouse gas emissions. However, the microbes, processes, and factors controlling greenhouse gas cycling in these systems have not been described. Here, we systematically compared the composition, functions, and activities of the microbes in paired fenced and unfenced cattle farm dams in southeastern Australia. RESULTS: We found that in situ methane (CH4) and nitrous oxide (N2O) emissions were strongly reduced in fenced dams. Even though methanogen abundance was higher in fenced dams, fencing increased levels of aerobic methanotrophs, including two previously uncharacterised, metabolically flexible species profiled via metagenome-assembled genomes (MAGs). In contrast, we provide gene- and genome-centric evidence that N2O emissions are likely higher in unfenced dams due to increased production (via denitrification) rather than decreased consumption. Manure likely increases CH4 and N2O emissions primarily by driving nutrient-induced eutrophication and hypoxia that, respectively, stimulate denitrifiers and inhibit methanotrophs. However, we also provide evidence that manure-associated methanogens and bacteria occur in farm dams, where they potentially enhance emissions. CONCLUSIONS: Our findings highlight how anthropogenic activities such as livestock farming can impact microbial communities and biogeochemical cycling, thereby increasing greenhouse gas emissions from freshwater systems, and how simple management actions like fencing can mitigate such emissions. Video Abstract.

Animals

Rate constants for nitrification and denitrification in soils.

Rate constants for reactions in flowing solutions in soil can be calculated from extents of reactions as functions of depth, rates of flow, effective biomass of microbes and independent measurements of hydrodynamic dispersion. Constants have been calculated from data in the literature and are shown to be arbitrary unless all of these quantities have been evaluated. Good agreement of constants obtained in laboratory columns and in the field have been obtained for nitrification and denitrification in a few cases.

Bacteria

Denitrification by fungi.

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.

Anaerobiosis