Nitric Oxide Reductase Research Articles

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH4+–N and NO2−–N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

Revisiting the metal sites of nitrous oxide reductase in a low-dose structure from Marinobacter nauticus

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl–. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding.Graphical The [4Cu:2S] CuZ site in M. nauticus N 2O reductase. The electron density map shown is contoured at the 5 σ level, highlighting the presence of two sulfide ligands. 705x677mm (72 x 72 DPI)

Estimation of Electrostatic Interaction Energies on a Trapped-Ion Quantum Computer.

We present the first hardware implementation of electrostatic interaction energies by using a trapped-ion quantum computer. As test system for our computation, we focus on the reduction of NO to N2O catalyzed by a nitric oxide reductase (NOR). The quantum computer is used to generate an approximate ground state within the NOR active space. To efficiently measure the necessary one-particle density matrices, we incorporate fermionic basis rotations into the quantum circuit without extending the circuit length, laying the groundwork for further efficient measurement routines using factorizations. Measurements in the computational basis are then used as inputs for computing the electrostatic interaction energies on a classical computer. Our experimental results strongly agree with classical noise-less simulations of the same circuits, finding electrostatic interaction energies within chemical accuracy despite hardware noise. This work shows that algorithms tailored to specific observables of interest, such as interaction energies, may require significantly fewer quantum resources than individual ground state energies would require in the straightforward supermolecular approach.

Impact of aerobic granular sludge sizes and dissolved oxygen concentration on greenhouse gas N2O emission

Aerobic granular sludge (AGS) at wastewater treatment plants (WWTPs) are known to produce nitrous oxide (N2O), a greenhouse gas which has a ∼300 times higher global warming potential than carbon dioxide. In this research, we studied N2O emissions from different sizes of AGS developed at a dissolved oxygen (DO) level of 2 mgO2/L while exposing them to disturbances at various DO concentrations ranging from 1 to 4 mgO2/L. Five different AGS size classes were studied: 212–600 µm, 600–1000 µm, 1000–1400 µm, 1400–2000 µm, and > 2000 µm. Metagenomic data showed N2O reductase genes (nosZ) were more abundant in the smaller AGS sizes which aligned with the observation of higher N2O reduction rates in small AGS under anaerobic conditions. However, when oxygen was present, the activity measurements of N2O emission showed an opposite trend compared to metagenomic data, smaller AGS (212 to 1000 µm) emitted significantly higher N2O (p < 0.05) than larger AGS (1000 µm to >2000 µm) at DO of 2, 3, and 4 mgO2/L. The N2O emission rate showed positive correlation with both oxygen levels and nitrification rate. This pattern indicates a connection between N2O emission and nitrification. In addition, the data suggested the penetration of oxygen into the anoxic zone of granules might have hindered nitrous oxide reduction, resulting in incomplete denitrification stopping at N2O and consequently contributing to an increase in N2O emissions. This work sets the stage to better understand the impacts of AGS size on N2O emissions in WWTPs under different disturbance of DO conditions, and thus ensure that wastewater treatment will comply with possible future regulations demanding lowering greenhouse gas emissions in an effort to combat climate change.

N2O consumption, uptake, and microbial reduction processes in flooded sandy loamy paddy soils

AbstractSandy loamy soils are widely distributed in fluvial floodplains and experience flooding events frequently, resulting in a large amount of nitrous oxide (N2O) emissions. This case is more serious when the soil use is changed to paddies. It is of great significance to figure out the N2O consumption and its influencing factors in sandy loamy paddy soils to mitigate N2O emissions. In this study, three sandy loamy paddy soils (0–5 cm) originated from lake deposits were selected (S1, S2, and S3) as objectives. A certain concentration of exogenous N2O was added at the bottom of the flooded soil column to monitor the dynamics of N2O and nitrogen (N2) on the soil surface. Total N2O consumption, N2O uptake, and N2 production were quantified, and the abundance of nitrous oxide reductase genes (nosZI, and nosZII) and other soil properties (ammonium‐nitrogen, nitrate‐nitrogen, and dissolved organic carbon [DOC] content) were analyzed. The results showed that the sandy loamy paddy soil column with a depth of 0–5 cm could intercept more than 95% of the exogenous N2O under the flooded anaerobic condition, indicating that the three sandy loamy paddy soils all had extremely strong N2O consumption capacities. And the increment of N2 accounted for 68.73%–76.09% of the total N2O consumption, which had a stronger relationship with the increase of nosZI gene abundance than nosZII gene. In addition, the total N2O consumption and N2 increment of S1 and S3 soils were significantly higher than those of S2 soil. This difference was mainly related to soil organic matter content, total nitrogen content, DOC consumption, and the increase of nosZI gene abundance (p &lt; 0.05). The strong N2O consumption potential of sandy loamy soils can provide feasible solutions for regulating N2O emissions in a wide range of similar environments in fluvial floodplains.

In situ membrane separation drives nitrous oxide enrichment from nitrate denitrification for energy recovery

Efficiently enriching nitrous oxide (N2O) can provide a foundation for energy recovery during wastewater treatment. In this study, gas-liquid separation membranes were placed in a sequencing batch reactor fed with acidic nitrate-containing wastewater to determine the feasibility of enriching N2O by in situ separation of dissolved gases. Gas separation across the membrane (GSM) had no obvious effect on chemical oxygen demand and nitrate removal, but N2O production efficiency (NPE) increased from 11.0 % to 82.6 %. Denitrifying bacteria with low N2O yield such as Thauera, Diaphorobacter, and Flavobacterium were eliminated, and Propioniclava (46.79 %) and Candidatus_Competibacter (22.53 %) became dominant. Metagenomic sequencing showed that the abundance of the nosZ gene encoding N2O reductase decreased from 17.3 % to 6.1 %. In conclusion, GSM prevented the further reduction of N2O, eliminating denitrifying bacteria carrying the nosZ gene, which the enriched Propioniclava likely lacked. This demonstrates that GSM is an alternative strategy for N2O enrichment through nitrate denitrification.

Complete genome sequence of Afipia carboxidovorans strain SH125, a non-denitrifying nitrous oxide-reducing bacterium isolated from anammox biomass.

Here, we report a genome sequence of Afipia carboxidovorans strain SH125 isolated from an anammox reactor. This facultative anaerobic strain possesses the clade I-type nitrous oxide (N2O) reductase gene, devoid of nitrite- and nitric oxide reductase genes. Deciphering the genome will help explore N2O reducers instrumental in N2O mitigation.

Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen-deficient zones with diverse metabolic potential.

Archaea from the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota) superphylum have diverse metabolic capabilities and participate in multiple biogeochemical cycles. While metagenomics and enrichments have revealed that many DPANN are characterized by ultrasmall genomes, few biosynthetic genes, and episymbiotic lifestyles, much remains unknown about their biology. We report 33 new DPANN metagenome-assembled genomes originating from the three global marine oxygen-deficient zones (ODZs), the first from these regions. We survey DPANN abundance and distribution within the ODZ water column, investigate their biosynthetic capabilities, and report potential roles in the cycling of organic carbon, methane, and nitrogen. We test the hypothesis that nitrous oxide reductases found within several ODZ DPANN genomes may enable ultrasmall episymbionts to serve as nitrous oxide consumers when attached to a host nitrous oxide producer. Our results indicate DPANN archaea as ubiquitous residents within the anoxic core of ODZs with the potential to produce or consume key compounds.

Impacts of Climate Change and Agricultural Practices on Nitrogen Processes, Genes, and Soil Nitrous Oxide Emissions: A Quantitative Review of Meta-Analyses

Microbial-driven processes, including nitrification and denitrification closely related to soil nitrous oxide (N2O) production, are orchestrated by a network of enzymes and genes such as amoA genes from ammonia-oxidizing bacteria (AOB) and archaea (AOA), narG (nitrate reductase), nirS and nirK (nitrite reductase), and nosZ (N2O reductase). However, how climatic factors and agricultural practices could influence these genes and processes and, consequently, soil N2O emissions remain unclear. In this comprehensive review, we quantitatively assessed the effects of these factors on nitrogen processes and soil N2O emissions using mega-analysis (i.e., meta-meta-analysis). The results showed that global warming increased soil nitrification and denitrification rates, leading to an overall increase in soil N2O emissions by 159.7%. Elevated CO2 stimulated both nirK and nirS with a substantial increase in soil N2O emission by 40.6%. Nitrogen fertilization amplified NH4+-N and NO3−-N contents, promoting AOB, nirS, and nirK, and caused a 153.2% increase in soil N2O emission. The application of biochar enhanced AOA, nirS, and nosZ, ultimately reducing soil N2O emission by 15.8%. Exposure to microplastics mostly stimulated the denitrification process and increased soil N2O emissions by 140.4%. These findings provide valuable insights into the mechanistic underpinnings of nitrogen processes and the microbial regulation of soil N2O emissions.

Distinct microbial characteristics of the robust single-stage coupling system during the conversion from anammox-denitritation to anammox-denitratation patterns

Simultaneous anammox-denitrification is effectively operated in two types, i.e., the anammox-denitritation (SAD pattern) and the anammox-denitratation (PDA pattern). The nitrate derived from inevitable nitrite oxidization likely determines the practical operational pattern of the coupling system, while little information is available regarding the microbial characteristics during the pattern conversion. Here, the single-stage bioreactor coupling anammox with denitrification was operated under conditions with a changed ratio of influent nitrite and nitrate. Results showed that the bioreactor exhibited a robust performance during the conversion from SAD to PDA patterns, corresponding with the total nitrogen removal efficiency ranging from 89.5% to 92.4%. Distinct community structures were observed in two patterns, while functional bacteria including the genera Denitratisoma, Thauera, Candidatus Brocadia, and Ca. Jettenia steadily co-existed. Meanwhile, the high transcription of hydrazine synthase genes demonstrated a stable anammox process, while the up-regulated transcription of nitrite and nitrous oxide reductase genes indicated that the complete denitrification process was enhanced for total nitrogen removal during the PDA pattern. Ecologically, stochastic processes dominantly governed the community assembly in two patterns. The PDA pattern improved the interconnectivity of communities, especially for the cooperative behaviors between dominant denitrifying bacteria and low-abundant species. These findings deepen our understanding of the microbial mechanism underlying the different patterns of the coupling system and potentially expand its engineering application.

PH selects for distinct N2O-reducing microbiomes in tropical soil microcosms.

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH < 5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02mM) and high (2mM) N2O assessed N2O reduction at pH4.5 and 7.3. All microcosms consumed N2O, with lag times of up to 7months observed in microcosms with 2mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. At pH4.5, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen high-quality metagenome-assembled genomes (MAGs) recovered from the N2O-reducing microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the Clade II type nosZ and lacking nitrite reductase genes (nirS/K). Five of the eight MAGs recovered from pH4.5 microcosms represent novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

The regulation effect of preventing soil nitrogen loss using microbial quorum sensing inhibitors

Preventing soil nitrogen (N) losses driven by microbial nitrification and denitrification contributes to improving global environmental concerns caused by NO3−-N leaching and N2O emission. Quorum sensing (QS) signals regulate nitrification and denitrification of N-cycling bacteria in pure culture and water treatment systems, and mediate the composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in activated sludge. However, whether disrupting QS could prevent soil N losses remains unclear. This study explored the feasibility of applying quorum sensing inhibitors (QSIs) as an innovative strategy to reduce N losses from agricultural soils. The two QSIs, penicillic acid and 4-iodo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-benzeneacetamide (4-iodo PHL), were more effective in reducing N losses than traditional inhibitors, including N-(n-butyl) thiophosphoric triamide and 3,4-dimethylpyrazole phosphate. After 36 days of aerobic incubation, penicillic acid and 4-iodo PHL inhibited nitrification by 39% and 68%, respectively. The inhibitory effects are attributed to the fact that 4-iodo PHL decreased the abundance of archaeal and bacterial amoA genes, as well as the relative abundance of Candidatus Nitrocosmicus (AOA), Candidatus Nitrososphaera (AOA), and Nitrospira (nitrite-oxidizing bacteria/comammox), while penicillic acid reduced archaeal amoA abundance and the relative abundance of Nitrosospira (AOB) and the microbes listed above. Penicillic acid also strongly inhibited denitrification (33%) and N2O emissions (61%) at the peak of N2O production (day 4 of anaerobic incubation) via decreasing nitrate reductase gene (narG) abundance and increasing N2O reductase gene (nosZ) abundance, respectively. Furthermore, the environmental risks of QSIs to microbial community structure and network stability, CO2 emissions, and soil animals were acceptable. Overall, QSIs have application potential in agriculture to reduce soil N losses and the associated effect on climate change. This study established a new method to mitigate N losses from the perspective of QS, and can serve as important basis of decreasing the environmental risks of agricultural non-point source pollution.

New insights into the effect of EDTA on pyrite oxidation and N2O emission during pyrite autotrophic denitrification

Pyrite autotrophic denitrification (PAD) is crucial for removing nitrate in groundwater and advanced wastewater treatment. While disulfide is typically seen as the electron donor, the role of iron in pyrite oxidation is often ignored. In this study, Ethylene Diamine Tetraacetic Acid (EDTA) had been proven to have the potential to facilitate pyrite oxidation by promoting the solubility of iron in solution, thereby enhancing the kinetics of pyrite autotrophic denitrification. Firstly, EDTA could improve PAD performance, i.e. the nitrate removal rate at EDTA addition of 5 mM was 0.69 mg N/L•h and twice that of the control. However, high concentrations of EDTA inhibited the activity of the nitrous oxide reductase, resulting in the maximum accumulation of N2O reaching 13.26 mg N/L with an EDTA concentration of 15 mM. Furthermore, the X-ray Photoelectron Spectroscopy (XPS) result indicated that the EDTA addition promoted the production of bioavailable sulfur, with the concentration of S0 at 15 mM EDTA being more than twice higher than that of the control. Transmission electron microscopy (TEM) analysis indicated that EDTA could relieve the damage of iron encrustation on cell activity because it was difficult for EDTA-chelated ferrous iron to penetrate the cell membrane. Microbial community analyses showed an increase abundance of iron oxidizing bacteria, suggesting that EDTA addition promoted the bioavailability of iron for Fe(II) autotrophic denitrification. Overall, this study will provide insights into the understanding of oxidation mechanism of PAD process and the practical application for efficient nitrogen removal and N2O mitigation.

No-tillage with total green manure mulching: A strategy to lower N2O emissions

ContextGreen manure application in agricultural soil is known to enhance nitrogen use efficiency and crop yield. However, limited research exists on the potential effects of green manure returning on N2O emissions. ObjectiveThis study aims to investigate the effects and underlying mechanisms of N2O emissions in maize fields under different green manure management practices. MethodsA field experiment was conducted using common vetch as a green manure crop following the harvest of spring wheat. The study employed a completely randomized block design and examined four treatments: tillage with total green manure incorporation, no-tillage with total green manure mulching (NTG), tillage with green manure root incorporation (T), and no-tillage with aboveground green manure removal (NT). The study explored both non-biological and biological factors affecting nitrogen conversion to elucidate the mechanisms in N2O emissions. ResultsN2O emissions were 17.7% lower in TG and NTG compared to T and NT (P < 0.01), with NTG showing a 12.2% reduction compared to TG (P < 0.05). NTG also exhibited the lowest average N2O fluxes at 24.2 μg N m−2 h−1. In terms of non-biological factors, N2O emissions were negatively correlated only with aggregate NH4+-N and NO3－-N (P < 0.05). Despite increasing soil mineral nitrogen, NTG had approximately 72.3%− 92.4% of it in the form of aggregate nitrogen. In addition, under NTG, the aggregates have better structure and stability. q-PCR analysis revealed higher nos Z gene abundance in NTG than in other treatments (P < 0.05). Regression analysis showed that N2O emissions were negatively correlated with nos Z's abundance and N2O reductase diversity (P < 0.05). ConclusionsNo-tillage with total green manure mulching (NTG) effectively reduced N2O emissions by enhancing mineral nitrogen in soil aggregates and increasing the abundance of the nos Z gene. ImplicationsOur findings offer valuable insights for long-term agricultural management strategies aimed at improving nitrogen retention and mitigating climate change impacts.

Triazenide-supported [Cu4S] structural mimics of CuZ that mediate N2O disproportionation rather than reduction.

As part of the nitrogen cycle, environmental nitrous oxide (N2O) undergoes the N2O reduction reaction (N2ORR) catalyzed by nitrous oxide reductase, a metalloenzyme whose catalytic active site is a tetranuclear copper-sulfide cluster (CuZ). On the other hand, heterogeneous Cu catalysts on oxide supports are known to mediate decomposition of N2O (deN2O) by disproportionation. In this study, a CuZ model system supported by triazenide ligands is characterized by X-ray crystallography, NMR and EPR spectroscopies, and electronic structure calculations. Although the triazenide-ligated Cu4(μ4-S) clusters are closely related to previous formamidinate derivatives, which differ only in replacement of a remote N atom for a CH group, divergent reactivity with N2O is observed. Whereas the formamidinate-ligated clusters were previously shown to mediate single-turnover N2ORR, the triazenide-ligated clusters are found to mediate deN2O, behavior that was previously unknown to natural or synthetic copper-sulfide clusters. The reaction pathway for deN2O by this model system, including previously unidentified transition state models for N2O activation in N-O cleavage and O-O coupling steps, are included. The divergent reactivity of these two related but subtly different systems point to key factors influencing behavior of Cu-based catalysts for N2ORR (i.e., CuZ) and deN2O (e.g., CuO/CeO2).

Denitrification genotypes of endospore-forming Bacillota.

Denitrification is a key metabolic process in the global nitrogen cycle and is performed by taxonomically diverse microorganisms. Despite the widespread importance of this metabolism, challenges remain in identifying denitrifying populations and predicting their metabolic end-products based on their genotype. Here, genome-resolved metagenomics was used to explore the denitrification genotype of Bacillota enriched in nitrate-amended high temperature incubations with confirmed N2O and N2 production. A set of 12 hidden Markov models (HMMs) was created to target the diversity of denitrification genes in members of the phylum Bacillota. Genomic potential for complete denitrification was found in five metagenome-assembled genomes from nitrate-amended enrichments, including two novel members of the Brevibacillaceae family. Genomes of complete denitrifiers encode N2O reductase gene clusters with clade II-type nosZ and often include multiple variants of the nitric oxide reductase gene. The HMM set applied to all genomes of Bacillota from the Genome Taxonomy Database identified 17 genera inferred to contain complete denitrifiers based on their gene content. Among complete denitrifiers it was common for three distinct nitric oxide reductases to be present (qNOR, bNOR, and sNOR) that may reflect the metabolic adaptability of Bacillota in environments with variable redox conditions.

Cu site differentiation in tetracopper(i) sulfide clusters enables biomimetic N2O reduction.

Copper clusters feature prominently in both metalloenzymes and synthetic nanoclusters that mediate catalytic redox transformations of gaseous small molecules. Such reactions are critical to biological energy conversion and are expected to be crucial parts of renewable energy economies. However, the precise roles of individual metal atoms within clusters are difficult to elucidate, particularly for cluster systems that are dynamic under operating conditions. Here, we present a metal site-specific analysis of synthetic Cu4(μ4-S) clusters that mimic the Cu Z active site of the nitrous oxide reductase enzyme. Leveraging the ability to obtain structural snapshots of both inactive and active forms of the synthetic model system, we analyzed both states using resonant X-ray diffraction anomalous fine structure (DAFS), a technique that enables X-ray absorption profiles of individual metal sites within a cluster to be extracted independently. Using DAFS, we found that a change in cluster geometry between the inactive and active states is correlated to Cu site differentiation that is presumably required for efficient activation of N2O gas. More precisely, we hypothesize that the Cu δ+⋯Cu δ- pairs produced upon site differentiation are poised for N2O activation, as supported by computational modeling. These results provide an unprecedented level of detail on the roles of individual metal sites within the synthetic cluster system and how those roles interplay with cluster geometry to impact the reactivity function. We expect this fundamental knowledge to inform understanding of metal clusters in settings ranging from (bio)molecular to nanocluster to extended solid systems involved in energy conversion.

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a β-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Metabolic mechanism of Cr(VI) pollution remediation by Alicycliphilus denitrificans Ylb10

Cr(VI) is a well-known toxic pollutant and its remediation has attracted great attention. It is important to continuously discover and explore new high-efficiency Cr(VI) reducing bacteria to further improve the efficiency of Cr(VI) pollution remediation. In this paper, metabolic mechanism of Cr(VI) reduction in a new highly efficient Cr(VI) reducing bacterium, Alicycliphilus denitrificans Ylb10, was investigated. The results showed that Ylb10 could tolerate and completely reduce 450 mg/L Cr(VI). Cr(VI) can be reduced in the intracellular compartment, membrane and the extracellular compartment, with the plasma membrane being the main active site for Cr(VI) reduction. With the addition of NADH, the reduction efficiency of cell membrane components for Cr(VI) increased 2.3-fold. The omics data analysis showed that sulfite reductase CysJ, thiosulfate dehydrogenase TsdA, nitrite reductase NrfA, nitric oxide reductase NorB, and quinone oxidoreductase ChrR play important roles in the reduction of Cr(VI), in the intracellular, and the extracellular compartment, and the membrane of Ylb10, and therefore Cr(VI) was reduced by the combined action of several reductases at these three locations.

Optimizing waste molasses utilization to enhance electron transfer via micromagnetic carriers: Mechanisms and high-nitrate wastewater denitrification performance

The biological denitrification of high-nitrate wastewater (HNW) is primarily hindered by insufficient carbon sources and excessive nitrite accumulation. In this study, micromagnetic carriers with varying micromagnetic field (MMF) strengths (0.0, 0.3, 0.6, 0.9 mT) were employed to enhance the denitrification of HNW using waste molasses (WMs) as a carbon source. The results revealed that 0.6 mT MMF significantly improved the total nitrogen removal (TN) efficiency at 96.3%. A high nitrate (NO3−-N) removal efficiency at 99.3% with a low nitrite (NO2−-N) accumulation at 25.5 mg/L was achieved at 0.6 mT MMF. The application of MMF facilitated the synthesis of adenosine triphosphate (ATP) and stimulated denitrifying enzymes (e.g., nitrate reductase (NAR), nitrite reductase (NIR), and nitric oxide reductase (NOR)), which thereby promoting denitrification. Moreover, the effluent chemical oxygen demand (COD), tryptophan and fulvic-like substances exhibited their lowest levels at 0.6 mT MMF. Analysis through 16S ribosomal ribonucleic acid gene sequencing indicated a significant enrichment of denitrifying bacteria including Castellaniella Klebsiella under the influence of MMF. Besides, the proliferation of Acholeplasma, Klebsiella and Proteiniphilum at 0.6 mT MMF promoted the hydrolysis and acidification of WMs. This study offers new insights into the enhanced utilization of WMs and the denitrification of HNW through the application of MMF.