Nitrite Reductase Research Articles

Temperature differentially regulates estuarine microbial N2O production along a salinity gradient

Nitrous oxide (N2O) is atmospheric trace gas that contributes to climate change and affects stratospheric and ground-level ozone concentrations. Ammonia oxidizers and denitrifiers contribute to N2O emissions in estuarine waters. However, as an important climate factor, how temperature regulates microbial N2O production in estuarine water remains unclear. Here, we have employed stable isotope labeling techniques to demonstrate that the N2O production in estuarine waters exhibited differential thermal response patterns between nearshore and offshore regions. The optimal temperatures (Topt) for N2O production rates (N2OR) were higher at nearshore than offshore sites. 15N-labeled nitrite (15NO2-) experiments revealed that at the nearshore sites dominated by ammonia-oxidizing bacteria (AOB), the thermal tolerance of 15N-N2OR increases with increasing salinity, suggesting that N2O production by AOB-driven nitrifier denitrification may be co-regulated by temperature and salinity. Metatranscriptomic and metagenomic analyses of enriched water samples revealed that the denitrification pathway of AOB is the primary source of N2O, while clade II N2O-reducers dominated N2O consumption. Temperature regulated the expression patterns of nitrite reductase (nirK) and nitrous oxide reductase (nosZ) genes from different sources, thereby influencing N2O emissions in the system. Our findings contribute to understanding the sources of N2O in estuarine waters and their response to global warming.

Plant growth and nitrate absorption and assimilation of two sweet potato cultivars with different N tolerances in response to nitrate supply

In sweet potato, rational nitrogen (N) assimilation and distribution are conducive to inhibiting vine overgrowth. Nitrate (NO3-) is the main N form absorbed by roots, and cultivar is an important factor affecting N utilization. Herein, a hydroponic experiment was conducted that included four NO3- concentrations of 0 (N0), 4 (N1), 8 (N2) and 16 (N3) mmol L-1 with two cultivars of Jishu26 (J26, N-sensitive) and Xushu32 (X32, N-tolerant). For J26, with increasing NO3- concentrations, the root length and root surface area significantly decreased. However, no significant differences were observed in these parameters for X32. Higher NO3- concentrations upregulated the expression levels of the genes that encode nitrate reductase (NR2), nitrite reductase (NiR2) and nitrate transporter (NRT1.1) in roots for both cultivars. The trends in the activities of NR and NiR were subject to regulation of NR2 and NiR2 transcription, respectively. For both cultivars, N2 increased the N accumulated in leaves, growth points and roots. For J26, N3 further increased the N accumulation in these organs. Under higher NO3- nutrition, compared with X32, J26 exhibited higher expression levels of the NiR2, NR2 and NRT1.1 genes, a higher influx NO3- rate in roots, and higher activities of NR and NiR in leaves and roots. Conclusively, the regulated effects of NO3- supplies on root growth and NO3- utilization were more significant for J26. Under high NO3- conditions, J26 exhibited higher capacities of NO3- absorption and distributed more N in leaves and in growth points, which may contribute to higher growth potential in shoots and more easily cause vine overgrowth.

Acid-catalyzed Transformation of Nitrite to Nitric Oxide on Copper(II)-Cobalt(II) Centers in a Bimetallic Complex.

Nitrite (NO2 -) serves as a pool of nitric oxide (NO) in biological systems under hypoxic conditions, and it is transformed to NO by nitrite reductase (NiR) enzyme in the presence of acid (H+ ions). However, NO synthases (NOSs) generate NO via L-arginine oxidation in normoxic conditions. Previously, acid-induced NO2 - reduction chemistry was modeled on mono-metallic 3d-metals, generating metal-nitrosyls or NO(g) with H2O or H2O2 products. Herein, to understand the relative potency of a bimetallic system, we report the acid-induced reductive conversion of η2-bound NO2 - to NO on CuII-CoII centers of a hetero-bimetallic CuII-nitrito-CoII complex, [(LN8H)CuII-NO2 --CoII]3+ (CuII-NO2 --CoII, 2) bearing an octadentate N8-cryptand ligand (LN8H). The CuII-NO2 --CoII generates [CuII(LN8H)CoII]4+ (1) upon reaction with one equiv. acid (HClO4, H+ ions source) with NO(g) via a presumed transient nitrousacid (ONOH) intermediate species. Likewise, this NO2 - reduction was found to form H2O, which is believed to be from the decomposition of H2O2, an intermediate species. In addition, complex 2, in the presence of more than one equiv. H+ ions also showed the formation of NO(g) with H2O. Mechanistic investigations, using 15N-labeled-15NO2 -, 18O-labeled-18O14N16O- and 2H-labeled-DClO4 (D+ source), revealed that the N-atom and O-atom in the 14/15NO and 14N18O gases are derived from NO2 - ligand and H-atom in H2O derived from H+-source, respectively.

Enhanced nitrogen removal and greenhouse gas reduction via activated carbon coupled iron-based constructed wetlands

Constructed wetlands (CWs) have been widely used to remove nitrogen in the effluent from sewage plants. Still, the removal capacity of nitrate (NO3−-N) in CWs is unsatisfactory due to the insufficient electron donor. This study explored the role of activated carbon (AC) in nitrogen removal and greenhouse gas (GHG) emissions reduction in different iron-based CWs. Long-term experimental results showed that pyrite and sponge iron-based CWs had higher NO3−-N removal rates (98.18 % and 94.10 %). Compared with the control group, AC promoted their total nitrogen (TN) removal efficiency (78.87–87.59 %) and reduced GHG emissions (CO2: 3.79–4.11 %, N2O: 2.97–8.78 %, CH4: 9.93–10.73 %). Moreover, AC not only promoted the values of electron transfer system activity (ETSA), the activity of nitrate reductase and nitrite reductase, but also significantly increased the microbial diversity and abundance of the substrate layer in pyrite/SI- based CWs. The relative abundance of the dominant bacteria in CWs with pyrite coupling with AC (PC-CW) was the highest (nirS: 77.60 %, nosZ: 88.98 %) compared with the other CWs. The higher relative abundance of Dechloromonas and Thiobacillus in PC-CW indicated that the composite substrates of pyrite and AC promoted the extracellular electron transfer of denitrifying microorganisms. This study provides new insights into optimizing inorganic electron donors to improve the removal of NO3−-N and the reduce of GHG emissions in CWs for low C/N wastewater treatment.

Nitrite reductase-mimicking catalysis temporally regulating nitric oxide concentration gradient adaptive for antibacterial therapy.

The unique bacterial infection microenvironment (IME) usually requires complicated design of nanomaterials to adapt to IME for enhancing antibacterial therapy. Here, an alternative IME adaptative nitrite reductase-mimicking nanozyme is constructed by in situ growth of ultrasmall copper sulfide clusters on the surface of a nanofibrillar lysozyme assembly (NFLA/CuS NHs), which can temporally regulate nitric oxide (NO) gradient concentration to kill bacteria initially and promote tissue regeneration subsequently. Benefiting from a copper nitrite reductase (CuNIR)-inspired structure with CuS cluster as active center and NFLA as skeleton, NFLA/CuS NHs efficiently boost the catalytic reduction of nitrite to NO. The inherent supramolecular fibrillar networks displays excellent bacterial capture capability, facilitating initial high-concentration NO attacks on the bacteria. The subsequent catalytic release of low-concentration NO by NFLA/CuS NHs-mediated nitrite reduction remarkably promotes cell migration and angiogenesis. This work paves the way for dynamically eliminating MDR bacterial infection and promoting tissue regeneration in a simple and smart way through CuNIR-mimicking catalysis.

Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Unraveling the impact of perfluorooctanoic acid on sulfur-based autotrophic denitrification process

PFOA has garnered heightened scrutiny for its impact on denitrification, especially given its frequent detection in secondary effluent discharged from wastewater treatment plants. However, it is still unclear what potential risk PFOA release poses to a typical advanced treatment process, especially the sulfur-based autotrophic denitrification (SAD) process. In this study, different PFOA concentration were tested to explore their impact on denitrification kinetics and microbial dynamic responses of the SAD process. The results showed that an increase PFOA concentration from 0 to 1000 μg/L resulted in a decrease in nitrate removal rate from 9.52 to 7.73 mg-N/L·h. At the same time, it increased nitrite accumulation and N2O emission by 6.11 and 2.03 times, respectively. The inhibitory effect of PFOA on nitrate and nitrite reductase activity in the SAD process was linked to the observed fluctuations in nitrate and nitrite levels. It is noteworthy that nitrite reductase was more vulnerable to the influence of PFOA than nitrate reductase. Furthermore, PFOA showed a significant impact on gene expression and microbial community. Metabolic function prediction revealed a notable decrease in nitrogen metabolism and an increase in sulfur metabolism under PFOA exposure. This study highlights that PFOA has a considerable inhibitory effect on SAD performance.

Methionine represses gray mold of tomato by keeping nitric oxide at an appropriate level via ethylene synthesis and signal transduction

Methionine (Met) can inhibit plant diseases caused by phytopathogens. However, the effect of Met on gray mold resulted from Botrytis cinerea in tomato is still unclear. This study showed 5 mM Met alleviated disease development of gray mold, enhanced chitinase (CHI) and β-1, 3-glucanase (GNS) activities and the expression of SlCHI, SlGNS, SlPR1 and SlNPR1 in tomatoes, rather than inhibited the growth of B. cinerea directly. Moreover, ethylene biosynthesis and signal transduction before pathogen inoculating were induced by 5 mM Met. Interestingly, Met reduced the nitrosylation levels of ACS4 and ACO6, enhanced the activities of nitric oxide synthase, nitrite reductase (NR) and S-nitrosoglutathione reductase (GSNOR) and the expression of SlNR and SlGSNOR. Tomatoes treated with aminoethoxyvinylglycine and carboxy-PTIO exhibited lower resistance to B. cinerea. These results indicate 5 mM Met promoted ethylene biosynthesis and signal transduction to facilitate NO synthesis and metabolism, enhancing the resistance of tomatoes to B. cinerea.

Insights into the evolutionary and ecological adaption strategies of nirS- and nirK-type denitrifying communities.

Denitrification is a crucial process in the global nitrogen cycle, in which two functionally equivalent genes, nirS and nirK, catalyse the critical reaction and are usually used as marker genes. The nirK gene can function independently, whereas nirS requires additional genes to encode nitrite reductase and is more sensitive to environmental factors than nirK. However, the ecological differentiation mechanisms of those denitrifying microbial communities and their adaptation strategies to environmental stresses remain unclear. Here, we conducted metagenomic analysis for sediments and bioreactor samples from Lake Donghu, China. We found that nirS-type denitrifying communities had a significantly lower horizontal gene transfer frequency than that of nirK-type denitrifying communities, and nirS gene phylogeny was more congruent with taxonomy than that of nirK gene. Metabolic reconstruction of metagenome-assembled genomes further revealed that nirS-type denitrifying communities have robust metabolic systems for energy conservation, enabling them to survive under environmental stresses. Nevertheless, nirK-type denitrifying communities seemed to adapt to oxygen-limited environments with the ability to utilize various carbon and nitrogen compounds. Thus, this study provides novel insights into the ecological differentiation mechanism of nirS and nirK-type denitrifying communities, as well as the regulation of the global nitrogen cycle and greenhouse gas emissions.

Effects of Long-Term Application of Nitrogen Fertilizer on Soil Acidification and Biological Properties in China: A Meta-Analysis.

Soil acidification is a global environmental problem with significant impacts on agricultural production, environmental protection, and ecosystem health. Soil acidification is widespread in China, affecting crop yields, agricultural product quality, and biodiversity. Since the 1980s, much work has been done on acidic soils in China, but it is controversial whether excessive nitrogen fertilizer application can lead to soil acidification mechanisms. To address the above issues, we conducted a meta-analysis of 115 published papers to integrate and analyze the effects of N fertilizer application on soil acidification and biological properties from 1980 to 2024. We also quantified the effect of nitrogen fertilization on soil acidification and biological changes under different climatic conditions. The results showed that under long-term application of nitrogen fertilizers in China from 1980 to 2024, soil pH decreased by an average of 15.27%, and the activities of soil urease, nitrate reductase, nitrite reductase, catalase, glutamate dehydrogenase, and glutamate synthetase decreased by an average of 9.82-22.37%. The soil microbial community richness (Chao1 index) increased by 6.53%, but the community diversity (Shannon index) decreased by 15.42%. Among the dominant soil microorganisms, the relative abundance of bacteria decreased by an average of 9.67-29.38% and the abundance of gene expression of nifH, amoA-AOA, amoA-AOB, and qnorB decreased by 9.92-19.83%. In addition, we found that the mean annual temperature and rainfall impacted soil acidification via their effect on soil microbial diversity and community composition. This study provides a scientific basis for an in-depth understanding of the spatial and temporal variation of soil acidification and biological properties in China.

Short-Term Effects of Cenchrus fungigraminus/Potato or Broad Bean Interplanting on Rhizosphere Soil Fertility, Microbial Diversity, and Greenhouse Gas Sequestration in Southeast China.

Cenchrus fungigraminus is a new species and is largely used as forage and mushroom substrate. However, it can usually not be planted on farmland on account of local agricultural land policy. Interplanting Cenchrus fungigraminus with other crops annually (short-term) is an innovative strategy to promote the sustainable development of the grass industry in southern China. To further investigate this, C. fungigraminus mono-planting (MC), C. fungigraminus-potato interplanting (CIP) and C. fungigraminus-broad bean interplanting (CIB) were performed. Compared to MC, soil microbial biomass carbon (SMBC), soil organic matter (SOM), ammoniacal nitrogen (AMN), pH and soil amino sugars had a positive effect on the rhizosphere soil of CIP and CIB, as well as enhancing soil nitrogenase, nitrite reductase, and peroxidase activities (p < 0.05). Moreover, CIP improved the root vitality (2.08 times) and crude protein (1.11 times). In addition, CIB enhanced the crude fiber of C. fungigraminus seedlings. These two interplanting models also improved the microbial composition and diversity (Actinobacteria, Firmicutes, and Bacteroidota, etc.) in the rhizosphere soil of C. fungigraminus seedlings. Among all the samples, 189 and 59 genes were involved in methane cycling and nitrogen cycling, respectively, which improved the presence of the serine cycle, ribulose monophosphate, assimilatory nitrate reduction, methane absorption, and glutamate synthesis and inhibited denitrification. Through correlation analysis and the Mantel test, the putative functional genes, encoding functions in both nitrogen and methane cycling, were shown to have a significant positive effect on pH, moisture, AMN, SOM, SMBC, and soil peroxidase activity, while not displaying a significant effect on soil nitrogenase activity and total amino sugar (p < 0.05). The short-term influence of the interplanting model was shown to improve land use efficiency and economic profitability per unit land area, and the models could provide sustainable agricultural production for rural revitalization.

Inorganic carbon metabolism enhanced hydrogen-driven denitrification: Evaluation of carbon fixation pathways and microbial traits

H2-driven denitrification offers a promising alternative for treating low C/N wastewater amidst global nitrogen pollution, due to its advantages of high removal efficiency, low energy consumption, and independence from exogenous organic carbon source. While inorganic carbon assimilation is crucial for the growth and reproduction of autotrophic microorganisms, it remains uncertain whether the fixation of inorganic carbon could impact H2-driven denitrification. This work demonstrated that the supplement of inorganic carbon efficiently improved the efficiency of nitrate removal to 90.3%, compared to 78.0% in the control. Further analysis indicated that inorganic carbon supplementation shifted the microbial structure from autotrophic-dominated (e.g., Hydrogenophaga and Rhodococcus with abundance of 2.3% and 2.6%) to mixotrophic-dominated (e.g., Thauera and Microscillaceae sp. with abundance of 5.6% and 32.8%). Additionally, although H2 boosted the carbon fixation via the reductive TCA cycle and Wood-Ljungdahl pathway, the Calvin-Benson cycle emerged as the predominant pathway, significantly boosted under inorganic carbon supplementation, along with the upregulation of critical enzyme expression including RuBisCo (EC 4.1.1.39), PGK (EC 2.7.2.3) and GAPDH (EC 1.2.1.12). Moreover, the expressions and activities of functional enzymes involved in nitrogen transformation, including nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR), as well as microbial electron transfer ability, were remarkably enhanced under sufficient inorganic carbon conditions. This work contributed to understanding the carbon metabolism mechanism in H2-driven denitrification, thereby facilitating the promotion of the efficient and low-carbon approach for nitrogen removal from wastewater.

Suitability of coconut bran and biochar as a composite substrate for lettuce cultivation in aquaponic systems

Growth substrates are essential for aquaponic systems and play an important role in vegetable growth and water quality. In this study, we explored an innovative combination of coconut bran and coconut shell biochar (CSB) as a composite growth substrate for lettuce cultivation in aquaponic systems. The study included the control (100 % coconut bran as the growth substrate) and treatment groups (T1–T5; containing 10 %, 20 %, 30 %, 40 %, and 50 % CSB as the growth substrate, respectively). The substrate properties; lettuce growth performance; and soil enzyme activity, nitrogen content, and abundance of microbial communities in the substrate were analyzed to determine the optimal substrate. Our findings indicated that CSB incorporation significantly altered the properties of the substrate, resulting in increased dry and bulk densities, pH, and water-holding capacity, and decreased electrical conductivity, water-absorption capacity, and porosity. Furthermore, the fresh weight of lettuce was notably increased in the treatment groups. The activities of fluorescein diacetate hydrolase, urease, nitrate reductase, and hydroxylamine reductase initially increased and further decreased, reaching the maximum in the T3 group. Conversely, the activity of nitrite reductase and contents of available nitrogen, nitrate-nitrogen, and ammonium-nitrogen in the substrates initially decreased and further increased, with the minimum values observed in the T3 group. The microbial sequencing results indicated that CSB incorporation significantly increased the microbial diversity and relative abundance of microorganisms associated with nitrogen transformation. Moreover, 30 % CSB incorporation exhibited the greatest effect on lettuce growth, with a 34.5 % and 31.6 % increase in fresh weight compared to the control during the growth and harvest periods, respectively. This study indicated the enormous potential of biochar in the research and development of green technologies for substrate amendment in aquaponic systems.

Microplastic exposure inhibits nitrate uptake and assimilation in wheat plants

Microplastic (MP) contamination in soil severely impairs plant growth. However, mechanisms underlying the effects of MPs on plant nutrient uptake remain largely unknown. In this study, we revealed that NO3− content was significantly decreased in shoots and roots of wheat plants exposed to high concentrations (50–100 mg L−1) of MPs (1 μm and 0.1 μm; type: polystyrene) in the hydroponic solution. Isotope labeling experiments demonstrated that MP exposure led to a significant inhibition of NO3− uptake in wheat roots. Further analysis indicated that the presence of MPs markedly inhibited root growth and caused oxidative damage to the roots. Additionally, superoxide dismutase and peroxidase activities in wheat roots decreased under all MP treatments, whereas catalase and ascorbate peroxidase activities significantly increased under the 100 mg L−1 MP treatment. The transcription levels of most nitrate transporters (NRTs) in roots were significantly downregulated by MP exposure. Furthermore, exposure to MPs distinctly suppressed the activity of nitrate reductase (NR) and nitrite reductase (NiR), as well as the expression levels of their coding genes in wheat shoots. These findings indicate that a decline in root uptake area and root vitality, as well as in the expression of NRTs, NR, and NiR genes caused by MP exposure may have adverse effects on NO3− uptake and assimilation, consequently impairing normal growth of plants.

Microbial response to the chronic toxicity effect of graphene and graphene oxide nanomaterials within aerobic granular sludge systems

Nanomaterials present in wastewater can pose a significant threat to aerobic granular sludge (AGS) systems. Herein, we found that compared to graphene nanomaterials (G-NMs), the long-term presence (95 days) of graphene oxide nanomaterials (GO-NMs) resulted in an increased proliferation of filamentous bacteria, poorer sedimentation performance (SVI30 of 74.1 mL/g) and smaller average particle size (1224.4 µm) of the AGS. In particular, the GO-NMs posed a more significant inhibitory effect to the total nitrogen removal efficiency of AGS (decreased by 14.3 %), especially for the denitrification process. The substantial accumulation of GO-NMs within the sludge matrix resulted in a higher level of reactive oxygen species in AGS compared to G-NMs, thereby inducing lactate dehydrogenase release, and enhancing superoxide oxidase and catalase activities. Such excessive oxidative stress could potentially result in a significant reduction in the activity of nitrogen metabolism enzymes (e.g., nitrate reductase and nitrite reductase) and the expression of key functional genes (e.g., nirS and nirK). Altogether, compared to G-NMs, prolonged exposure to GO-NMs had a more significant chronic toxicity effect on AGS systems. These findings implied that the presence of G-NMs and GO-NMs is a hidden danger to biological nitrogen removal and should receive more attention.

Salinity responsive mechanisms of sulfur-based mixotrophic denitrification and ectoine induced tolerance enhancement

Increasing discharge of saline wastewater poses a challenge for heterotrophic and sulfur autotrophic denitrification (HSAD) in nitrogen removal. This study explored the response mechanism of mixotrophic denitrifying microorganisms to high-salt stress and feasible mitigation strategies. Results showed that denitrification efficiency was promoted at 2 % salinity but significantly decreased at 6 %, with NO3–-N removal rate decreased from 95.77 % to 38.01 %. The contribution of sulfur autotrophic denitrification (SAD) remained higher than heterotrophic denitrification (HD). High salinity stimulation led to an increase in reactive oxygen species (ROS) content, while the nicotinamide adenine dinucleotide (NADH) content, adenosine triphosphate (ATP) level, and electron transport system activity (ETSA) decreased by 10.74 %, 46.6 % and 56.28 % at 6 % salinity, respectively. Additionally, high salt inhibited the activity of nitrate reductase (NAR) and nitrite reductase (NIR), reducing the abundance of denitrifying bacteria. Notably, the addition of 250 mg/L of ectoine at 6 % salinity alleviated stress, promoted the secretion of extracellular polymeric substances (EPS) to protect the microorganisms, reduced microbial oxidative stress, and maintained metabolic and denitrification enzyme activities. Ectoine acted as an osmotic pressure regulator and protective agent, maintaining intracellular enzymes and macromolecular structure, thus enhancing denitrification efficiency. Microbial community analysis demonstrated a 3.99 % increase in HD functional bacteria abundance with ectoine addition, highlighting its crucial role in regulation community succession and stability. Structural equations modeling analysis identified the regulatory relationship paths involved in the response and mitigation of HSAD to high salinity stress. This study deepens understanding of the inhibition mechanism of HSAD caused by high-salt wastewater and provides a feasible technical solution for salt stress alleviation in sulfur-based mixotrophic denitrification.

High nitrite accumulation in hydrogenotrophic denitrification at low temperature: Transcriptional regulation and microbial community succession

High Pressure Hydrogenotrophic Denitrification (HPHD) provided a promising alternative for efficient and clean nitrate removal. In particular, the denitrification rates at low temperature could be compensated by elevated H2 partial pressure. However, nitrite reduction was strongly inhibited while nitrate reduction was barely affected at low temperature. In this study, the nitrate reduction gradually recovered under long-term low temperature stress, while nitrite accumulation increased from 0.1 to 41.0 mg N/L. The activities of the electron transport system (ETS), nitrate reductase (NAR), and nitrite reductase (NIR) decreased by 45.8 %, 27.3 %, and 39.3 %, respectively, as the temperature dropped from 30 °C to 15 °C. Real time quantitative PCR analysis revealed that the denitrifying gene expression rather than gene abundance regulated nitrogen biotransformation. The substantial nitrite accumulation was attributed to the significant up-regulation by 54.7 % of narG gene expression and down-regulation by 73.7 % of nirS gene expression in hydrogenotrophic denitrifiers. In addition, the nirS-gene-bearing denitrifiers were more sensitive to low temperature compared to those bearing nirK gene. The dominant populations shifted from the genera Paracoccus to Hydrogenophaga under long-term low temperature stress. Overall, this study revealed the microbial mechanism of high nitrite accumulation in hydrogenotrophic denitrification at low temperature.

Reaction Discovery Using Spectroscopic Insights from an Enzymatic C-H Amination Intermediate.

Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH2OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV-vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagents─nitrite (NO2-), nitric oxide (NO), and nitroxyl (HNO)─that are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C-H amination inspired by the nitrite reductase pathway. This study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.

Catalytic enhancement of microbial denitrification by FeVO4@biochar: Insight into the extra cellular polymeric matrix mechanism

The biological denitrification is being considered as one of the best methods for nitrate removal from wastewaters, however it suffers with lower efficiency which needs to be improved effectively. The present study was aimed to prepare FeVO4@biochar and investigated its role as electron shuttle in enhancing the biological denitrification through various batch experiments. Significantly higher rates of denitrification were observed after addition of FeVO4@biochar in different concentrations and the highest efficiency was observed in 15 wt% FeVO4@biochar (BF-15) where 87.6 % denitrification was achieved after 7 days (from 100 mg/L to 12.7 mg/L of nitrate) compared to pure biochar (65.7 mg/L; 34.3 %) and sludge (72.7 mg/L; 27.3 %). FeVO4@biochar increased the transfer of external electron to bacteria and enhanced the denitrification activity. The variations in NAR (nitrate reductase), NIR (nitrite reductase) and ETSA (electron transport system activity) were investigated to understand the changes in microbial-community during denitrification. The FeVO4@biochar and extracellular polymeric substance (EPS) both acted as the media for electron transport along with the decreased repulsions between nitrate and the catalyst surface that further provides higher nitrate reduction efficiency. The electroactive bacteria could easily utilize the photogenerated electrons from FeVO4@biochar and facilitate excellent denitrification. The microbial-diversity analysis revealed Bacteroidota, Firmicutes, Proteobacteria, Chloroflexi, Desulfobacterota, Acidobacteriota, Actinobacteriota, and Gemmatimonadota were observed as the most predominant phyla actively participated in enhanced denitrification. This study suggested that the addition of photoactive catalyst strongly enhanced the electron transfer during denitrification and provides new ideas to the biological denitrification

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.