Nitrate Pathways Research Articles

A Cytochrome P450 TxtE Model System with Mechanistic and Theoretical Evidence for a Heme Peroxynitrite Active Species

AbstractThe cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L‐tryptophan to 4‐nitro‐L‐tryptophan, using nitric oxide (NO) and dioxygen (O2) as co‐substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)‐peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)FeIII(O2−⋅)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4‐nitroindole, the product analogous to that obtained with TxtE. Moreover, 15N and 18O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings.

Selecting an optimal sorghum cultivar can improve nitrogen availability and wheat yield in crop rotation.

Sorghum (Sorghum bicolor L. Moench) is a cereal crop known for its biological nitrification inhibition (BNI) capacity, a plant-mediated activity limiting nitrification pathway. The use of BNI-producing plants represents an environmentally friendly and cost-effective approach to reduce nitrogen (N) losses, such as nitrate (NO3 -) leaching and nitrous oxide (N2O) gas emissions. The present study aimed to test the effectiveness of different S. bicolor cultivars in rotation to retain ammonium (NH4 +) in soils and promote N availability for the subsequent wheat crop. A two-year field rotation was established with four sorghum cultivars followed by winter wheat (Triticum aestivum L.). Urea alone or combined with the urease inhibitor N-(n-butyl) thiophosphoric triamide was applied to promote a NH4 +-based fertilization regimes. AddingN-(n-butyl) thiophosphoric triamide maintained higher soil NH4 + content and reduced ammonia-oxidizing bacteria population during sorghum cultivation. However, the benefits of the inhibitor on sorghum growth were cultivar-dependent. Notably, the further reduction in ammonia-oxidizing bacteria abundance for sorghum Voyenn and the increased soil NH4 + content for Vilomene suggested a BNI potential for these cultivars. Importantly, the Vilomene precedent enhanced wheat yield for both fertilization regimes. Overall, the present study confirms that sorghum is a suitable catch crop and emphasizes the importance of selecting the proper sorghum cultivar to maximize the yield of the target wheat crop, at the same time as minimizing N losses. Furthermore, developing combined strategies with selected sorghum cultivars and the application of urease inhibitors enables to enhance sorghum productivity as forage, achieving added value to the rotation. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of reactive oxygen species (ROS) produced by pyridine and quinoline on NH4+-N removal under phenol stress: The shift of nitrification pathway and its potential mechanisms

Pyridine and quinoline are typical nitrogenous heterocyclic compounds with different structures that are found in coking wastewater. However, neither the corresponding mechanism nor its effect on the degradation of NH4+-N under phenol stress is known. In this study, the effects of pyridine and quinoline degradation on NH4+-N removal under phenol stress were evaluated using three lab-scale sequencing batch reactors. The average NH4+-N removal efficiencies of the reactors were 99.46 %, 88.86 %, and 98.64 %. With the increased concentration of pyridine and quinoline, NH4+-N and NO3--N accumulated to 58.37 mg/L and 141.37 mg/L, respectively, due to the lack of an electron donor and anaerobic environment. The addition of pyridine and quinoline significantly improved antioxidant response and altered the nitrification pathway. The nitrification process shifted from the mediation of amo and hao to the mediation of Ncd2 due to oxidative stress induced by pyridine and quinoline. Furthermore, oxidative stress interferes with the metabolism of carbon sources, resulting in decreased biomass. These results provide a new perspective for coking wastewater treatment processes.

A Cytochrome P450 TxtE Model System with Mechanistic and Theoretical Evidence for a Heme Peroxynitrite Active Species.

The cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L-tryptophan to 4-nitro-L-tryptophan, using nitric oxideand dioxygenas co-substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)-peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)FeIII(O2-•)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4-nitroindole, the product analogous to that obtained with TxtE. Moreover, 15N and 18O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings.

Effect of nitrogen reduction by chemical fertilization with green manure (Vicia sativa L.) on soil microbial community, nitrogen metabolism and and yield of Uncaria rhynchophylla by metagenomics.

Uncaria rhynchophylla is an important herbal medicine, and the predominant issues affecting its cultivation include a single method of fertilizer application and inappropriate chemical fertilizer application. To reduce the use of inorganic nitrogen fertilization and increase the yield of Uncaria rhynchophylla, field experiments in 2020-2021 were conducted. The experimental treatments included the following categories: S1, no fertilization; S2, application of chemical NPK fertilizer; and S3-S6, application of chemical fertilizers and green manures, featuring nitrogen fertilizers reductions of 0%, 15%, 30%, and 45%, respectively. The results showed that a moderate application of nitrogen fertilizer when combined with green manure, can help alleviate soil acidification and increase urease activity. Specifically, the treatment with green manure provided in a 14.71-66.67% increase in urease activity compared to S2. Metagenomics sequencing results showed a decrease in diversity in S3, S4, S5, and S6 compared to S2, but the application of chemical fertilizer with green manure promoted an increase in the relative abundance of Acidobacteria and Chloroflexi. In addition, the nitrification pathway displayed a progressive augmentation in tandem with the reduction in nitrogen fertilizer and application of green manure, reaching its zenith at S5. Conversely, other nitrogen metabolism pathways showed a decline in correlation with diminishing nitrogen fertilizer dosages. The rest of the treatments showed an increase in yield in comparison to S1, S5 showing significant differences (p < 0.05). In summary, although S2 demonstrate the ability to enhance soil microbial diversity, it is important to consider the long-term ecological impacts, and S5 may be a better choice.

Efficient heterotrophic nitrification by a novel bacterium Sneathiella aquimaris 216LB-ZA1-12T isolated from aquaculture seawater

High concentration of ammonia poses a common threat to the healthy breeding of marine aquaculture organisms. Since aquaculture water is rich in organic matter, heterotrophic nitrifying bacteria might play a crucial role in ammonia removal. However, their roles in ammonia oxidation remain unknown. Here, we report a novel strain isolated from shrimp aquaculture seawater, identified as Sneathiella aquimaris 216LB-ZA1–12T, capable of heterotrophic nitrification. It is the first characterized heterotrophic nitrifier of the order Sneathiellales in the class Alphaproteobacteria. It exhibits high activity in heterotrophic nitrification, removing nearly 94% of ammonium-N under carbon-constrained conditions in 8 days with no observed nitrite accumulation. The heterotrophic nitrification pathway, inferred based on detection and genomic data was as follows: NH4+→NH2OH→NO→NO2-→NO3-. While this pathway aligns with the classical nitrification pathway, while the significant difference lies in the absence of classical HAO and HOX encoding genes in the genome, which is common in heterotrophic nitrifying bacteria. In summary, this bacterium is not only valuable for studying the nitrifying mechanism, but also holds potential for practical applications in ammonia removal in marine aquaculture systems and saline wastewater.

Enhanced nitrogen removal in a pilot-scale AOA-SBFASR system treating municipal wastewater: Process efficiency, microbial community and nitrogen metabolic pathways

In case of no advanced treatment units and supplement of carbon sources with high dose, removal of TN remains as challenge for wastewater treatment plants. In this study, a 67.5 m3/day Anoxic-Oxic-Anoxic/ Sequencing Batch Fixed-biofilm and Activated Sludge Reactor (AOA-SBFASR) was developed and operated for 150 days. During the trial, removal efficiencies of COD, NH4+-N and TN reached 92.55 %, 98.75 %, and 75.64 %, respectively. Reconfiguration of microbial community was observed during the trial, and the abundances of nitrifying bacteria, denitrifying bacteria and refractory organic degrading bacteria were increased, indicating that AOA-SBFASR might has great potentials for enhanced nitrogen removal and refractory organics conversion and utilization. The analysis of nitrogen metabolism showed that the reduced nitrogen was oxidized through complete nitrification pathway and the nitrogen reduction was mainly performed by pathways of denitrification and dissimilatory nitrate reduction.

Decoupling the simultaneous effects of NO2−, pH and free nitrous acid on N2O and NO production from enriched nitrifying activated sludge

In the pursuit of energy and carbon neutrality, nitrogen removal technologies have been developed featuring nitrite (NO2−) accumulation. However, high NO2− accumulations are often associated with stimulated greenhouse gas (i.e., nitrous oxide, N2O) emissions. Furthermore, the coexistence of free nitrous acid (FNA) formed by NO2− and proton (pH) makes the consequence of NO2− accumulation on N2O emissions complicated. The concurrent three factors, NO2−, pH and FNA may play different roles on N2O and nitric oxide (NO) emissions simultaneously, which has not been systematically studied. This study aims to decouple the effects of NO2− (0-200 mg N/L), pH (6.5-8) and FNA (0-0.15 mg N/L) on the N2O and NO production rates and the production pathways by ammonia oxidizing bacteria (AOB), with the use of a series of precisely executed batch tests and isotope site-preference analysis. Results suggested the dominant factors affecting the N2O production rate were NO2− and FNA concentrations, while pH alone played a relatively insignificant role. The most influential factor shifted from NO2− to FNA as FNA concentrations increased from 0 to 0.15 mg N/L. At concentrations below 0.0045 mg HNO2-N/L, nitrite rather than FNA played a significant role stimulating N2O production at elevated nitrite concentrations. The inhibition effect of FNA emerged with further increase of FNA between 0.0045-0.015 mg HNO2-N/L, weakening the promoting effect of increased nitrite. While at concentrations above 0.015 mg HNO2-N/L, FNA inhibited N2O production especially from nitrifier denitrification pathway with the level of inhibition linearly correlated with the FNA concentration. pH and the nitrite concentration regulated the production pathways, with elevated pH promoting the nitrifier nitrification pathway, while elevated NO2− concentrations promoting the nitrifier denitrification pathway. In contrast to N2O, NO emission was less susceptible to FNA at concentrations up to 0.015 mg N/L but was stimulated by increasing NO2− concentrations. This study, for the first time, distinguished the effects of pH, NO2− and FNA on N2O and NO production, thereby providing support to the design and operation of novel nitrogen removal systems with NO2− accumulation.

Nitrogen removal capability and mechanism of a novel heterotrophic nitrification–aerobic denitrification bacterium Halomonas sp. DN3

Recently, the functional microorganisms capable of eliminating nitrogenous waste have been applied in mariculture systems. As a potential candidate for treating mariculture wastewater, strain DN3 eliminated 100% of ammonia and nitrate and 96.61%–100% of nitrite within 72 h, when single nitrogen sources at concentrations of 0–50 mg/L. Strain DN3 also exhibited the efficient removal performance of mixed-form nitrogen (ammonia, nitrate, and nitrite) at salinity 30 ‰, C/N ratio 20, and 180 rpm. The nitrogen assimilation pathway dominated inorganic nitrogen metabolism, with less nitrogen (14.23%-25.02% of TN) lost into the air via nitrification and denitrification, based on nitrogen balance analysis. Moreover, the bacterial nitrification pathway was explored by enzymatic assays and inhibition assays. These complex nitrogen assimilation and dissimilation processes were further revealed by bacterial genome analysis. These results provide important insight into nitrogen metabolism of Halomonas sp. and theoretical support for treating mariculture wastewater with strain DN3.

Nitrite pathway in A2/O WWTPs: Modelling organic matter reduction, operational cost and N2O emissions

Deciding whether the implementation of the nitrite pathway is useful for the treatment of a given wastewater depends on several criteria such as organic matter availability, N and P removal, N2O emissions and operational costs. This work is a simulation-based study with a conventional Anaerobic/Anoxic/Oxic (A2/O) WWTP where the nitrite pathway can be implemented. The outcomes are general correlations to calculate the minimum COD requirements for a certain influent and a practical decision tree on the opportunities of nitrite pathway in A2/O WWTPs as a function of the influent wastewater composition (for P and N concentrations in the ranges 3–11 mgPO4−3-P·L−1 and 20–60 mg NH4+-N·L−1). This study shows that the implementation of the nitrite pathway reduces the COD requirements (depending on the influent, between 9 and 68%). It also can lead to a reduction in aeration costs. For an equivalent COD in the influent, the implementation of the nitrite pathway compared to the nitrate pathway results in a reduction of 41-47% in aeration costs and similar sludge production, leading to a reduction in the operational cost index of 10-16%. However, it is essential to note that this strategy can also lead to increased N2O emissions, with an emission factor for the nitrite pathway in the range of 2.5–17 times that of the nitrate pathway.

Mechanism of nitro-byproducts formation during persulfate-based electrokinetic in situ oxidation for remediation of anthracene contaminated soil

Persulfate-based electrokinetic (EK) chemical oxidation appears to be a novel and viable strategy for the in situ remediation of polycyclic aromatic hydrocarbons (PAHs) polluted soil; however, the possible toxic byproducts of PAHs have been overlooked. In this study, we systematically investigated the formation mechanism of the nitro-byproducts of anthracene (ANT) during the EK process. Electrochemical experiments revealed that NH4+ and NO2- originating from nitrate electrolyte or soil substrates were oxidized to NO2• and NO• in the presence of SO4•-. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS) analysis with 15N labeling revealed the formation of nitro-byproducts (14 kinds), including 1-hydroxy-4-nitro-anthraquinone and its similar derivatives, 4-nitrophenol, and 2,4-dinitrophenol. The nitration pathways of ANT have been proposed and described, mainly including the formation of hydroxyl-anthraquinone-oxygen and phenoxy radicals and the subsequent addition of NO2• and NO•. ANT-based formation of nitro-byproducts during EK, which is usually underestimated, should be further investigated due to their enhanced acute toxicity, mutagenic effects, and potential threat to the ecosystem.

Nitration of Phenols by Reaction with Aqueous Nitrite: A Pathway for the Formation of Atmospheric Brown Carbon

Nitrophenols are a major component of light-absorbing atmospheric organic aerosols, commonly referred to as brown carbon (BrC). Most nitrophenol formation pathways involve reactions of phenolic compounds with OH, NO3, and NO2 in the gas phase. In this study, an aqueous nitrophenol production pathway is investigated that can proceed in the dark without apparent OH radical formation. Using high-performance liquid chromatography–mass spectrometry, we demonstrate that catechol reacts in acidic solutions with dissolved nitrite to form nitrocatechol. The rate of nitration increases significantly from pH 4.4 to pH 3.4 such that nitrocatechol is susceptible to second-generation reactions under the most acidic conditions producing chromophores that absorb in the visible region (peak at 425 nm). Increases in the N:C ratio of the reaction solution, as detected by aerosol mass spectrometry, and enhanced absorption from 300 to 500 nm of wood and peat smoke aqueous extracts exposed to nitrite suggest that this is a general nitration pathway. The atmospheric conditions under which this BrC formation process may occur are discussed.

Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3−-N, NO2−-N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3−-N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3−-N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3−→NO2−→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2−→NO3−, but the nitrification pathway of NH4+→ NO2− needs further studies.

Molecular analysis of microbial nitrogen transformation and removal potential in the plant rhizosphere of artificial tidal wetlands across salinity gradients

This study explored the microbial nitrogen transformation and removal potential in the plant rhizosphere of seven artificial tidal wetlands under different salinity gradients (0–30‰). Molecular biological and stable isotopic analyses revealed the existence of simultaneous anammox (anaerobic ammonium oxidation), nitrification, DNRA (dissimilatory nitrate reduction to ammonium) and denitrification processes, contributing to nitrogen loss in rhizosphere soil. The microbial abundances were 2.87 × 103–9.12 × 108 (nitrogen functional genes) and 1.24 × 108–8.43 × 109 copies/g (16S rRNA gene), and the relative abundances of dissimilatory nitrate reduction and nitrification genera ranged from 6.75% to 24.41% and from 0.77% to 1.81%, respectively. The bacterial 16S rRNA high-throughput sequencing indicated that Bacillus, Zobellella and Paracoccus had obvious effects on nitrogen removal by heterotrophic nitrifying/aerobic denitrifying process (HN-AD), and autotrophic nitrification (Nitrosomonas, Nitrospira and Nitrospina), conventional denitrification (Bradyrhizobium, Burkholderia and Flavobacterium), anammox (Candidatus Brocadia and Candidatus Scalindua) and DNRA (Clostridium, Desulfovibrio and Photobacterium) organisms co-existed with HN-AD bacteria. The potential activities of DNRA, nitrification, anammox and denitrification were 1.23–9.23, 400.03–755.91, 3.12–35.24 and 30.51–300.04 nmolN2·g−1·d−1, respectively. The denitrification process contributed to 73.59–88.65% of NOx− reduction, compared to 0.71–13.20% and 8.20–15.42% via DNRA and anammox, as 83.83–90.74% of N2 production was conducted by denitrification, with the rest through anammox. Meanwhile, the nitrification pathway accounted for 95.28–99.23% of NH4+ oxidation, with the rest completed by anammox bacteria. Collectively, these findings improved our understanding on global nitrogen cycles, and provided a new idea for the removal of contaminants in saline water treatment.

Metagenomic analyses reveal nitrogen metabolism responses to copper and chromium contamination in sludge-based microbial communities

In this study, we used metagenomic sequencing to explore the effects of chromium (Cr) and copper (Cu) on microbial communities and function by analyzing pathway of nitrogen metabolism based on KEGG annotation. High concentrations of Cr (VI) and Cu (II) reduced the removal efficiency of total nitrogen and ammonium in the system. Metagenomic analyses showed that the taxonomic composition of nitrogen metabolism-associated microorganisms (NMAM) was mainly composed of Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Sphingobacteria, Deltaproteobacteria, Flavobacteria and Nitrospira. Different concentrations of Cr (VI) and Cu (II) led to great changes in NMAM in activated sludge and enriched dominant microbes, respectively, suggesting specific response strategies in microbial communities. NMDS (Non-metric multidimensional scaling) analyses revealed the impact of different Cr (VI) and Cu (II) concentrations on NMAM β diversity, and indicated that Cr (VI) treatment induced greater variations in NMAM communities than Cu (II). We also identified a significant association (p < 0.05) between enzymes of nitrogen metabolism pathway and corresponding NMAM. This study revealed that under Cu (II) and Cr (VI) stress, the sludge nitrification pathway was inhibited, especially for ammonia monooxygenase and hydroxylamine dehydrogenase. These combined effects led to a decrease in sludge efficiency for nitrogen removal. These findings underscore the influence of heavy metals in water treatment.

The effect of beet juice-based foods on the performance of athletes in cyclic sports

BACKGROUND: Modern data from the scientific literature indicate that nitrate, when added to food, can serve as a precursor of nitric oxide (NO). According to research results, the concentrations of nitrates and nitrites in plasma increase when subjects are taking dietary supplements containing nitrates. Such an increase not only contributes to training performance by activating the nitrate pathway of energy supply to muscle activity but also generally improves physical performance.&#x0D; AIMS: To evaluate the effect of consumption of beet juice, as a source of nitrates, on the performance of biathletes-sprinters.&#x0D; MATERIALS AND METHODS: Forty male biathletes aged from 18 to 22 years took part in the study. The study took place in a training camp in the summer of 2017. The athletes were divided into an experimental group and a control group. The experimental group was given a preparation containing beet juice of a special variety twice daily after each training session, and the athletes of the control group did not consume anything after training sessions. Before the experiment and after 20 days of intervention, load testing before work refusal was carried out, as well as biochemical and general blood analysis.&#x0D; RESULTS: In the athletes of the experimental group, who were consuming beet juice, there was a decrease in oxygen uptake per kilogram of body weight and an increase in heart rate at the moment of anaerobic threshold, as well as an increase in load time, which indicates an increase in endurance during anaerobic exercise. This parameter is one of the limiting factors of performance in cyclic sports. In addition, increases in blood iron content, transferrin saturation, hemoglobin concentration, and average erythrocyte volume were observed in participants who consumed the beet juice preparation.&#x0D; CONCLUSIONS: The observed changes in indicators of the oxygen-transport system of the blood may be one of the mechanisms through which exogenous nitrates realize their positive effect on performance in the anaerobic phase.

Greywater treatment in SBR-SND reactor - optimization of hydraulic retention time, volumetric exchange ratio and sludge retention time

ABSTRACT In this study, simultaneous nitrification and denitrification-sequencing batch reactor (SND-SBR) process was investigated to treat greywater. The effect of three process parameters, including hydraulic retention time (HRT), volumetric exchange ratio (VER) and sludge retention time (SRT), was optimised using a 23 full factorial design. The statistic model was developed for two response variables, i.e. chemical oxygen demand (COD) and ammonia (NH3-N) removal. The optimum conditions were 6.8 h HRT (anaerobic/aerobic/anoxic: 1.77 h/2.77 h/2.27 h), 0.7 VER and 7.94 d SRT, which resulted in 93.9% COD and 84.6% NH3-N removal efficiency. SRT was the most significant factor, followed by HRT and VER for COD and NH3-N removal. The interaction effect of VER and SRT was significant in COD removal. On the other hand, the interaction effects of HRT-VER and HRT-SRT were significant in NH3-N removal. The removal efficiencies of 89.6 ± 1.1% and 83.7 ± 2.3% were observed for TKN and TN, respectively, in the optimised SND-SBR system. NH3-N removal was obtained via nitrate pathway in the SND-SBR system. The PO4 3--P removal of 74.2 ± 3.4% was obtained via aerobic phosphorus uptake and post anoxic denitrification at the optimal condition. To enhance PO4 3--P removal, adsorption (using corn cob adsorbent) was integrated with SBR by adding the optimum adsorbent dose (0.5 g/L). The PO4 3--P removal efficiency in the SBR-adsorption system was found to be 80 ± 1.5%. The biodegradation of emerging contaminants (ECs) was also carried out in the SND-SBR system, and the results showed removal rate of 58.9 ± 2.3% benzophenone-3 (BP) and 80.1 ± 2.2% anionic surfactant (AS).

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide.

TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO2-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO2-Trp under single- or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (FeII) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet-visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity.

Understanding the effect of free ammonia on microbial nitrification mechanisms in suspended activated sludge bioreactors

During nitrification, the varieties of microbial structures, metabolic pathways and functional profiles in four parallel laboratory-scale sequencing batch reactors (SBRs) with 0.5, 5, 10 and 15 mg/L of free ammonia (FA) concentrations were analyzed by high-throughput sequencing of the 16S rRNA gene. The SBRs were named S0.5, S5, S10 and S15, respectively. Ammonia removal via the nitrate pathway was achieved in S0.5 and S5 throughout the whole experimental period, while ammonia removal via the nitrite pathway was established in S10 and S15 after 89 and 146 day, respectively. The key finding of this study is that both the microbial diversity and richness were significantly affected (p < 0.05) by the FA concentration at different taxonomic levels. The most dominant taxa of S5, S10 and S15 were same, and mainly included Thauera while S0.5 was mainly composed of Zoogloea. Linear discriminant analysis (LDA) effect size (LEfSe) analysis was used to identify unique biomarkers in SBR activated sludge (AS) sample. The functional genera and enzyme in the four SBRs are similar but different in abundance and they are responsible for the removal of organics and nitrogen. Moreover, metabolic pathways are similar by PICRUSt analysis. The relative proportions of pathway-specific genes involved in some metabolic pathways differed to some extent. The ammonia oxidation rate was positively linked to Nitrosomonas and amo (both Spearman correlation coefficients (ρ) = 0.777) while the nitrite oxidation rate was positively linked to Nitrospira (ρ = 0.777) by co-occurrence network analysis. This work deciphered the response of microbial characteristics to different FA constraints in AS process and could provide helpful information for revealing the biological mechanism of FA inhibition on nitrogen removal.