Hydroxylamine Reductase Research Articles

Cathodic poised potential stimulated the electron-sensitive C-P lyase pathway in glyphosate biodegradation

Glyphosate, the most widely used herbicide globally, is accumulating in the environment and poses significant potential eco- and bio-toxicity risks. While natural attenuation of glyphosate has been reported, the efficacy varies considerably and the dominant metabolite, aminomethylphosphonic acid (AMPA), is potentially more persistent and toxic. This study investigated the bioelectrochemical system (BES) for glyphosate degradation under anaerobic, reductive conditions. Atomistic simulations using density functional theory (DFT) predicted increased thermodynamic favorability for the non-dominant C-P lyase degradation pathway under external charge, which suppressed AMPA production. Experimental results confirmed that cathodic poised potential (-0.4 V vs. Ag/AgCl) enhanced glyphosate degradation (75 % in BES vs. ∼40 % in the control conditions after 37 days), and lowered the AMPA yield (0.52 mol AMPA yield per mol glyphosate removed in BES vs. 0.77–0.86 mol mol-1 in the control conditions). Geobacter lovleyi was likely the active species driving the C-P lyase pathway, as evidenced by the increase of its relative abundance, the upregulation of its extracellular electron transfer genes (most notably mtr) and the up-regulation of its phnJ and hcp genes (encoding C-P layse and hydroxylamine reductase respectively).

Regulatory roles of extracellular polymeric substances in uranium reduction via extracellular electron transfer by Desulfovibrio vulgaris UR1

The pathway of reducing U(VI) to insoluble U(IV) using electroactive bacteria has become an effective and promising approach to address uranium-contaminated water caused by human activities. However, knowledge regarding the roles of extracellular polymeric substances (EPS) in the uranium reduction process involving in extracellular electron transfer (EET) mechanisms is limited. Here, this study isolated a novel U(VI)-reducing strain, Desulfovibrio vulgaris UR1, with a high uranium removal capacity of 2.75 mM/(g dry cell). Based on a reliable EPS extraction method (45 °C heating), manipulation of EPS in D. vulgaris UR1 suspensions (removal or addition of EPS) highlighted its critical role in facilitating uranium reduction efficiency. On the second day, U(VI) removal rates varied significantly across systems with different EPS contents: 60.8% in the EPS-added system, 48.5% in the pristine system, and 22.2% in the EPS-removed system. Characterization of biogenic solids confirmed the reduction of U(VI) by D. vulgaris UR1, and the main products were uraninite and UO2 (2.88–4.32 nm in diameter). As EPS formed a permeable barrier, these nanoparticles were primarily immobilized within the EPS in EPS-retained/EPS-added cells, and within the periplasm in EPS-removed cells. Multiple electroactive substances, such as tyrosine/tryptophan aromatic compounds, flavins, and quinone-like substances, were identified in EPS, which might be the reason for enhancement of uranium reduction via providing more electron shuttles. Furthermore, proteomics revealed that a large number of proteins in EPS were enriched in the subcategories of catalytic activity and electron transfer activity. Among these, iron-sulfur proteins, such as hydroxylamine reductase (P31101), pyruvate: ferredoxin oxidoreductase (A0A0H3A501), and sulfite reductase (P45574), played the most critical role in regulating EET in D. vulgaris UR1. This work highlighted the importance of EPS in the uranium reduction by D. vulgaris UR1, indicating that EPS functioned as both a reducing agent and a permeation barrier for access to heavy metal uranium.

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.

Innovative fertilizer management system maintains higher maize productivity with lower environmental costs in the Loess Plateau region of China

ContextInnovative fertilizer management is the most effective strategy for increasing crop yields and reducing environmental pollution. ObjectivesDeep fertilization reduces NH3 emissions and increases crop yield, but its effects on N2O emissions are still unclear. Nitrification or urease inhibitors trigger cross-contamination of N2O and NH3. Little is known about whether innovative fertilizer management strategies can reduce nitrogen emissions while maintaining higher yields. MethodsWe conducted field experiments using maize in the semi-humid and drought-prone areas of the Loess Plateau in China from 2019 to 2021. To further verify the research results, experiments were conducted in semi-arid and drought-prone areas in 2022 and 2023 using surface fertilization (SF), surface fertilization with nitrification (SNI) or urease inhibitor (SUI), deep fertilization (DF), and deep fertilization with urease and nitrification inhibitor (DUN) as experimental treatments. ResultsThe trends were similar at the three experimental locations over five years, where the ability of DF to reduce emissions was equivalent to those of SNI for N2O and SUI for NH3. However, the increase in the crop productivity was significantly higher under DF than SUI and SNI. Compared with SF, DF significantly reduced the N2O and NH3 emissions by 39.85 % and 46.08 %, respectively, and significantly the increased maize yield, N uptake, and nitrogen use efficiency (NUE) by 16.86 %, 15.83 %, and 32.79 %. The innovative fertilizer management strategies combined the advantages of inhibitors and deep fertilization, and they obtained the highest maize yield and NUE, as well as the lowest N2O and NH3 emissions and gaseous nitrogen loss intensity (GNLI) among all treatments. Compared with SF, DUN reduced the urease, nitrate reductase, nitrite reductase, and hydroxylamine reductase activities by 22.03 %, 58.81 %, 52.72 %, and 50.27 %, respectively, the soil NO3–-N and NH4+-N contents by 82.94 % and 53.71 %, and N2O and NH3 emissions by 80.79 %, and 79.33 %. DUN increased the maize yield and NUE by 23.30 % and 46.91 %, respectively, and decreased the GNLI by 84.69 %. ConclusionsDUN reduced the concentration of substrates for producing gaseous nitrogen by regulating the activities of soil nitrogen circulating enzymes to significantly reduce the nitrogen loss, and it achieved the highest maize yield (11,818.10 kg ha–1) and NUE (46.62 %) with the lowest GNLI (0.71 g N kg–1 grain). Innovative fertilizer management strategies can help to balance economic and environmental benefits in the Loess Plateau region of China. ImplicationsOur findings provide a reference for obtaining higher maize productivity with low environmental pollution in the Loess Plateau region of China.

The Impact of an Alien Snail Pomacea canaliculata Invading Coastal Saline Soils on Soil Chemical and Biological Properties

Recent studies have indicated that the invasive apple snail (Pomacea canaliculata) exhibits tolerance to the salinity levels present in coastal agricultural soils, suggesting that apple snails could potentially invade salt-affected coastal agricultural areas. However, the effects of the alien snail Pomacea canaliculata invasion on coastal saline soils, such as in terms of soil properties, microbial diversity, and abundance, remain poorly understood. To fill this gap, we conducted experiments involving three salinity levels (0, 2‰, and 5‰, w/w), coupled with varying snail densities (0, 5, and 10 snails per box), applied to agricultural soil. We analyzed soil chemical properties, enzyme activities, and bacterial communities. The findings revealed that heightened soil salinity increased soil electrical conductivity (EC) (exceeding 1312.67 μS cm−1). Under saline conditions, snail treatments significantly increased the soil organic matter (SOM) content from 15.82 mg kg−1 to 18.69 mg kg−1, and concurrently diminished the dissolved organic carbon (DOC) from 47.45 mg kg−1 to 34.60 mg kg−1. Both snail and salinity treatments resulted in ammonia nitrogen (NH4+-N) accumulation, while nitrate nitrogen (NO3−-N) concentrations remained low in salt-affected soils. A notable positive correlation existed between the EC and the activities of hydroxylamine reductase (HR) and peroxidase (POD), where HR exhibited a positive correlation with NH4+-N, and POD displayed a negative correlation with NO3−-N. Salinity substantially decreased the diversity and altered the composition of soil bacterial community, with the phyla Bacteroidota, Proteobacteria, and Firmicutes adapting to salt-affected soil environment and proliferating. Structural equation modeling (SEM) analysis indicated that snails exerted a direct influence on soil-available nitrogen (including NO3−-N and NH4+-N), while salinity impacted available nitrogen by modulating soil enzyme activities and bacterial communities. Our findings provide insights into how soil responds to the concurrent impacts of snail invasion and soil salinization, establishing some references for future research.

Class III hybrid cluster protein homodimeric architecture shows evolutionary relationship with Ni, Fe-carbon monoxide dehydrogenases

Hybrid cluster proteins (HCPs) are Fe-S-O cluster-containing metalloenzymes in three distinct classes (class I and II: monomer, III: homodimer), all of which structurally related to homodimeric Ni, Fe-carbon monoxide dehydrogenases (CODHs). Here we show X-ray crystal structure of class III HCP from Methanothermobacter marburgensis (Mm HCP), demonstrating its homodimeric architecture structurally resembles those of CODHs. Also, despite the different architectures of class III and I/II HCPs, [4Fe-4S] and hybrid clusters are found in equivalent positions in all HCPs. Structural comparison of Mm HCP and CODHs unveils some distinct features such as the environments of their homodimeric interfaces and the active site metalloclusters. Furthermore, structural analysis of Mm HCP C67Y and characterization of several Mm HCP variants with a Cys67 mutation reveal the significance of Cys67 in protein structure, metallocluster binding and hydroxylamine reductase activity. Structure-based bioinformatics analysis of HCPs and CODHs provides insights into the structural evolution of the HCP/CODH superfamily.

Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation

In forest ecosystems, variations in aboveground litter input caused by global changes, substantially alter soil N cycling. In field-grown plants, few studies have directly measured root exudation rates and quantified their effects on N transformations under litter manipulation. We quantified soil N transformation rate responses to litter manipulation in a Pinus massoniana plantation, and unravelled the effect of root exudation on soil N transformations. We measured in situ P. massoniana root exudation rates as well as soil microbial biomass, soil C and N concentrations, the activities of four soil enzymes involved in soil N transformations, and net N mineralization and net nitrification rates after experimental litter removal and litter addition treatments. Litter removal and litter addition treatments had little impact on soil C and N concentrations, microbial biomass, soil enzyme (urease, hydroxylamine reductase, nitrate reductase, and nitrite reductase) activity, and net N mineralization rates. However, both litter removal and addition increased net N nitrification rates. Additionally, litter removal significantly decreased root C exudation rates (in April 2021 and annually), whereas litter addition had no significant effects on root C exudation rates across all seasons. Furthermore, root C exudation rates were positively associated with urease and nitrate reductase activities, but negatively associated with hydroxylamine reductase and nitrite reductase activities, as well as net N nitrification rate. Overall, we demonstrated that root exudates may be an important physiological adjustment by which trees respond to changes in litter input caused by global environmental changes, regulating underground N biochemical processes. Furthermore, we provide new evidence from root exudates for understanding the potential influence of litter inputs on soil N cycling. A strong correlation exists between root exudates and N transformation, shedding new light on the dynamics of rhizosphere nutrient cycling crucial for maintaining forest ecosystem stability and productivity under changing environmental conditions.

Identification and examination of nitrogen metabolic genes in Lelliottia amnigena PTJIIT1005 for their ability to perform nitrate remediation

Lelliottia amnigena PTJIIT1005 is a bacterium that utilizes nitrate as the sole nitrogen source and can remediate nitrate from media. The annotation was done related to nitrogen metabolic genes using the PATRIC, RAST tools, and PGAP from the genome sequence of this bacterium. Multiple sequence alignments and phylogenetic analysis of respiratory nitrate reductase, assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, hydroxylamine reductase, nitric oxide reductase genes from PTJIIT1005 were done to find out sequence identities with the most similar species. The identification of operon arrangement in bacteria was also identified. The PATRIC KEGG feature mapped the N-metabolic pathway to identify the chemical process, and the 3D structure of representative enzymes was also elucidated. The putative protein 3D structure was analyzed using I-TASSER software. It gave good quality protein models of all nitrogen metabolism genes and showed good sequence identity with reference templates, approximately 81–99%, except for two genes; assimilatory nitrate reductase and nitrite reductase. This study suggested that PTJIIT1005 can remove N-nitrate from water because of having N-assimilation and denitrification genes.

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.

Nitrogen occurrence forms and bacterial community in sediment influenced by Bellamya purificata bioturbation

Endogenous water pollution and excessive nitrogen in ponds have always been prominent problems threatening aquaculture. To solve this problem, a 70-day indoor simulation experiment was conducted. The snail Bellamya purificata was stocked at four different densities: 0, 15, 30, and 60 individuals/tank, represented as CON (control), LD (low density), MD (medium density), and HD (high density), respectively, to investigate the effects of B. purificata on the nitrogen occurrence forms and bacterial communities in the sediment. At the end of the experiment, the nitrate concentration was significantly higher, while the total nitrogen content was significantly lower in the MD group than in the other three groups. Ammonia monooxygenase activity was significantly lower in the CON group than in the other three groups, and hydroxylamine oxidase activity was significantly higher in the HD group than in the other three groups. The CON and MD groups showed the highest and lowest values in nitrate reductase activity, respectively. The hydroxylamine reductase activity decreased significantly with increasing density. Through 16S ribosomal RNA (rRNA) high-throughput sequencing, significantly affected bacterial communities by B. purificata were found. Alpha diversity results showed that, a significantly lower Shannon index was observed in the MD group than in the other three groups. The LD and MD groups showed the highest and lowest Chao1 index values, respectively. Phyla Nitrospinae and family Nitrosomonadaceae were significantly enriched in the HD and MD groups, respectively. Redundancy analysis (RDA) indicated a significant correlation between differential bacterial taxa and TN content. Predicted functional analysis based on FAPROTAX (Functional annotation of prokaryotic taxa) database showed that functional groups aerobic ammonia oxidation and aerobic nitrite oxidation were significantly enriched in the MD group. Overall, B. purificata significantly altered the bacterial community composition, increased hydroxylamine oxidase and ammonia monooxygenase activities, enhanced the bacterial nitrification process, and promoted the transformation of total nitrogen to nitrate. Moreover, B. purificata stocked at 30 and 60 individuals/tank appeared to have a significant promotion effect on the bacterial community and nitrogen occurrence forms in the sediment. Hence, co-culturing B. purificata may be a feasible and effective ecological restoration method to alleviate excess nitrogen and reduce water eutrophication.

Impacts of Biochar-Based Controlled-Release Nitrogen Fertilizers on Soil Prokaryotic and Fungal Communities

Controlled-release Nitrogen Fertilizers (CRNFs) are an effective fertilization technique by minimizing nutrient loss and making Nitrogen (N) available to plants as they grow. Biochar-based CRNF (BCRNF) technologies have been demonstrated very promising in increase of corn yield. Despite the beneficial effects of BCRNFs, their impacts on prokaryotic and fungal soil communities are not well evaluated. Different formulations of BCRNF were developed to investigate their effects on corn productivity. We analyzed the soil microbes and their functional potential under different BCRNF regimes using amplified V3–V4 region of 16s rRNA for determining prokaryotic, and ITS genes for fungal communities. The soil prokaryotic diversity was similar across the treatments, with differences in prokaryotic genera with relative abundance of 0.1% or less in the soil (p < 0.05). In contrast, the fungal community diversity was different only for unfertilized soil. It had a high relative abundance for Aspergillus. Genus level comparison showed that Pseudofabraea was higher in Bioasphalt-based BCRNF compared to other treatments. Moreover, the N-fixing communities in soil were also similar across the treatments. At genus level, Microvirga, Azospirillum, and Methyloprofundus were highest in no-fertilizer control. The functional potential predictions using PICRUSt2 portrayed a consistent N-cycling functions across the treatments. However, the predicted gene functions related to nitrous-oxide reductase (nosZ) and hydroxylamine reductase (hcp) were significantly lower in soil receiving BCRNF containing biosolid. Overall, BCRNF treatments previously identified to increase corn yield displayed minimal shifts in the soil microbial communities. Thus, such novel fertilization would enable increased crop yield without affecting soil communities leading to sustainable crop production.

Successive corn stover and biochar applications mitigate N2O emissions by altering soil physicochemical properties and N-cycling-related enzyme activities: A five-year field study in Northeast China

As the most important source of commodity grain in China, Northeast China plays a crucial role in guaranteeing national food security. The application of biochar in agricultural ecosystems has shown immense potential in ensuring crop yields while mitigating N2O emissions. However, the long-term impacts and mechanisms of successive application of corn stover and its derived biochar on soil N2O emission in the corn monocropping region, such as Northeast China, remain unclear. A five-year field experiment was conducted in Northeast China to fill this research gap regarding the soil nitrogen (N) cycle. Three experimental treatments were established: a control with no corn stover or biochar application (CK), corn stover returned yearly at a rate of 7.5 t ha−1 (ST), and biochar added yearly at a rate of 2.6 t ha−1 (BC) corresponding to 35% of the stover addition rate in ST. Identical mineral fertilizers were applied in all three treatments. The total accumulated N2O emissions across five analyzed growing seasons within the CK, ST, and BC treatments were 260.2, 184.0, and 151.0 mg N2O m−2, respectively, with significant 29.3% and 42.0% decreases observed in ST and BC treatments, respectively, compared with the control. Based on the annual average from 2013 to 2017, ST and BC treatments significantly increased soil pH and total N (TN) content and significantly decreased the levels of NO3––N, NH4+-N, and soil bulk density (BD). Additionally, BC treatment remarkably lowered the average NH4+-N content than that under ST treatment by 24.1%. Both ST and BC showed a significant inhibitory effect on nitrate reductase (NR), nitrite reductase (NiR), and hydroxylamine reductase (HyR) activities related to denitrification. Soil N2O emission was significantly positively correlated with BD, NO3–-N, NH4+-N, NR, NiR, and HyR activities. Path analysis suggested that N2O emissions were primarily driven by NR and HyR activities and NO3–-N content. Overall, the applications of corn stover and its derived biochar are a feasible means of mitigating soil N2O emissions in the context of continuous corn cropping systems in mid-latitude regions like Northeast China, with biochar being more beneficial than stover application.

Impact of Modified Atmospheres on Growth and Metabolism of Meat-Spoilage Relevant Photobacterium spp. as Predicted by Comparative Proteomics.

Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O2, 30% CO2, red and white meats) and oxygen-free MAP (70% N2, 30% CO2, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of Photobacterium carnosum and P. phosphoreum was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for P. carnosum. Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose (P. phosphoreum) or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro, and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

Different Forms and Proportions of Exogenous Nitrogen Promote the Growth of Alfalfa by Increasing Soil Enzyme Activity

Nitrogen fertilization is a simple and effective field management strategy for increasing plant productivity, but the regulatory mechanisms of nitrogen forms and proportions on soil nutrients and plant growth remain unclear. Therefore, we investigated soil enzyme activities and nutrient contents of alfalfa under different forms and proportions of exogenous nitrogen addition. Results showed that nitrogen input significantly increased the activity of three oxidoreductases (hydroxylamine reductase, nitrate reductase, and nitrite reductase) while having no significant effects on urease. A high proportion of ammonium nitrogen significantly increased neutral protease activity. The amylase activity markedly increased under mixed-nitrogen addition but decreased under single-nitrogen addition. Additionally, the contents of soil nutrients (soil organic matter, total nitrogen, nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium) were significantly increased under different exogenous nitrogen inputs, which drove the changes in enzyme activities. Further, nitrogen addition also improved the biomass and nitrogen content of alfalfa. These findings indicated that applying different forms and proportions of exogenous nitrogen may stimulate soil enzyme activities, which will accelerate the transformation of nutrients and then promote alfalfa growth.

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.

Enhanced nitrogen removal from low-temperature wastewater by an iterative screening of cold-tolerant denitrifying bacteria.

The biological process to remove nitrogen in winter effluent is often seriously compromised due to the effect of low temperatures (< 13°C) on the metabolic activity of microorganisms. In this study, a novel heterotrophic nitrifying-aerobic denitrifying bacterium with cold tolerance was isolated by iterative domestication and named Moraxella sp. LT-01. The LT-01 maintained almost 60% of its maximal growth activity at 10°C. Under initial concentrations of 100mg/L, the removal efficiencies of ammonium, nitrate, nitrite by LT-01 were 70.3%, 65.4%, 61.7% respectively for 72h incubation at 10°C. Nitrogen balance analysis showed that about 46% of TN was released as gases and 16% of TN was assimilated for cell growth. The biomarker genes involved in nitrification and denitrification pathways were identified by gene-specific PCR and revealed that the LT-01 has nitrite reductase (NirS) but not hydroxylamine reductase (HAO), which implies the involvement of other genes in the process. The study indicates that LT-01 has the potential for use in low-temperature regions for efficient sewage treatment.

Impacts of silver nanoparticles on enzymatic activities, nitrifying bacteria, and nitrogen transformation in soil amended with ammonium and nitrate

Silver nanoparticles (AgNPs) are effective antimicrobial compounds that are used in a myriad of applications. Soil microorganisms play crucial roles in nitrogen cycling, but there is a lack of comprehensive understanding of the effects of AgNPs on enzymatic activity in the nitrogen cycle, nitrifying bacteria, and nitrogen transformation in soil. Herein, enzyme activities were determined following the addition of different forms of nitrogen, ammonium nitrogen ((NH4)2SO4), nitrate nitrogen (KNO3), and amide nitrogen (urea, CO(NH2)2) at 200 mg N kg–1, into the soil amended with AgNPs at 0, 10, 50, and 100 mg kg–1. After 7 d of incubation with 10 mg kg–1 AgNPs, the activities of urease, nitrite reductase (NiR), nitrate reductase (NaR), and hydroxylamine reductase (HyR) were reduced by 12.5%, 25.0%, 12.2%, and 24.2%, respectively. Of particular note, more than 53.5%, 61.7%, and 34.7% of NaR, NiR, and HyR activities, respectively, were inhibited at 100 mg kg–1 AgNPs. The abundance (most probable number) of ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively) was measured using real-time quantitative polymerase chain reaction (qPCR) and the Cochran method. The abundance of AOB and NOB decreased when AgNPs were present in the soil. The NH4NO3 amendment increased copy numbers of bacterial and archaeal amoA nitrification functional genes, by 38.3% and 12.4%, respectively, but AgNPs at 50 mg kg–1 decreased these values by 70% and 56.4%, respectively. The results of 15N enrichment (atom% excess) of NH4+ and NO3– experiments illustrated the influence of AgNPs on soil nitrogen transformation. According to the 15N atom% excess detected, the conversion of 15N-labeled NH4+ to NO3– was significantly inhibited by the different levels of AgNPs in soil. The reduced gross nitrification rate further confirmed this finding. Overall, this study revealed considerable evidence that AgNPs inhibited nitrogen cycle enzyme activity, the number of nitrifying bacteria, the abundance of the amoA gene, and the gross nitrification rate. Silver nanoparticles inhibited nitrogen transformation, and the rate of nitrification was also negatively correlated with AgNP levels.

A Mechanistic Investigation of Cytochrome c Nitrite Reductase Catalyzed Reduction of Nitrite to Ammonia: The Search for Catalytic Intermediates

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were done with the wild type enzyme (wtccNiR) as well as with R103Q, Y206F, and H257Q variants. A UV/Vis stopped-flow investigation of the reaction between nitrite-loaded wtccNiR and TMPD revealed for the first time that the reaction proceeds via a transient 1-electron reduced intermediate. Generation of this species is pH-independent, whereas its decay to a previously characterized 2-electron reduced intermediate is fastest at pH 6.8 and significantly slower at higher pH. The pH dependence is ascribed to the rate-limiting cleavage of the nitrite N – O bond, which requires a prior di-protonation predicted to be slow in earlier computational studies. Under steady-state conditions, S. oneidensis ccNiR catalyzed the slow 1-electron reduction of nitrite to nitric oxide, which was monitored by tracking the concomitant appearance of the colored TMPD+ radical product. The rate of TMPD+ formation was found to be directly proportional to the concentration of TMPD+ at low TMPD concentrations, providing important insights about the mechanism of NO˙ release. The steady-state studies also showed that nitrite is a substrate inhibitor of NO˙ release when TMPD is the electron source, probably because it blocks exit by NO˙ through the nitrite entry channel. The H257Q variant of ccNiR was found to have 1/400th of the wild type enzyme's nitrite reductase activity in the standard assay in which methyl viologen monocation radical is the electron source, but nearly normal hydroxylamine reductase activity. This demonstrated that H257 is essential for nitrite reduction but not for reduction of hydroxylamine, a putative intermediate in the catalytic process. UV/Vis spectropotentiometry showed that the nitrite-loaded active site of H257Q still reduced at fairly high applied potential, but the reduction was by one electron, whereas the wild type is reduced in a concerted 2-electron step. The experiments with the variant, coupled with the pH-dependent stopped flow experiments with wtccNiR, confirm H257's importance in facilitating nitrite reduction, but suggest that its role is to modulate the pKa values of one or more of the waters that form a complex hydrogen bonding network in the active site. This view is more conservative than earlier theoretical predictions of direct histidine involvement in nitrite diprotonation leading to N – O bond cleavage.

Nitrogen process in stormwater bioretention: the impact of alternate drying and rewetting on nitrogen migration and transformation.

Nitrogen migration and transformation in the stormwater bioretention system were studied in laboratory experiments, in which the effects of drying-rewetting were particularly investigated. The occurrence and distribution of nitrogen in the plants, the soil, and the pore water were explored under different drying-rewetting cycles. The results clearly showed that bioretention system could remove nitrogen efficiently in all drying-rewetting cycles. The incoming nitrogen could be retained in the topsoil (0-10 cm) and accumulated in the planted layer. However, the overlong dry periods (12 and 22 days) cause an increase in nitrate in the pore water. In addition, nitrogen is mostly stored in the plants' stem tissues. Up to 23.26% of the inflowing nitrogen can be immobilized in plant tissues after a dry period of 22 days. In addition, the relationships between nitrogen reductase activity in the soil and soil nitrogen content were explored. The increase of soil TN content could enhance the activity of nitrate reductase. Meanwhile, the activity of hydroxylamine reductase (HyR) could be enhanced with the increase of soil NO3- content. These results provide a reference for the future development of nitrogen transformation mechanism and the construction of stormwater bioretention systems.

Influence of Antecedent Dry Days on Nitrogen Removal in Bioretention Systems

Bioretention systems have become an optimal technology during the construction of the sponge city, but its nitrogen removal performance can be affected by antecedent dry days (ADD). This study was designed to investigate the effects of different lengths of ADD (1,2,3, 5, 7, 12, and 22 d) on nitrogen removal performance using a series of laboratory-scale bioretention systems to form seven constant alternate drying-rewetting regimes. The influence mechanism was further investigated by analyzing the spatial distribution of nitrogen reductase activity and microbial community structure under different drying-rewetting regimes. The results showed that the ammonium removal efficiency was not significantly affected by ADD, while exhibiting high variation depending on the hydraulic permeability of the filler and plant growth conditions. The nitrate and total nitrogen removal efficiency decreased as the length of ADD increased form 7 d to 22 d. In addition, the spatial distribution of nitrate reductase (NaR), nitrite reductase (NiR), and hydroxylamine reductase (HyR) were affected by ADD to some extent. It was found that the soil moisture of submerged layer (SL) regulated the nitrogen processes. The nitrate dissimilatory reduction to ammonium (DNRA) can occur in the SL through secondary catalytic reduction by nitrogen reductases, thus affecting the removal of ammonium. The soil microbial community structure and its spatial distribution could be altered by ADD significantly, and the removal of multiple nitrogen species was partly affected. Thereinto, under shorter ADD values of 1, 2, 3, and 5 d, the dominant phylum was Firmicutes, a group of denitrifying microbes, and its dominant genus, Clostridium_sensu_stricto_1, also had the function of DNRA. The results of the study confirmed that ADD has a certain effect on the nitrogen removal capacity and nitrogen reductase activity, while resulting in spatial changes in the microbial community structure in the bioretention system under constant drying-rewetting conditions.