Flux balance analysis of the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC19718 unravels specific metabolic activities while degrading toxic compounds.

The ammonia-oxidizing bacterium Nitrosomonas europaea has been widely recognized as an important player in the nitrogen cycle as well as one of the most abundant members in microbial communities for the treatment of industrial or sewage wastewater. Its natural metabolic versatility and extraordinary ability to degrade environmental pollutants (e.g., aromatic hydrocarbons such as benzene and toluene) enable it to thrive under various harsh environmental conditions. Constraint-based metabolic models constructed from genome sequences enable quantitative insight into the central and specialized metabolism within a target organism. These genome-scale models have been utilized to understand, optimize, and design new strategies for improved bioprocesses. Reduced modeling approaches have been used to elucidate Nitrosomonas europaea metabolism at a pathway level. However, genome-scale knowledge about the simultaneous oxidation of ammonia and pollutant metabolism of N. europaea remains limited. Here, we describe the reconstruction, manual curation, and validation of the genome-scale metabolic model for N. europaea, iGC535. This reconstruction is the most accurate metabolic model for a nitrifying organism to date, reaching an average prediction accuracy of over 90% under several growth conditions. The manually curated model can predict phenotypes under chemolithotrophic and chemolithoorganotrophic conditions while oxidating methane and wastewater pollutants. Calculated flux distributions under different trophic conditions show that several key pathways are affected by the type of carbon source available, including central carbon metabolism and energy production.

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase.

Ammonia (NH3)-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N2O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH3 oxidation to nitrite (NO2-) via a single obligate intermediate, hydroxylamine (NH2OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH2OH to NO2- We provide evidence that HAO oxidizes NH2OH by only three electrons to NO under both anaerobic and aerobic conditions. NO2- observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O2 under aerobic conditions. Our present study implies that aerobic NH3 oxidation by AOB occurs via two obligate intermediates, NH2OH and NO, necessitating a mediator of the third enzymatic step.

Kinetics of NH3 -oxidation, NO-turnover, N2 O-production and electron flow during oxygen depletion in model bacterial and archaeal ammonia oxidisers.

Ammonia oxidising bacteria (AOB) are thought to emit more nitrous oxide (N2 O) than ammonia oxidising archaea (AOA), due to their higher N2 O yield under oxic conditions and denitrification in response to oxygen (O2 ) limitation. We determined the kinetics of growth and turnover of nitric oxide (NO) and N2 O at low cell densities of Nitrosomonas europaea (AOB) and Nitrosopumilus maritimus (AOA) during gradual depletion of TAN (NH3 + NH4+) and O2 . Half-saturation constants for O2 and TAN were similar to those determined by others, except for the half-saturation constant for ammonium in N. maritimus (0.2 mM), which is orders of magnitudes higher than previously reported. For both strains, cell-specific rates of NO turnover and N2 O production reached maxima near O2 half-saturation constant concentration (2-10 µM O2 ) and decreased to zero in response to complete O2 -depletion. Modelling of the electron flow in N. europaea demonstrated low electron flow to denitrification (≤1.2% of the total electron flow), even at sub-micromolar O2 concentrations. The results corroborate current understanding of the role of NO in the metabolism of AOA and suggest that denitrification is inconsequential for the energy metabolism of AOB, but possibly important as a route for dissipation of electrons at high ammonium concentration.

Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4-dimethylpyrazole phosphate.

Soil ecosystem represents the largest contributor to global nitrous oxide (N2 O) production, which is regulated by a wide variety of microbial communities in multiple biological pathways. A mechanistic understanding of these N2 O production biological pathways in complex soil environment is essential for improving model performance and developing innovative mitigation strategies. Here, combined approaches of the 15 N-18 O labelling technique, transcriptome analysis, and Illumina MiSeq sequencing were used to identify the relative contributions of four N2 O pathways including nitrification, nitrifier-induced denitrification (nitrifier denitrification and nitrification-coupled denitrification) and heterotrophic denitrification in six soils (alkaline vs. acid soils). In alkaline soils, nitrification and nitrifier-induced denitrification were the dominant pathways of N2 O production, and application of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) significantly reduced the N2 O production from these pathways; this is probably due to the observed reduction in the expression of the amoA gene in ammonia-oxidizing bacteria (AOB) in the DMPP-amended treatments. In acid soils, however, heterotrophic denitrification was the main source for N2 O production, and was not impacted by the application of DMPP. Our results provide robust evidence that the nitrification inhibitor DMPP can inhibit the N2 O production from nitrifier-induced denitrification, a potential significant source of N2 O production in agricultural soils.

Regulation of membrane fixation and energy production/conversion for adaptation and recovery of ZnO nanoparticle impacted Nitrosomonas europaea.

The ZnO nanoparticle (NP) effects on typical ammonia-oxidizing bacteria, Nitrosomonas europaea in a chemostat bioreactor, and the cells' toxicity adaptation and recovery potentials were explored. Hardly any inhibition was observed when the NP concentration was high up to 10 mg/L. The cells exposed to 50 mg/L ZnO NPs displayed time-dependent impairment and recovery potentials in terms of cell density, membrane integrity, nitrite production rate, and ammonia monooxygenase activity. The 6-h NP stress impaired cells were nearly completely restored during a 12-h recovery incubation, while the longer exposure time would cause irretrievable cell damage. Microarray analysis further indicated the transcriptional adaptation of N. europaea to NP stress. The regulations of genes encoding for membrane permeability or osmoprotectant, membrane integrity preservation, and inorganic ion transport during NP exposure and cell recovery revealed the importance of membrane fixation and the associated metabolisms for cells' self-protection and the following recovery from NP stress. The oxidative phosphorylation, carbon assimilation, and tricarboxylic acid (TCA) cycling pathways involved in the cells' antitoxicity activities and would promote the energy production/conversion efficiency for cell recovery. The heavy metal resistance, histidine metabolism, toxin-antitoxin defense, glycolysis, and sulfate reduction pathways were also suggested to participate in the cell detoxication and recovery processes. All these findings provided valuable insights into the mechanisms of cell-mediated ZnO NP cytotoxicity and their potential impacts on wastewater nitrogen removal system.

Effects of the chemical characteristics and concentration of inorganic suspended solids on nitrification in freshwater.

The effect of inorganic suspended solids (ISS) on nitrification in freshwater samples has been described inconsistently and remains unclear. This study therefore investigated the effects of the chemical characteristics and concentration of ISS on the nitrification rate by focusing on Nitrosomonas europaea and Nitrobacter winogradskyi as the two most dominant nitrification species in freshwater. Batch-wise experiments were conducted using three chemically well-characterized ISS (i.e. the clay minerals montmorillonite, sericite, and kaolinite in the concentration range 0-1,000 mg L-1). The results show that the ammonium oxidation rate constant (kNH4) was significantly affected by the ISS type, whereas changes in the ISS concentration had an insignificant effect on kNH4, except for kaolinite. The highest kNH4 was observed in samples containing sericite (kNH4, 0.067 L mg-1 day-1), followed by samples containing montmorillonite (kNH4, 0.044 L mg-1 day-1). The ammonium oxidation rate was low in the control and kaolinite samples. Nitrite oxidation was enhanced in the presence of all types of ISS. The rate constants of ISS-mediated nitrite oxidation (kNO2, 0.13-0.21 L mg-1 day-1) were not significantly different among the three types of ISS, but kNO2 was significantly affected by ISS concentration. Overall, our study indicated various effects of the ISS type and concentration on nitrification and, in particular, a notable positive effect of sericite.

Functionally Distinct Bacterial Cytochrome c Peroxidases Proceed through a Common (Electro)catalytic Intermediate.

The diheme cytochrome c peroxidase from Shewanella oneidensis (So CcP) requires a single electron reduction to convert the oxidized, as-isolated enzyme to an active conformation. We employ protein film voltammetry to investigate the mechanism of hydrogen peroxide turnover by So CcP. When the enzyme is poised in the active state by incubation with sodium l-ascorbate, the graphite electrode specifically captures a highly active state that turns over peroxide in a high potential regime. This is the first example of an on-pathway catalytic intermediate observed for a bacterial diheme cytochrome c peroxidase that requires reductive activation, consistent with the observed voltammetric response from the diheme cytochrome c peroxidase from Nitrosomonas europaea (Ne), which is constitutively active and does not require the same one electron activation. Mutational analysis at the active site of So CcP confirms that the rate-limiting step involves a proton-coupled single electron reduction of a high valent iron species centered on the low-potential heme, consistent with the same mutation in Ne CcP. The pH dependence of catalysis for wild-type So CcP suggests that reduction shifts the pK(a)'s of at least two amino acids. Mutation of His81 in "loop 1", a surface exposed loop thought to shift conformation during the reductive activation process, eliminated one of the pH dependent features, confirming that the loop 1 shifts, changing the environment of His81 during the rate-limiting step. The observed catalytic intermediate has the same electron stoichiometry and similar pH dependence to that previously reported for Ne CcP, which is constitutively active and therefore hypothesized to follow a different catalytic mechanism. The prominent similarities between the rate-limiting steps of differing mechanistic classes of bCcPs suggest unexpected similarities in the intermediates formed.

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea.

UNLABELLED: Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production. IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.

Monochloramine Cometabolism by Mixed-Culture Nitrifiers under Drinking Water Conditions.

Chloramines are the second most used secondary disinfectant by United States water utilities. However, chloramination may promote nitrifying bacteria. Recently, monochloramine cometabolism by the pure culture ammonia-oxidizing bacteria, Nitrosomonas europaea, was shown to increase monochloramine demand. The current research investigated monochloramine cometabolism by nitrifying mixed cultures grown under more relevant drinking water conditions and harvested from sand-packed reactors before conducting suspended growth batch kinetic experiments. Four types of batch kinetic experiments were conducted: (1) positive controls to estimate ammonia kinetic parameters, (2) negative controls to account for biomass reactivity, (3) utilization associated product (UAP) controls to account for UAP reactivity, and (4) cometabolism experiments to estimate cometabolism kinetic parameters. Kinetic parameters were estimated in AQUASIM with a simultaneous fit to the experimental data. Cometabolism kinetics were best described by a first-order model. Monochloramine cometabolism kinetics were similar to those of ammonia metabolism, and monochloramine cometabolism accounted for 30% of the observed monochloramine loss. These results demonstrated that monochloramine cometabolism occurred in mixed cultures similar to those found in drinking water distribution systems; therefore, monochloramine cometabolism may be a significant contribution to monochloramine loss during nitrification episodes in drinking water distribution systems.

Global metabolomic responses of Nitrosomonas europaea 19718 to cold stress and altered ammonia feeding patterns.

The model ammonia-oxidizing bacterium Nitrosomonas europaea represents one of the environmentally and biotechnologically significant microorganisms. Genome-based studies over the last decade have led to many intriguing discoveries about its cellular biochemistry and physiology. However, knowledge regarding the regulation of overall metabolic routes in response to various environmental stresses is limited due to a lack of comprehensive, time-resolved metabolomic analyses. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling was performed to characterize the temporal variations of N. europaea 19718 intercellular metabolites in response to varied temperature (23 and 10 °C) and ammonia feeding patterns (shock loading and continuous feeding of 20 mg N/L). Approximately 87 metabolites were successfully identified and mapped to the existing pathways of N. europaea 19718, allowing interpretation of the influence of temperature and feeding pattern on metabolite levels. In general, varied temperature had a more profound influence on the overall metabolism than varied feeding patterns. Total extracellular metabolite concentrations (relative to internal standards and normalized to biomass weight) were lower under cold stress and shock loading conditions compared with the control (continuous feeding at 23 °C). Cold stress caused the widespread downregulation of metabolites involved in central carbon metabolism, amino acid, and lipid synthesis (e.g., malonic acid, succinic acid, putrescine, and phosphonolpyruvate). Metabolites that showed differences under varied feeding patterns were mainly involved in nucleotide acid, amino acid, and lipid metabolism (e.g., adenine, uracil, and spermidine). This study highlighted the roles of central carbon and nitrogen metabolism in countering cold stress and altered ammonia availability. In addition, transcriptomic, proteomic, and metabolomic data from three studies on N. europaea were compared to achieve a holistic view of some important synergy and interconnectivity among different cellular components (RNA, protein, and metabolites) during ammonia starvation.

Optimization of fertilization characteristics of urine by addition of Nitrosomonas europaea bio-seed.

BACKGROUND: Because of the high concentration of nutrients in human urine, its utilization as an organic fertilizer has been notable throughout history. However, the nitrogen compounds in urine are not stable. Therefore, to convert urine into a suitable fertilizer, it is important to stabilize and adjust unstable nitrogen compounds such as ammonia. Because nitrification can influence the nitrogen profile, the use of nitrifying microorganisms can be useful for stabilizing the nitrogen profile of urine. This study investigated the changes in nitrogen compounds in pure urine and examined the effect of adding Nitrosomonas europaea bio-seed solution on these changes. RESULTS: It was found that the addition of bio-seed could reduce nitrogen loss as well as the time required to stabilize the nitrogen profile. Furthermore, the optimum concentration of bio-seed (6 × 10(5) N. europaea cells L(-1) ) that not only leads to the least nutrient loss but also results in an adequate nitrate/ammonium ratio and regulates the amount of nitrate produced, thereby preventing over-fertilization, was determined. CONCLUSION: At this concentration, no dilution or dewatering is required, thus minimizing water and energy consumption. Usage of the optimum of concentration of bio-seed will also eliminate the need for inorganic chemical additives. © 2016 Society of Chemical Industry.

The Crystal Structure of Nitrosomonas europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into Sucrose Metabolism in Prokaryotes.

UNLABELLED: In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the nonphotosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure, from the plant Arabidopsis thaliana, was in a closed conformation. Comparative structural analysis revealed a "hinge-latch" combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family was shown to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, and Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded. IMPORTANCE: We obtained biochemical and structural evidence of sucrose metabolism in nonphotosynthetic bacteria. Until now, only sucrose synthases from photosynthetic organisms have been characterized. Here, we provide the crystal structure of the sucrose synthase from the chemolithoautotroph N. europaea. The structure supported that the enzyme functions with an open/close induced fit mechanism. The enzyme prefers as the substrate adenine-based nucleotides rather than uridine-based like the eukaryotic counterparts, implying a strong connection between sucrose and glycogen metabolism in these bacteria. Mutagenesis data showed that the catalytic mechanism must be conserved not only in sucrose synthases but also in all other retaining GT-B glycosyltransferases.

Kinetic parameter estimation in N. europaea biofilms using a 2-D reactive transport model.

Biofilms of the ammonia oxidizing bacterium Nitrosomonas europaea were cultivated to study microbial processes associated with ammonia oxidation in pure culture. We explored the hypothesis that the kinetic parameters of ammonia oxidation in N. europaea biofilms were in the range of those determined with batch suspended cells. Oxygen and pH microelectrodes were used to measure dissolved oxygen (DO) concentrations and pH above and inside biofilms and reactive transport modeling was performed to simulate the measured DO and pH profiles. A two dimensional (2-D) model was used to simulate advection parallel to the biofilm surface and diffusion through the overlying fluid while reaction and diffusion were simulated in the biofilm. Three experimental studies of microsensor measurements were performed with biofilms: i) NH3 concentrations near the Ksn value of 40 µM determined in suspended cell tests ii) Limited buffering capacity which resulted in a pH gradient within the biofilms and iii) NH3 concentrations well below the Ksn value. Very good fits to the DO concentration profiles both in the fluid above and in the biofilms were achieved using the 2-D model. The modeling study revealed that the half-saturation coefficient for NH3 in N. europaea biofilms was close to the value measured in suspended cells. However, the third study of biofilms with low availability of NH3 deviated from the model prediction. The model also predicted shifts in the DO profiles and the gradient in pH that resulted for the case of limited buffering capacity. The results illustrate the importance of incorporating both key transport and chemical processes in a biofilm reactive transport model.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Methionine ligand lability of homologous monoheme cytochromes c.

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

Characterizing the metabolic trade-off in Nitrosomonas europaea in response to changes in inorganic carbon supply.

The link between the nitrogen and one-carbon cycles forms the metabolic basis for energy and biomass synthesis in autotrophic nitrifying organisms, which in turn are crucial players in engineered nitrogen removal processes. To understand how autotrophic nitrifying organisms respond to inorganic carbon (IC) conditions that could be encountered in engineered partially nitrifying systems, we investigated the response of one of the most extensively studied model ammonia oxidizing bacteria, Nitrosomonas europaea (ATCC19718), to three IC availability conditions: excess gaseous and excess ionic IC supply (40× stoichiometric requirement), excess gaseous IC supply (4× stoichiometric requirement in gaseous form only), and limiting IC supply (0.25× stoichiometric requirement). We found that, when switching from excess gaseous and excess ionic IC supply to excess gaseous IC supply, N. europaea chemostat cultures demonstrated an acclimation period that was characterized by transient decreases in the ammonia removal efficiency and transient peaks in the specific oxygen uptake rate. Limiting IC supply led to permanent reactor failures (characterized by biomass washout and failure of ammonia removal) that were preceded by similar decreases in the ammonia removal efficiency and peaks in the specific oxygen uptake rate. Notably, both excess gaseous IC supply and limiting IC supply elicited a previously undocumented increase in nitric and nitrous oxide emissions. Further, gene expression patterns suggested that excess gaseous IC supply and limiting IC supply led to consistent up-regulation of ammonia respiration genes and carbon assimilation genes. Under these conditions, interrogation of the N. europaea proteome revealed increased levels of carbon fixation and transport proteins and decreased levels of ammonia oxidation proteins (active in energy synthesis pathways). Together, the results indicated that N. europaea mobilized enhanced IC scavenging pathways for biosynthesis and turned down respiratory pathways for energy synthesis, when challenged with excess gaseous IC supply and limiting IC supply.

Revision of N2O-producing pathways in the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC 19718.

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2 tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2 (-)) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2 tensions. Anoxic resting-cell assays and measurements of instantaneous NO2 (-) reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.

Modelling ammonium-oxidizing population shifts in a biofilm reactor.

The dynamic reactor behaviour of a nitrifying inverse turbulent bed reactor, operated at varying loading rate, was described with a one-dimensional two-step nitrification biofilm model. In contrast with conventional biofilm models, this model includes the competition between two genetically different populations of ammonia-oxidizing bacteria (AOB), besides nitrite-oxidizing bacteria (NOB). Previously gathered experimental evidence showed that different loading rates in the reactor resulted in a change in the composition of the AOB community, besides a different nitrifying performance. The dissolved oxygen concentration in the bulk liquid was put forward as the key variable governing the experimentally observed shift from Nitrosomonas europaea (AOB1) to Nitrosomonas sp. (AOB2), which was confirmed by the developed one-dimensional biofilm model. Both steady state and dynamic analysis showed that the influence of microbial growth and endogenous respiration parameters as well as external mass transfer limitation have a clear effect on the competition dynamics. Overall, it was shown that the biomass distribution profiles of the coexisting AOB reflected the ecological niches created by substrate gradients.

Disinfectant-induced ammonia oxidation disruption in microbial N-cycling process in aquatic ecosystem after the COVID-19 outbreak.

Anthropogenic activities significantly impact the elemental cycles in aquatic ecosystems, with the N-cycling playing a critical role in potential nutrient turnover and substance cycling. We hypothesized that measures to prevent COVID-19 transmission profoundly altered the nitrogen cycle in riverine ecosystems. To investigate this, we re-analyzed metagenomic data and identified 60 N-cycling genes and 21 host metagenomes from four urban reaches (one upstream city, Wuhan, and two downstream cities) along the Yangtze River. Our analyses revealed a marked decrease in the abundance of bacterial ammonia monooxygenase genes, as well as in the host, ammonia-oxidizing autotrophic Nitrosomonas, followed by a substantial recovery post-pandemic. We posited that discharge of sodium hypochlorite (NaOCl) disinfectant may be a primary factor in the reduction of N-cycling process. To test this hypothesis, we exposed pure cultures of Nitrosomonas europaea to NaOCl to explore the microbial stress response. Results indicated that NaOCl exposure rapidly compromised the cell structure and inhibited ammonia oxidation of N. europaea, likely due to oxidative stress damage and reduced expression of nitrogen metabolism-related ammonia monooxygenase. Using the functional tagging technique, we determined that NaOCl directly destroyed the ammonia monooxygenase protein and DNA structure. This study highlights the negative impacts of chlorine disinfectants on the function of aquatic ecosystems and elucidates potential mechanisms of action.

Identification of function and mechanistic insights of guanine deaminase from Nitrosomonas europaea: role of the C-terminal loop in catalysis.

NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10(5) M(-1) s(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 Å. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.