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The ammonia monooxygenase (AMO) enzyme catalyses the first step of ammonia oxidation into hydroxylamine, and copper (Cu) is the main co-factor in its functioning. We hypothesise that bioavailable Cu in soil is a limiting factor for nitrification. An increase in bioavailable Cu will increase nitrification, while a reduction in bioavailable Cu, induced through the application of Cu complexing compounds, will reduce nitrification. Hence, this study aimed to characterise the relationship between bioavailable Cu concentration and nitrification rate in dairy grazed pasture systems. An incubation study was undertaken using three contrasting soils (Pumice, Pallic and Recent soil) and high molecular weight organic acids (HMWOAs). The three soils were spiked with five levels of Cu (0.1, 0.3, 0.5, 1, and 3 mg kg−1) and incubated for 14 days before amendment with treatments. Four treatments were separately applied to each Cu level: urea only (300 mg N kg−1), dicyandiamide (DCD) (10 mg kg−1) + urea, calcium lignosulphonate (Ca-lig) (120 mg kg−1) + urea, and co-poly acrylic-maleic acid (PA-MA) (10 mg kg−1) + urea. After 4 and 8 days of incubation, the soil was extracted and analysed for bioavailable Cu and mineral N (nitrate, ammonia) to establish the effect of bioavailable Cu on nitrification rate. The dose response of nitrification rate to Cu was also calculated. The nitrification rates at day-8 significantly (P

Nitrification is a key step in the global nitrogen cycle. Compared with autotrophic nitrification, heterotrophic nitrification remains poorly understood. In this study, Halomonas venusta MA-ZP17-13, isolated from seawater in shrimp aquaculture (Penaeus vannamei), could simultaneously undertake nitrification and denitrification. With the initial ammonium concentration at 100 mg/L, the maximum ammonium-nitrogen removal rate reached 98.7% under the optimal conditions including C/N concentration ratio at 5.95, pH at 8.93, and NaCl at 2.33%. The corresponding average removal rate was 1.37 mg/(L·h) (according to nitrogen) in 3 d at 11.2°C. By whole genome sequencing and analysis, nitrification- and denitrification-related genes were identified, including ammonia monooxygenase, nitrate reductase, nitrite reductase, nitric oxide dioxygenase and nitric oxide synthase; while no gene encoding hydroxylamine oxidase was identified, it implied the existence of a novel nitrification pathway from hydroxylamine to nitrate. These results indicate heterotrophic bacterium H. venusta MA-ZP17-13 can undertake simultaneous nitrification and denitrification at low temperature and has potential for NH 4 + -N/NH3-N removal in marine aquaculture systems.

The type, functions, and mechanisms of biological nitrification inhibitors (BNIs) from rice were investigated using a combination of chemical and molecular techniques, bacterial bioassays, and soil microcosm experiments. We report the discovery of an effective nitrification inhibitor, syringic acid, in the root exudates of rice. Nitrification inhibition activity by syringic acid was verified in both weakly acidic and neutral pure cultures of Nitrosomonas europaea, and was superior to the widely used synthetic nitrification inhibitor, dicyandiamide (DCD). Moreover, syringic acid exhibited a dual inhibitory effect on ammonia monooxygenase (AMO), active in ammonium/ammonia oxidation, and on urease, active in urea hydrolysis. Nitrification inhibition by syringic acid was also demonstrated in a paddy soil system, and the abundance of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) was significantly inhibited under all syringic acid treatments. A synergistic effect of syringic acid and another rice BNI, 1,9-decanediol, on nitrification was found in two pure Nitrosomonas cultures and a paddy soil. Together, our results enhance our understanding of BNI production by rice and enable the design of natural inhibitor formulations that regulate soil N transformation in a concerted manner.

A pilot-scale biological pre-treatment reactor filled with porous polyurethanes carriers (BioNET) was operated over 500 days under different hydraulic retention times (HRT) (1.3 to 0.5 h) to evaluate the feasibility and efficiency of ammonia removal for a slightly contaminated source water. Under 0.5 h HRT, 84 % nitrification efficiency and 0.42 kg-N/m3/day ammonia removal rate could be achieved with influent ammonia concentration of 10.4 mg N/L. Results of batch tests indicated that the effect of aeration on nitrification of BioNET was more significant than temperature and components in raw water. Specific ammonia oxidization rate for batches with aeration were 4.5–9.1 times higher than those without aeration. In addition, simultaneous nitrification and denitrification were observed with batch tests using BioNET obtained from the reactor. Ammonia monooxygenase subunit A (amoA) gene of ammonia oxidizing archaea and bacteria (AOA and AOB) and 16S rRNA gene of total bacteria, Nitrospira, and Nitrobacter were quantified using real time quantification polymerase chain reaction (qPCR). AOB and Nitrospira were dominant in the bioreactor. Higher nitrification efficiency (>60 %) could be achieved at AOB abundance higher than 1.25 × 108 copy/BioNET and AOB/TB higher than 1.2. This study demonstrated that the BioNET system can be a promising technology for removing ammonia from slightly polluted river water at a low HRT condition.

Aquaponic systems are sustainable solutions for food production combining fish growth (aquaculture) and production of vegetables (hydroponic) in one recirculating system. In aquaponics, nitrogen-enriched wastewater from fish in the aquaculture serves as fertilizer for the plants in the hydroponics, while the nitrogen-depleted and detoxified water flows back to the aquaculture. To investigate bacterial nitrogen-cycling in such an aquaponic system, measurements of nitrogen species were coupled with time-resolved 16S rRNA gene profiling and the functional capacity of organisms was studied using metagenomics. The aquaponic system was consistently removing ammonia and nitrite below 23 µM and 19 µM, and nitrate to steady-state concentrations of about 0.5 mM. 16S rRNA gene amplicon sequencing of sediments exposed in the pump sump revealed that typical signatures of canonical ammonia-oxidising microorganisms were below detection limit. However, one of the most abundant operational taxonomic units (OTU) was classified as a member of the genus Nitrospira with a relative abundance of 3.8%. For this genus, also genome scaffolds were recovered encoding the only ammonia monooxygenase genes identified in the metagenome. This study indicates that even in highly efficient aquaponic systems, comammox Nitrospira were found to participate in ammonium removal at low steady-state ammonia concentrations.

Bacteria capable of complete ammonia oxidation (comammox) are widespread and contribute to nitrification in wastewater treatment facilities. However, their roles in partial nitrification-anaerobic ammonium oxidation (anammox) systems remain unclear. In this study, a bench-scale bioreactor with continuous stirring was operated for more than 1000 days with limited oxygen supply to achieve efficient nitrogen removal (70.1 ± 2.7%) at a low ammonium loading of 35.2 mg-N/L/day. High-throughput amplicon sequencing analysis of the comammox ammonia monooxygenase subunit A (amoA) gene revealed seven sequence types from two clusters in clade A of comammox Nitrospira. Quantitative polymerase chain reaction analyses suggested that the comammox species dominated the ammonia-oxidizing community, with an abundance as high as 89.2 ± 7.9% in total prokaryotic amoA copies. Multiple linear regression further revealed the substantial contribution of the comammox Nitrospira to ammonia oxidation in the bioreactor. The investigation with bioreactor and batch experiments consistently showed that activities of comammox Nitrospira were inhibited by free ammonia far more severely than other ammonia-oxidizing microbes. Overall, this study provided new insight into the ecology of comammox Nitrospira under hypoxic conditions and suggested comammox-associated partial nitrification-anammox as a potential method for treating low-strength ammonium-containing wastewater.

Agricultural production and associated applications of nitrogen (N) fertilizers have increased dramatically in the last century, and current projections to 2050 show that demands will continue to increase as the human population grows. Applied in both organic and inorganic fertilizer forms, N is an essential nutrient in crop productivity. Increased fertilizer applications, however, create the potential for more N loss before plant uptake. One strategy for minimizing N loss is the use of enhanced efficiency fertilizers, fortified with a nitrification inhibitor, such as nitrapyrin. In soils and water, nitrapyrin inhibits the activity of ammonia monooxygenase, a microbial enzyme that catalyzes the first step of nitrification from ammonium to nitrite. Potential benefits of using nitrification inhibitors range from reduced nitrate leaching and nitrous oxide emissions to increased crop yield. The extent of these benefits, however, depends on environmental conditions and management practices. Thus, such benefits are not always realized. Additionally, nitrapyrin has been shown to transport off-field, and it is unknown what effects environmental nitrapyrin could have on nontarget organisms and the ecological nitrogen cycle. Here, we review the agronomic and environmental benefits and costs of nitrapyrin use and present a series of research questions and considerations to be addressed with future nitrification inhibitor research.

Perfluorooctane sulfonic acid (PFOS) has been widely detected in wastewater treatment plants (WWTPs). Here, we investigated the nitrogen removal efficiency of activated sludge and related stress mechanisms in a sequencing batch reactor (SBR) after 8 h exposure to PFOS at 10, 100, and 1000 μg L−1. At 10 μg L−1, no significant effect (p > 0.05) was observed on total nitrogen (TN) removal. However, the TN removal efficiency significantly increased from 79.24% (control) to 81.99% and 86.21% at high concentrations (100 and 1000 μg L−1), respectively. In addition, the activities of nitrogen removal enzymes such as ammonia monooxygenase, nitrite oxidoreductase, nitrate reductase, and nitrite reductase were promoted by PFOS, which had variation trends similar to the TN removal rates. High throughput sequencing revealed that acute-term exposure to PFOS had little influence on the microbial richness (Chao) and diversity (Shannon) of activated sludge, but the relative abundance of most nitrogen-removing bacteria increased (Paracoccus, Thauera, and Comamonas increased from 0.43%, 1.78%, and 1.52% in the control to 0.60%, 2.13%, and 1.74% in SBR3, respectively), which could explain the increased nitrogen removal efficiency. This might be due to the protection of relevant bacteria by extracellular polymeric substances (EPSs), because the EPS evidently increased at 100 and 1000 μg L−1. However, an extremely significant (p < 0.01) increase of reactive oxygen species production and lactate dehydrogenase release at 100 and 1000 μg L−1 PFOS showed that PFOS caused oxidative damage and destroyed the integrity of microbial cell membranes. This indicated that PFOS has an acute bio-augmentation effect on the nitrogen removal process in WWTPs, but causes cytotoxicity after acute exposure.

This study investigated the effect of low and high chemical oxygen demand (COD):N ratios on biological nitrogen removal and microbial distributions in full-scale step-feed (SF) municipal wastewater treatment plants (WWTPs) in Thailand (SF1) and Taiwan (SF2). The SF1 WWTP had a low COD:N (4:1) ratio, a long solids retention time (SRT) (> 60 d), and low dissolved oxygen (DO) conditions (0.2 mg L− 1 in anoxic tank and 0.9 mg L− 1 in aerobic tank). The total nitrogen (TN) removal efficiency was 48%. The SF2 WWTP had a high COD:N (10:1) ratio, a short SRT (7 d), and high DO (0.6 mg L− 1 in anoxic tank and 1.8 mg L− 1 in aerobic tank). The TN removal efficiency was 61%. The nitrification and denitrification rates from these two plants were inadequate. Using a quantitative polymerase chain reaction (qPCR) technique, the populations of ammonium oxidizing bacteria (AOB) and ammonium oxidizing archaea were quantified. Measurement of ammonia monooxygenase (amoA) gene abundances identified these AOB: Nitrosomonas sp., Nitrosospira sp., Nitrosoccus sp. and Zoogloea sp. Higher amounts of the archaeal-amoA gene were found with long SRT, lower DO and COD:N ratios. Abundance of Nitrobacter sp. was slightly higher than Nitrospira sp. at the SF1, while abundance of Nitrobacter sp. was two orders of magnitude greater than Nitrospira sp. at the SF2. More denitrifying bacteria were of the nirS-type than the nirK-type, especially at higher COD:N ratio. Most bacteria belong to the phyla Acidobacteria, Actinobacteria Bacteroidetes, Chloroflexi, Proteobacteria. The results from this work showed that insufficient carbon sources at the SF1 and high DO concentration in anoxic tank of SF2 adversely affected nitrogen removal efficiencies. In further research work, advanced techniques on the next generation sequencing with different variable regions should be recommended in full-scale WWTPs.

Abstract Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract

Photoinhibition of ammonia oxidation occurs widely in aquatic environments and could suppress the nitrification rate, lead to the composition variation of inorganic nitrogen and influence the stability of aquatic ecosystems. Both ammonia-oxidizing bacteria (AOB) and archaea are sensitive to light. The extent of photoinhibition and the time required for recovery depend on light wavelength, intensity, photon quantity and strains. Strong evidence indicates that photoinhibition in AOB by visible light is mainly caused by irreversible damage to ammonia monooxygenase (AMO) and the degradation of AMO is beneficial to AOB recovery. This review discusses photoinhibition in metabolic pathways used by ammonia oxidizers.

The ammonia oxidizing bacterial consortium (AMOPCU-1) has been under use for efficient biological removal of ammonia from the recirculating aquaculture system. In this context a metaproteomic approach to the study of the consortium was undertaken to analyse the proteins involved in nitrification. Ammonia removal efficiency was monitored while amplifying the consortium in 2 L fermenter under standard conditions, and the consortium kept under substrate starvation served as the control. When the bacterial consortium attained peak ammonia removal efficiency, their total proteins were extracted. Protein profiling was done using one dimensional and two-dimensional SDS-PAGE. Eleven visible protein spots, which appeared during peak ammonia oxidation were identified by MALDI-TOF analysis. The proteins could be linked to metabolic processes that were active during nitrification. These included enzymes that influenced ammonia oxidation by regulating intracellular cytoplasmic pH of bacterial cells and transcription of ammonia monooxygenase, proteins having key roles in electron transport chain, ATP generation and fatty acid synthesis, and bacterial signal proteins that respond to environmental nitrite and nitrate and nitrous oxide reductase, a key enzyme in complete nitrate reduction. The present study is the first of its kind undertaken to investigate the metaproteome of ammonia oxidising consortium through which relevant proteins involved in efficient nitrification-denitrification could be revealed.

Ammonia oxidizing archaea (AOA) inhabiting soils have a central role in the global nitrogen cycle. Copper (Cu) is central to many enzymes in AOA including ammonia monooxygenase (AMO), the enzyme involved in the first step of ammonia oxidation. This study explored the physiological response of the AOA soil isolate, Nitrososphaera viennensis (EN76T) to Cu-limiting conditions in order to approach its limiting threshold under laboratory conditions. The chelator TETA (1,4,8,11-tetraazacyclotetradecane N, N′, N″, N‴-tetraacetic acid hydrochloride hydrate) with selective affinity for Cu2+ was used to lower bioavailable Cu2+ in culture experiments as predicted by thermodynamic speciation calculations. Results show that N. viennensis is Cu-limited at concentrations ≤10−15 mol L−1 free Cu2+ compared to standard conditions (10−12 mol L−1). This Cu2+ limiting threshold is similar to pure cultures of denitrifying bacteria and other AOA and AOB inhabiting soils, freshwaters and sewage (

The oxidation of ammonium to nitrate by biological process is known as nitrification. This process of nitrification is accomplished by Nitrosomonas spp. and Nitrobacter spp. Current intensive agricultural systems, characterized by heavy-nitrification systems, are prone to greater losses of nitrogen through nitrate leaching and emissions of greenhouse gases (GHGs) such as nitrous oxide, contributing to the increment of global temperature and ozone layer depletion. Furthermore, nitrate, under anoxic or partially anoxic conditions, escapes into the atmosphere as gaseous forms of nitrogen (N2 and N2O) through denitrification. Synthetic nitrification inhibitors (NIs) are used to regulate nitrification process in soil. The NIs are compounds that regulate this process by inhibiting ammonia monooxygenase (AMO) enzymes. Ability of certain plant species to exudate certain organic compounds from roots that have regulatory effect on performance of nitrifying bacteria has been known as biological nitrification inhibition (BNI). It inhibits both AMO and HAO (hydroxylamine oxidoreductase) enzymatic pathway in that process. The compounds are also stable in soil and production of BNI compounds are localized mainly to the rhizosphere and therefore, their modes of action may be more efficient. Brachiaria humidicola, a tropical pasture grass capable of growing in acidic soils and humid environments, is a well-known BNI-capable species that produces biochemical compound known as brachialactone. Cropping system adoption with current move toward ecologically intensified cropping with high nutrient-use efficiency is found to be novel strategy for controlling nitrification process.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that—like for AOB—ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

This article aims to illustrate and expand the information published in the article “Simultaneous nitrification and denitrification of hypersaline wastewater by a robust bacterium Halomonas salifodinae from a repeated-batch acclimation” [1] . The data present the salt tolerance of strain Y5 at different salinities (0%, 5%, 10%, 15%, and 20%). The effect of salinity on the morphology of bacteria was observed by scanning electron microscope. The influence of culture conditions including carbon source, C/N ratio, initial pH value, temperature, and shaking speed on bacterial growth and NH4+-N removal capability of strain Y5 was investigated by single factor experiments. The enzymatic activities of ammonia monooxygenase (AMO), hydroxylamine oxidase (HAO), nitrite reductase (NIR), and periplasm nitrate reductase (NAP) were measured by extracting the cell-free crude enzymes from strain Y5.

Inhibitors of urease and ammonia monooxygenase can limit the rate of conversion of urea to ammonia and ammonia to nitrate, respectively, potentially improving N fertilizer use efficiency and reducing gaseous losses. Winter wheat grown on a sandy soil in the UK was treated with urea fertilizer with the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT), the nitrification inhibitor dicyandiamide (DCD) or a combination of both. The effects on soil microbial community diversity, the abundance of genes involved in nitrification and crop yields and net N recovery were compared. The only significant effect on N-cycle genes was a transient reduction in bacterial ammonia monooxygenase abundance following DCD application. However, overall crop yields and net N recovery were significantly lower in the urea treatments compared with an equivalent application of ammonium nitrate fertilizer, and significantly less for urea with DCD than the other urea treatments. Electronic supplementary material The online version of this article (10.1007/s00374-019-01411-5) contains supplementary material, which is available to authorized users.

The abilities of three phylogenetically distant ammonia oxidizers, Nitrososphaera gargensis, an ammonia-oxidizing archaeon (AOA); Nitrosomomas nitrosa Nm90, an ammonia-oxidizing bacterium (AOB); and Nitrospira inopinata, the only complete ammonia oxidizer (comammox) available as a pure culture, to biotransform seven sulfonamides (SAs) were investigated. The removals and protein-normalized biotransformation rate constants indicated that the AOA strain N. gargensis exhibited the highest SA biotransformation rates, followed by N. inopinata and N. nitrosa Nm90. The transformation products (TPs) of sulfadiazine (SDZ), sulfamethazine (SMZ) and sulfamethoxazole (SMX) and the biotransformation mechanisms were evaluated. Based on the analysis of the TP formulas and approximate structures, it was found that during biotransformation, i) the AOA strain carried out SA deamination, hydroxylation, and nitration; ii) the AOB strain mainly performed SA deamination; and iii) the comammox isolate participated only in deamination reactions. It is proposed that deamination was catalyzed by deaminases while hydroxylation and nitration were mediated by nonspecific activities of the ammonia monooxygenase (AMO). Additionally, it was demonstrated that among the three ammonia oxidizers, only AOB contributed to the formation of pterin-SA conjugates. The biotransformation of SDZ, SMZ and SMX occurred only when ammonia oxidation was active, suggesting a cometabolic transformation mechanism. Interestingly, SAs could also be transformed by hydroxylamine, an intermediate of ammonia oxidation, suggesting that in addition to enzymatic conversions, a microbially induced abiotic mechanism contributes to SA transformation during ammonia oxidation. Overall, using experiments with pure cultures, this study provides important insights into the roles played by ammonia oxidizers in SA biotransformation.

The performance, nitrogen removal rate, microbial enzymatic activity and extracellular polymeric substances (EPS) of activated sludge were assessed under nickel (Ni(II)) stress. The organic matter and NH4+-N removal efficiencies were stable at less than 10 mg/L Ni(II) and subsequently decreased with the increment of Ni(II) concentration from 10 to 30 mg/L. The specific oxygen uptake rate and dehydrogenase activity kept stable at less than 5 mg/L Ni(II) and then declined at 5–30 mg/L Ni(II). Both specific ammonia-oxidizing rate (SAOR) and specific nitrite-oxidizing rate (SNOR) decreased with the increment of Ni(II) concentration. The changing trends of ammonia monooxygenase and nitrite oxidoreductase activities were matched those of SAOR and SNOR, respectively. The nitrite-reducing rate and nitrate-reducing rate illustrated a similar variation tendency to the nitrite reductase activity and nitrate reductase activity, respectively. Ni(II) impacted on the production, chemical composition and functional group of EPS. The relation between the sludge volume index and the EPS production exhibited a better linear function with a negative slope, demonstrating that Ni(II) improved the sludge settleability despite of the increase of EPS production.

Nitrogen cycling is dependent on ammonia-oxidizing bacteria and archaea transforming ammonia to nitrite. Copper is an essential cofactor for ammonia monooxygenase in ammonia-oxidizing archaea and bacteria. Copper is also essential for several electron carriers in ammonia-oxidizing archaea. Hence, copper acquisition is a critical step for complete enzyme maturation and metabolic function. In this work, we describe copper adsorption onto the bacterial species Nitrosococcus oceani C-107 (N. oceani) and Nitrosomonas europaea C-31 (N. europaea) and archaeal species Nitrosopumilus maritimus SCM1 (N. maritimus). Surface complexation models described copper adsorption onto bacterial species with one reaction, while N. maritimus required two reactions to adequately describe adsorption. Enthalpies of copper adsorption derived from isothermal titration calorimetry combined with surface complexation modeling revealed slightly exothermic enthalpies for N. maritimus and thermoneutral to endothermic enthalpies for the bacterial species. Entropies of copper adsorption were positive and indicative of entropy-driven inner-sphere complexation reactions. Thermodynamic parameters describing copper adsorption onto N. maritimus are consistent with adsorption onto a mixture of phosphorous-bearing anionic oxygen and thiol ligands. Thermodynamic parameters describing Cu(II) adsorption onto N. oceani and N. europaea were consistent with Cu(II) adsorption onto phosphorous-bearing anionic oxygen ligands. Modeling competitive copper complexation between N. maritimus and two organic acids, to mimic dissolved organic carbon, showed that N. maritimus could compete with nitrilotriacetic acid and triethylenetetramine under conditions similar to experimental measurements of copper adsorption. These results suggest that selectivity of the N. maritimus surface may confer some advantage in low-copper environments.

Methane oxidation plays a key role in carbon and nutrient cycling and has the potential to be applied in engineered bioprocesses, including wastewater and gas treatments. To provide insights into the dynamics of the methanotrophic community under microoxic and anoxic conditions, two sequencing batch reactors under microoxic (MO2-SBR) and anoxic (Anox-SBR) conditions were operated. The methane oxidation rate was higher under microoxic conditions (5.3 ± 0.9 mmol.batch cycle−1) than anoxic conditions (3.1 ± 0.8 mmol.batch cycle−1). Higher methane oxidation led to higher nitrate reduction rates (9.4 ± 2.5 mgN.batch cycle−1 and 4.0 ± 2.0 mg N.batch cycle−1 for MO2-SBR and Anox-SBR, respectively). 16S rDNA sequencing revealed reads corresponding to aerobic oxidizers (0.5% and 2.0% for Anox-SBR and MO2-SBR, respectively), to the Nitrosospira genus (26.6% and 28.3% for Anox-SBR and MO2-SBR, respectively), and to anaerobic methane-oxidizing archaea (ANME) (4.0% and 3.5% for Anox-SBR and for MO2-SBR, respectively). Nitrifying organisms are capable of oxidizing methane due to the homology between the enzymes ammonia monooxygenase and methane monooxygenase. These findings seem to indicate that methane oxidation is carried out by versatile metabolic pathways and couples with other biological processes, such as denitrification.

Nitrogen (N) application may lead to niche segregation of soil ammonia-oxidizing archaea (AOA) and bacteria (AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availability. However, the adaptive mechanisms of AOA and AOB under N enrichment remain poorly understood. Stable isotope probing (SIP) microcosm incubation was employed to reveal community changes of active AOA and AOB in a loess soil from a field experiment growing potatoes that received no N (control, CK), low N (LN, 75 kg N ha−1), and high N (HN, 375 kg N ha−1). The results showed that the soil potential nitrification rate (PNR) was measured by culturing of the soil samples from the field experiment. Soil PNR was significantly increased in HN by 87.5% and 67.5% compared with CK and LN, respectively. Compared with CK, the 13C-amoA genes of soil AOA and AOB in HN had 2.58 × 104 and 1.55 × 106 copies, representing 1.6- and 16.2-fold increase respectively. It was indicated that AOB dominated soil ammonia oxidation. A phylogenetic analysis of the 13C-amoA gene showed that N application significantly increased the proportion of 54d9-like AOA up to 90% in HN, while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared. Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like to Nitrosospira sp. TCH711-like. Our study provides compelling evidence for the emergence and maintenance of active nitrifying communities under the intensified N input to an agricultural ecosystem.

Physical and chemical treatments of Tungsten smelting wastewater, with high salt content and low C/N ratio, are often tedious. As a solution, this study suggested a simultaneous nitrification and denitrification membrane bioreactor (SND-MBR) for salinity gradient domestication. During the salinity acclimation period, we observed 20% and 11% removal of NH4+-N and Chemical Oxygen Demand (COD), respectively. However, the SND efficiency reached 95.55% after stable operation at 3.0% salinity. Through stoichiometric and kinetic analyses, we confirmed that increased salinity significantly inhibited electron transport system activity, nitrification, and denitrification, evidenced by the extremely low ammonia monooxygenase and nitrite reductase activities. Further high-throughput sequencing showed that Nitrosomonas dominated the functional microbial flora succession and denitrification in high salinity environments. In comparison with a control, the Kyoto Encyclopedia of Genes and Genomes analysis showed that wastewater salinity weakened the functional gene level of MBR microbial flora, and the enzyme key to the assimilation nitrate reduction changed from nitrate reductase to assimilation nitrate reductase.

The presence of toxic compounds in wastewater can cause problems for organic matter and nutrient removal. In this study, the long-term effect of a model xenobiotic, 2-fluorophenol (2-FP), on ammonia-oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and phosphate accumulating organisms (PAO) in aerobic granular sludge was investigated. Phosphate (P) and ammonium (N) removal efficiencies were high (>93%) and, after bioaugmentation with 2-FP degrading strain FP1, 2-FP was completely degraded. Neither N nor P removal were affected by 50 mg L−1 of 2-FP in the feed stream. Changes in the aerobic granule bacterial communities were followed. Numerical analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed low diversity for the ammonia monooxygenase (amoA) gene with an even distribution of species. PAOs, including denitrifying PAO (dPAO), and AOB were present in the 2-FP degrading granules, although dPAO population decreased throughout the 444 days reactor operation. The results demonstrated that the aerobic granules bioaugmented with FP1 strain successfully removed N, P and 2-FP simultaneously. HIGHLIGHTS Inoculation with strain FP1 led to 2-FP removal within the aerobic phase.; Nitrifiers and PAOs activities were not affected by 2-fluorophenol.; Bioaugmented granules with FP1 were able to simultaneously remove N, P and 2-FP.; Granular sludge is promising for the treatment of wastewaters containing toxics.

This work evaluated the fates of linear alkylbenzene sulfonate (LAS), chemical oxygen demand (COD), ammonia nitrogen ( N H 4 + − N ), and total nitrogen (TN) when treating greywater (GW) in an oxygen-based membrane biofilm reactor (O2-MBfR). An influent ratio of chemical oxygen demand to total nitrogen (COD/TN) of 20 g COD/g N gave the best removals of LAS, COD, N H 4 + − N and TN, and it also had the greatest EPS accumulation in the biofilm. Higher EPS and improved performance were linked to increases in the relative abundances of bacteria able to biodegrade LAS (Zoogloea, Pseudomonas, Parvibaculum, Magnetospirillum and Mycobacterium) and to nitrify (Nitrosomonas and Nitrospira), as well as to ammonia oxidation related enzyme (ammonia monooxygenase). The EPS was dominated by protein, which played a key role in adsorbing LAS, achieving short-time protection from LAS toxicity and allowed LAS biodegradation. Continuous high-efficiency removal of LAS alleviated LAS toxicity to microbial physiological functions, including nitrification, nitrate respiration, the tricarboxylic acid (TCA) cycle, and adenosine triphosphate (ATP) production, achieving the stable high-efficient simultaneous removal of organics and nitrogen in the O2-MBfR.

Carbon/nitrogen ratio is an important parameter during the biological wastewater treatment. Our study emphasizes revealing the mechanisms of chemical oxygen demand/total nitrogen (COD/TN) ratio dependent improved greywater (GW) treatment in an oxygen based membrane biofilm reactor (O2-MBfR). Results showed that reducing COD/TN ratio from 40 to 20 g COD/g N by supplementing NH4Cl to GW improved the relative abundance of genera related to LAS-biodegradation (from 8.39% to 35.7%), nitrification (from 0.20% to 0.62%) and denitrification (from 3.01% to 7.59%). Reducing COD/TN ratio also led to an increase in the ammonia monooxygenase (AMO) activity (from 7.56 to 10.2 mg N/g VSS-h), as well as improved ammoxidation and linear alkylbenzene sulfonate (LAS) mineralization although the dissolved oxygen (DO) concentration and pH decreased. Much higher NH4+ − N at lower COD/TN ratio (10 units) led to lower DO (0.13 ± 0.01 mg/L) and pH (6.72 ± 0.02), but the continuously increased AMO activity (up to 12.9 mg N/g VSS-h) enabled the cometabolism of ammoxidation and LAS mineralization, leading to the efficient removal of organics and nitrogen under the low DO condition.

The positive correlation between the nitrification activity of activated sludge and 17α-ethinylestradiol (EE2) removal has been widely reported. However, up to now the effect of the granulation of nitrifying activated sludge (NAS) on EE2 removal has not been determined. In this study, nitrifying granular sludge (NGS) exhibited more effective EE2 removal efficiency with 3.705 μgEE2∙(gMLSS∙h)−1 in a sequential batch reactor (SBR). Through the artificial neural network (ANN) model and Spearman correlation analysis, nitrite accumulation was demonstrated to be the key factor affecting EE2 removal. Notably, under the same aeration condition (0.15 L/min), nitrite accumulation was more easily achieved in NGS because of its dense structure. Full-length 16S rRNA gene sequencing suggested that EE2 could strongly influence the microbial communities of NAS and NGS. NGS exhibited an increase in community diversity and richness, but NAS exhibited a decrease. In addition, the relative abundance of Nitrosomonas (ammonia-oxidizing bacteria, AOB) decreased considerably in both NAS and NGS, whereas the expression of amoA and nirK genes in Nitrosomonas was upregulated. It was suggested that Nitrosomonas was forced to regulate its gene expression to resist the negative effects of EE2. Denitrifying bacteria, such as Comamonas, were enriched in both NAS and NGS, and there were more species of heterotrophs that can degrade micropollutants in NGS with exposure to EE2. The transformation pathways of EE2 were uniform in NAS and NGS. Ammonia monooxygenase (AMO) in AOB directly biotransformed EE2 while reactive species produced by AOB chemically transformed EE2. Heterotrophs degraded EE2 and its transformation products (TPs) generated by AOB. According to TPs and microbial structure, NGS exhibited better performance than NAS regarding the collaborative removal of EE2 by AOB and heterotrophs. These results provide important information for the development and application of NGS to treat wastewater containing estrogen and high-strength ammonium.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co- occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest substrate affinity of any characterized AOA, which are similar to previously determined affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values strongly supports the hypothesis that – like for AOB – ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

How to achieve stable mainstream deammonification is still a huge challenge. In this work, satisfactory nitrogen removal were achieved in a deammonification granular sludge reactor (R1, 0.42 ± 0.03 kg N / (m3·d)) and a mixed flocculent with granular sludge reactor (R2, 0.39 ± 0.04 kg N / (m3·d)) at ambient temperature (21–28 ℃) . The good adaptability of anammox bacteria (Candidatus Jettenia) to ambient temperature ensured its efficient activity (0.84–1.54 mg N/(g VSS·h)). The overexpression ammonia monooxygenase gene abundances in ammonia oxidizing bacteria (Nitrosomonas) was also predicted. The inhibition of hydrazine and the competition of denitrifying bacteria (Denitratisoma) to nitrite nitrogen, leading to a low Nitrospira relative abundances (0.2%-2.1%) . It was also found that R1 was more resistant to the unfavorable condition. For R2, higher Denitratisoma abundances (9.2%-18.5%) and predicted metabolic pathway abundances related to carbon metabolism were observed.

Organic micropollutants (OMPs) are a threat to aquatic environments, and wastewater treatment plants may act as a source or a barrier of OMPs entering the environment. Understanding the fate of OMPs in wastewater treatment processes is needed to establish efficient OMP removal strategies. Enhanced OMP biotransformation has been documented during biological nitrogen removal and has been attributed to the cometabolic activity of ammonia-oxidizing bacteria (AOB) and, specifically, to the ammonia monooxygenase (AMO) enzyme. Yet, the exact mechanisms of OMP biotransformation are often unknown. This critical review aims to fundamentally and quantitatively evaluate the role of ammonia oxidation in OMP biotransformation during wastewater treatment processes. OMPs can be transformed by AOB via direct and indirect enzymatic reactions: AMO directly transforms OMPs primarily via hydroxylation, while biologically produced reactive nitrogen species (hydroxylamine (NH2OH), nitrite (NO2-), and nitric oxide (NO)) can chemically transform OMPs through nitration, hydroxylation, and deamination and can contribute significantly to the observed OMP transformations. OMPs containing alkyl, aliphatic hydroxyl, ether, and sulfide functional groups as well as substituted aromatic rings and aromatic primary amines can be biotransformed by AMO, while OMPs containing alkyl groups, phenols, secondary amines, and aromatic primary amines can undergo abiotic transformations mediated by reactive nitrogen species. Higher OMP biotransformation efficiencies and rates are obtained in AOB-dominant microbial communities, especially in autotrophic reactors performing nitrification or nitritation, than in non-AOB-dominant microbial communities. The biotransformations of OMPs in wastewater treatment systems can often be linked to ammonium (NH4+) removal following two central lines of evidence: (i) Similar transformation products (i.e., hydroxylated, nitrated, and desaminated TPs) are detected in wastewater treatment systems as in AOB pure cultures. (ii) Consistency in OMP biotransformation (rbio, μmol/g VSS/d) to NH4+ removal (rNH4+, mol/g VSS/d) rate ratios (rbio/rNH4+) is observed for individual OMPs across different systems with similar rNH4+ and AOB abundances. In this review, we conclude that AOB are the main drivers of OMP biotransformation during wastewater treatment processes. The importance of biologically driven abiotic OMP transformation is quantitatively assessed, and functional groups susceptible to transformations by AMO and reactive nitrogen species are systematically classified. This critical review will improve the prediction of OMP transformation and facilitate the design of efficient OMP removal strategies during wastewater treatment.

Complete ammonia oxidizers (comammox), able to individually oxidize ammonia to nitrate, are considered to play a significant role in the global nitrogen cycle. However, the distribution of comammox Nitrospira in estuarine tidal flat wetland and the environmental drivers affecting their abundance and diversity remain unknown. Here, we present a large-scale investigation on the geographical distribution of comammox Nitrospira along the estuarine tidal flat wetlands of China, where comammox Nitrospira were successfully detected in 9 of the 16 sampling sites. The abundance of comammox Nitrospira ranged from 4.15 × 105 to 6.67 × 106 copies/g, 2.21- to 5.44-folds lower than canonical ammonia oxidizers: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Phylogenetic analysis based on the alpha subunit of the ammonia monooxygenase encoding gene (amoA) revealed that comammox Nitrospira Clade A, mainly originating from upstream river inputs, accounts for more than 80% of the detected comammox Nitrospira, whereas comammox Nitrospira clade B were rarely detected. Comammox Nitrospira abundance and dominant comammox Nitrospira OTUs varied within the estuarine samples, showing a geographical pattern. Salinity and pH were the most important environmental drivers affecting the distribution of comammox Nitrospira in estuarine tidal flat wetlands. The abundance of comammox Nitrospira was further negatively correlated with high ammonia and nitrite concentrations. Altogether, this study revealed the existence, abundance and distribution of comammox Nitrospira and the driving environmental factors in estuarine ecosystems, thus providing insights into the ecological niches of this recently discovered nitrifying consortium and their contributions to nitrification in global estuarine environments.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

The effects of zeolite and biochar addition on ammonia-oxidizing bacteria (AOB) and archaea (AOA) communities during agricultural waste composting were determined in this study. Four treatments were conducted as follows: Treatment A as the control with no additive, Treatment B with 5% of zeolite, Treatment C with 5% of biochar, and Treatment D with 5% of zeolite and 5% biochar, respectively. The AOB and AOA amoA gene abundance as well as the ammonia monooxygenase (AMO) activity were estimated by quantitative PCR and enzyme-linked immunosorbent assay, respectively. The relationship between gene abundance and AMO enzyme activity was determined by regression analysis. Results indicated that the AOB was more abundant than that of AOA throughout the composting process. Addition of biochar and its integrated application with zeolite promoted the AOB community abundance and AMO enzyme activity. Significant positive relationships were obtained between AMO enzyme activity and AOB community abundance (r2 = 0.792, P &lt, 0.01) and AOA community abundance (r2 = 0.772, 0.01), indicating that both bacteria and archaea played significant roles in microbial ammonia oxidation during composting. Using biochar and zeolite might promote the nitrification activity by altering the sample properties during agricultural waste composting.

The naturally occurring nitrogen (N) isotopes, 15N and 14N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO2–), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or εAMO, has been typically determined based on isotope kinetics during product formation (cumulative product, NO2–) alone, which may have overestimated εAMO due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH3/NH4+). Here, we analyzed 15N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH4+) and cumulative product (CP, NO2–) pools in pure cultures of the soil strain Nitrososphaera viennensis EN76 and in highly enriched cultures of the marine strain Nitrosopumilus adriaticus NF5, under non-limiting substrate conditions. We obtained εAMO values of 31.9–33.1‰ for both strains based on RS (δ15NH4+) and showed that estimates based on CP (δ15NO2–) give larger isotope fractionation factors by 6–8‰. Complementary analyses showed that, at the end of the growth period, microbial biomass was 15N-enriched (10.1‰), whereas nitrous oxide (N2O) was highly 15N depleted (−38.1‰) relative to the initial substrate. Although we did not determine the isotope effect of NH4+ assimilation (biomass formation) and N2O production by AOA, our results nevertheless show that the discrepancy between εAMO estimates based on RS and CP might have derived from the incorporation of 15N-enriched residual NH4+ after AMO reaction into microbial biomass and that N2O production did not affect isotope fractionation estimates significantly.

Heavy metal pollution affects soil ecological function. Biochar and compost can effectively remediate heavy metals and increase soil nutrients. The effects and mechanisms of biochar and compost amendments on soil nitrogen cycle function in heavy-metal contaminated soils are not fully understood. This study examined how biochar, compost, and their integrated use affected ammonia-oxidizing microorganisms in heavy metal polluted soil. Quantitative PCR was used to determine the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB). Ammonia monooxygenase (AMO) activity was evaluated by the enzyme-linked immunosorbent assay. Results showed that compost rather than biochar improved nitrogen conversion in soil. Biochar, compost, or their integrated application significantly reduced the effective Zn and Cd speciation. Adding compost obviously increased As and Cu effective speciation, bacterial 16S rRNA abundance, and AMO activity. AOB, stimulated by compost addition, was significantly more abundant than AOA throughout remediation. Correlation analysis showed that AOB abundance positively correlated with NO3−-N (r = 0.830, P

A microorganism isolated and identified as Acinetobacter courvalinii was found to be able to perform sequential free cyanide (CN&minus, ) degradation, simultaneous nitrification and aerobic denitrification (SNaD), this ability was associated with the multiphase growth profile of the microorganism when provided with multiple nitrogenous sources. The effect of CN&minus, on SNaD including enzyme expression, activity and protein functionality of Acinetobacter courvalinii was investigated. It was found that CN&minus, concentration of 1.9 to 5.8 mg CN&minus, /L did not affect the growth of Acinetobacter courvalinii. Furthermore, the degradation rates of CN&minus, and ammonium-nitrogen (NH4-N) were found to be 2.2 mg CN&minus, /L/h and 0.40 mg NH4-N/L/h, respectively. Moreover, five models&rsquo, (Monod, Moser, Generic Rate law, Haldane, and Andrews) ability to predict SNaD under CN&minus, conditions, indicated that, only the Rate law, Haldane and Andrew&rsquo, s models, were suited to predict both SNaD and CN&minus, degradation. The effect of CN&minus, on NH4-N, nitrate-nitrogen (NO3&minus, ) and nitrite-nitrogen (NO2&minus, ) oxidizing enzymes indicated that the CN&minus, did not affect the expression and activity of ammonia monooxygenase (AMO), albeit, reduced the expression and activity of nitrate reductase (NaR) and nitrite reductase (NiR). Nevertheless, a slow decrease in NO2&minus, was observed after the supplementation of CN&minus, to the cultures, thus confirming the activity of NaR and the activation of the denitrification pathway by the CN&minus, These special characteristics of the Acinetobacter courvalinii isolate, suggests its suitability for the treatment of wastewater containing multiple nitrogenous compounds in which CN&minus, is present.

Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase., Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, “Candidatus Nitrosocosmicus franklandus” C13 and “Candidatus Nitrosotalea sinensis” Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from “Ca. Nitrosocosmicus franklandus” and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits “Ca. Nitrosocosmicus franklandus” and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from “Ca. Nitrosocosmicus franklandus,” and it does not compete with NH3 for binding at the active site. IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Trichloroethylene (TCE) has been found to be highly potentially carcinogenic and extremely toxic. Due to the low solubility of methane gas, it is difficult to handle this primary substrate for methanotrophs during operation. Hence, using higher solubility of ammonia/ammonium instead of methane as primary substrate for nitrifying bacteria to cometabolize TCE by ammonia monooxygenase might increase the operation efficiencies in in-situ bioremediation of TCE. This study uses an enriched nitrifying culture to investigate the degradation efficiencies of TCE and its effects of inhibition to ammonia-oxidizing activity in cometabolism of TCE. Nitrificational oxygen uptake rate was analyzed by using the Gilson Model 5/6H Oxygraph respirometer. Nitrifying biomass was tested as mixed liquor volatile suspended solids and measured by using the Standard Methods. Both of the high initially applied TCE concentrations and low DO levels maintained in the batch system might cause less ammonia oxidation by nitrifying bacteria.

Ionic liquids (ILs) were widely applied because of their excellent properties. The present investigation studied the toxicity of the IL 1-octyl-3-methylimidazolium hexafluorophosphate ([Omim]PF6) to the soil microbial population and community diversity with dose (1.0, 2.0, 4.0, 6.0, and 8.0 mg kg-1) and exposure time (7, 10, and 13 d). The results show the IL was stable during the entire experimental period. The Biolog-ECO plate results indicated that the average well color development (AWCD) in the 6.0 and 8.0 mg kg-1 treatments was lower than these in the other treatments. The diversity indices of the Biolog analysis were significantly reduced. The abundance of the ammonia-oxidizing archaea (AOA-) and the ammonia-oxidizing bacteria (AOB-) ammonia monooxygenase (amoA) genes was measured by the real-time polymerase chain reaction (RT-PCR). In the treatments of 4.0, 6.0 and 8.0 mg kg-1, the abundance of amoA genes of the AOA- and AOB- were inhibited by IL [Omim]PF6.

The extensive release of cerium dioxide nanoparticles (CeO2 NPs) causes increasing potential health and environmental risks. In this study, acute and chronic responses of macrophyte and microorganisms were evaluated following exposure to CeO2 NPs in constructed wetlands (CWs). It was found that most of the CeO2 NPs were retained in the CW substrate, with minor amounts accumulated in plant tissues. Photosynthetic activity experiment indicated that environmentally relevant concentration (1 mg/L) of CeO2 NPs enhanced the net photosynthesis rate of plants, while chronic exposure to a high concentration (50 mg/L) of CeO2 NPs inhibited photosynthesis. The variations of the malondialdehyde (MDA) content and antioxidant enzyme activities in plant tissues revealed that acute exposure to CeO2 NPs induced oxidative stress to plants, while there was no obvious chronic effect. Moreover, the biomass increased marginally under exposure to 1 mg/L CeO2 NPs, whereas exposure to 50 mg/L CeO2 NPs reduced the total biomass of plants by 29%. The release of lactate dehydrogenase (LDH) suggested that chronic exposure to CeO2 NPs inhibited microbial viability. Metagenome and enzymatic activities analyses further revealed that CeO2 NPs significantly changed the microbial community structure and inhibited the activities of dehydrogenase, urease, ammonia monooxygenase, and phosphatase. This resulted in the decline of contaminant removal efficiency. Overall, acute and chronic exposure to CeO2 NPs had multiple effects on macrophyte and microorganisms in CWs, which significantly impacted the treatment performance of the ecological system.

Cattle manure composting was performed in an aerated vessel. Community structure and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) techniques targeting the ammonia monooxygenase alpha subunit (amoA) gene and the correlation between AOB and AOA communities and environmental factors was explored. Thirteen (13) AOB sequences were obtained, which were closely related to Nitrosomonas spp., Nitrosomonas eutropha, and Nitrosospira spp. and uncultured bacteria, among which Nitrosomonas spp. were predominant. Excessively high temperature and high ammonium concentration were not favorable for AOB growth. Five AOA sequences, belonging to Candidatus Nitrososphaera gargensis and to an uncultured archaeon, were obtained. During composting, community diversity of AOB and AOA fluctuated, with AOA showing a higher Shannon-Wiener index. The AOB community changed more dramatically in the mesophilic stage and the early thermophilic stage, whereas the most obvious AOA community succession occurred in the late thermophilic stage, the cooling stage and the maturity stage. Water content, total nitrogen (TN) and ammonium concentration were more relevant to the AOB community structure, while higher correlations were observed between ammonia, nitrate and TN and the AOA community. AOB community diversity was negatively correlated with pH (r = -0.938, p < 0.01) and water content (r = -0.765, p < 0.05), while positively correlated with TN (r = 0.894, p < 0.01). AOA community diversity was negatively correlated with ammonium concentration (r = -0.901, p < 0.01). Ammonium concentration played an important role in the succession of AOB and AOA communities during composting. Resumen Se llevó a cabo un compostaje de estiércol de ganado en un recipiente aireado. Se investigó la estructura de la comunidad y la diversidad de bacterias oxidantes del amoníaco (AOB) y las arqueas oxidantes del amoníaco (AOA) mediante el uso de las técnicas de reacción en cadena de la polimerasa y la electroforesis en gel con gradiente de desnaturalización (PCR-DGGE) dirigidas al gen de la subunidad alfa de la amonio monooxigenasa (amoA), y se exploró la correlación entre las comunidades AOB, AOA y los factores ambientales. Se obtuvieron 13 secuencias de AOB, las cuales se relacionaron estrechamente con Nitrosomonas spp., Nitrosomonas eutropha y Nitrosospira spp., y bacterias no cultivadas, entre las cuales fueron predominantes las Nitrosomonas spp. La temperatura excesivamente alta y la concentración de amonio elevada no fueron favorables para el crecimiento de las AOB. Se obtuvieron 5 secuencias de AOA, pertenecientes a Candidatus Nitrososphaera gargensis y un Archaeon no cultivado. Durante el compostaje, la diversidad de AOB y AOA fluctuó y las AOA mostraron un índice de Shannon-Wiener más alto. La comunidad de AOB cambió significativamente en la etapa mesofílica y la etapa termofílica temprana, mientras que la sucesión más obvia de la comunidad AOA ocurrió en la etapa termofílica tardía y las etapas de enfriamiento y de maduración. El contenido de agua, el nitrógeno total (TN) y la concentración de amonio fueron más relevantes para la estructura de la comunidad AOB, mientras que se observaron correlaciones mayores entre amoníaco, nitrato y TN, y la comunidad AOA. La diversidad de la comunidad AOB se correlacionó negativamente con el pH (r= -0,938; p < 0,01) y el contenido de agua (r = -0,765; p < 0,05), mientras que se relacionó positivamente con TN (r = 0,894; p < 0,01). La diversidad de la comunidad AOA se correlacionó negativamente con la concentración de amonio (r = -0,901; p < 0,01). La concentración de amonio desempenó un papel importante en la sucesión de las comunidades AOB y AOA durante el compostaje.

Benzylalkyldimethylethyl ammonium compounds are pervasive in natural environments and toxic at high concentrations. The changes in functional genes and microbial diversity in eutrophic lake samples exposed to benzyldimethyldodecyl ammonium chloride (BAC) were assessed. BAC exerted negative effects on bacteria abundance, particularly at concentrations of 100 μg L-1 and higher. A significant increase in the number of the quaternary ammonium compound-resistant gene qacA/B was recorded within the 10 μg L-1 treatment after the first day of exposure. Not all antibiotic resistance genes increased in abundance as the concentrations of BAC increased; rather, gene abundances were dependent on the gene type, concentrations of BAC, and contact time. The nitrogen fixation-related gene nifH and ammonia monooxygenase gene amoA were inhibited by high concentrations of BAC after the first day, whereas an increase of the nitrite reductase gene nirK was stimulated by exposure. Microbial communities within higher treatment levels (1000 and 10 000 μg L-1) exhibited significantly different community composition compared to other treatment levels and the control. Selective enrichment of Rheinheimera, Pseudomonas, and Vogesella were found in the higher treatment levels, suggesting that these bacteria have some resistance or degradation capacity to BAC. Genes related with RNA processing and modification, transcription, lipid transport and metabolism, amino acid transport and metabolism, and cell motility of microbial community function were involved in the process exposed to the BAC stress.

Ammonia oxidation plays a fundamental role in river nitrogen cycling ecosystems, which is normally governed by both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB). Co-contamination of typical emerging pollutant Polybrominated diphenyl ethers (PBDEs) and heavy metal on AOA and AOB communities in river sediments remains unknown. In this study, multiple analytical tools, including high-throughput pyrosequencing and real-time quantitative PCR (qPCR), were used to reveal the ammonia monooxygenase (AMO) activity, subunit alpha ( amo A) gene abundance, and community structures of AOA and AOB in river sediments. It was found that the inhibition of AMO activities was increased with the increase of decabromodiphenyl ether (BDE 209, 1–100 mg kg −1 ) and copper (Cu, 50–500 mg kg −1 ) concentrations. Moreover, the synergic effects of BDE 209 and Cu resulted in a higher AMO activity reduction than the individual pollutant BDE 209. The AOA amo A copy number declined by 75.9% and 83.2% and AOB amo A gene abundance declined 82.8% and 90.0% at 20 and 100 mg kg −1 BDE 209 with a 100 mg kg −1 Cu co-contamination, respectively. The pyrosequencing results showed that both AOB and AOA community structures were altered, with a higher change of AOB than that of AOA. The results demonstrated that the AOB microbial community may be better adapted to BDE 209 and Cu pollution, while AOA might possess a greater capacity for stress resistance. Our study provides a better understanding of the ecotoxicological effects of heavy metal and micropollutant combined exposure on AOA and AOB in river sediments.

Biological nitrogen removal is the most prevalent wastewater nitrogen removal process but nitrification limits the rate of the whole process mainly due to the low efficiency of oxygen transfer. In this study, clean-water oxygenation tests, batch tests, long-term operational tests and metagenomic analyses were applied to assess the effects of micro-nano aeration on nitrification. The oxygen transfer coefficient (KLa), oxygen transfer rate (OTR) and oxygen transfer efficiency (OTE) were determined to be 0.56 min−1, 0.36 kg·m−3·h−1 and 71.43%, respectively during micro-nano-bubble aeration. Impressively, these values were 15 times greater than those of conventional aeration. The results of batch tests and long-term operation experiments found that the ammonia removal rate of micro-nano aeration was 3.2-fold that of conventional aeration. The energy cost for micro-nano aeration was calculated to be 3694.5 mg NH4+-N/kW·h, a 50% energy saving in comparison to conventional aeration. In addition, the nitrite accumulation ratio in the Micro-nano (MN) reactor was 1.5 that of the Conventional (CV) reactor. Metagenomic analysis showed that after long-term operation in micro-nano aeration, the abundances of genes encoding ammonia monooxygenase (amoA) and hydroxylamine oxidoreductase (hao) was more than 8-fold and 4-fold of those in conventional aeration, respectively. The abundance of the gene encoding nitrite oxidoreductase (nxrA) was similar in both reactors. Read taxonomy revealed that abundance of AOB-Nitrosomonas increased significantly when using micro-nano aeration, while abundance of NOB-Nitrospira abundance was similar in both reactors. The results of this study indicated that the micro-nano aeration process will increase the ammonia oxidation performance by enhancing oxygen transfer but was also shown to be beneficial for enhancing partial nitrification by specific enrichment of ammonia oxidizing bacteria. This latter result demonstrates the potential benefits of the micro-nano aeration process as an alternative approach to establishing high-rate partial nitrification.

This study presents an innovative mainstream Anammox based on multiple NO2−-N supplement pathways to treat actual mature landfill leachate over 180 days. Desirable effluent quality of 11.8 mg/L total nitrogen (TN) and nitrogen removal efficiency of 98.8% were achieved despite fluctuation conditions of 1.5-fold influent substrates and 8.0-fold dissolved oxygen overload. Nitrogen mass balance confirmed Anammox was the dominant nitrogen removal pathway, contributing up to 87.9%. Functional genes of ammonia monooxygenase (amoA), hydrazine synthase (hzsB), and ratio of nitrate/nitrite reductase were highly detected. Anammox genera, Candidatus_Kuenenia (4.1%) and Candidatus_Brocadia (5.3%) were dominant in two functional systems, respectively, due to the different affinity of nitrite, oxygen, and organic carbon. As an economical and sustainable technology, the innovative process enabled a 95.1% decrease in organic carbon demand, a 61.5% reduction in aeration energy consumption, and 77.6% lower biomass production compared with traditional nitrification–denitrification process.

ABSRACT The differences of cultured organism species, aquaculture model and supervisor mode lead to different carbon/nitrogen ratios in mariculture wastewater. Therefore, the performance, microbial community and enzymatic activity of sequencing batch biofilm reactor were compared in treating synthetic mariculture wastewater at different chemical oxygen demand/nitrogen (COD/N) ratios. Compared with COD/N ratio of 6, the ammonia-oxidizing rate and nitrite-oxidizing rate at COD/N ratio of 5, 4 and 3 increased by 3.66 % and 3.08 %, 11.19 % and 14.95 %, and 24.50 % and 32.54 %, respectively. Similarly, the ammonia monooxygenase and nitrite oxidoreductase activities increased by 3.50 % and 6.76 %, 11.09 % and 16.22 %, and 25.43 % and 39.19 % at COD/N ratio at 5, 4 and 3, respectively. However, the denitrifying rate and denitrification enzymatic activity declined with the decrease of C/N ratio from 6 to 3. The production, protein content and polysaccharide content of loosely bound extracellular polymeric substances (LB-EPS) and tightly bound EPS (TB-EPS) reduced with the decrease of COD/N ratio from 6 to 3. The abundance of nitrifying genera increased with the decrease of COD/N ratio from 6 to 3, whereas most of denitrification genera displayed a decreasing trend. The microbial co-occurrence pattern, keystone taxa and significant difference were altered with the decrease of COD/N ratio. Among the keystone taxa, Thauera, Denitromonas, Nitrosomonas and Denitratisoma had a close link with nitrogen transformation. The present results can provide some theoretical basis for evaluating the effect of carbon/nitrogen ratio on the nitrogen removal of biological wastewater treatment systems.

Combined impacts of diclofenac (DCF) and divalent copper (Cu2+) on the nitrogen removal, enzyme activity and bacterial community from a sequencing batch reactor were examined. DCF and/or Cu2+ in the influent rendered the decrease of ammonia nitrogen and chemical oxygen demand elimination rates. The combined inhibition impacts of Cu2+ and DCF on ammonia monooxygenase (AMO), nitrite oxidoreductase (NOR), nitrite reductase (NIR) and nitrate reductase (NAR) contents exhibited synergetic, and the AMO and NOR activities reduced levels were lower than NAR and NIR as the mixed Cu2+ and DCF concentration increment. The decreased degrees of AMO activities were higher than NOR at different mixed Cu2+ and DCF concentrations. The contents of reactive oxygen species and lactate dehydrogenase at the mixed Cu2+ and DCF stress were higher than those at the alone Cu2+ and DCF stress, and they increased as the mixed Cu2+ and DCF concentration increase. In extracellular polymeric substances (EPS), the content of protein (PN) was more easily affected by Cu2+ and mixed Cu2+/DCF than polysaccharide (PS), while PN and PS were both sensitive to the stress of DCF. Compared to seeding sludge, the denitrifying bacteria and nitrifying bacteria relative abundance ratio was decreased at the stress of alone Cu2+ and DCF, while the ratio was increased as the mixed Cu2+ and DCF concentration increment.

The effect of aerobic/anoxic duration on the performance, microbial community and enzymatic activity of sequencing batch biofilm reactor (SBBR) were investigated in treating mariculture wastewater. The microbial oxygen uptake rate and nitrifying rate gradually decreased with the aerobic/anoxic duration from 120/210 to 30/300 min, whereas the nitrite reducing rate and nitrate reducing rate had the opposite results. The activities of dehydrogenase, ammonia monooxygenase and nitrite oxidoreductase gradually decreased with the aerobic/anoxic duration from 120/210 to 30/300 min, but the activities of nitrate reductase and nitrite reductase had a gradual increment. The microbial nitrogen removal rates had similar changing trends to their corresponding enzymatic activities at different aerobic/anoxic duration. The variation of aerobic/anoxic duration obviously affected the microbial richness and diversity of SBBR. The co-occurrence, keystone taxa and significant difference of microbial community had some changes with the aerobic/anoxic duration from 120/210 to 30/300 min.

Achieving stable nitrite accumulation at low temperature continues to be a challenging problem in partial nitrification (PN). This study proposes a strategy to achieve stable PN of thickened sludge at low temperatures through selective inactivation of enzymes by intermittent thermal treatment. This method was verified using a sequencing batch reactor (SBR) operated at 9 ± 1 °C in full nitrification mode. After thermally treating the activated sludge at 35 °C for 2 days, which was the optimal condition for selective inactivation of enzymes determined by batch tests, an average nitrite accumulation rate (NAR) of 90.18 ± 5.74% was rapidly achieved and the SBR was stably operated at 9 ± 1 °C for 80 days. This was consistent with a significant decrease of the specific nitrite uptake rate (SNUR), but no a reduction, or even an increase, of the specific ammonia uptake rate (SAUR). Also, there was no remarkable change in the nitrifying bacterial community except for a slight increase in both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). In addition, after thermal treatment at 35 °C for 2 days, the nitrite oxidoreductase (NOR) activity was significantly decreased, while those of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) showed an increasing trend. Thus, the achievement of stable PN at low temperatures with this strategy may be due to the selective inactivation of NOR, rather than NOB washout.