Thermally stable high-loading single Cu sites on ZSM-5 for selective catalytic oxidation of NH3.

Rigorous comparisons between single site- and nanoparticle (NP)-dispersed catalysts featuring the same composition, in terms of activity, selectivity, and reaction mechanism, are limited. This limitation is partly due to the tendency of single metal atoms to sinter into aggregated NPs at high loadings and elevated temperatures, driven by a decrease in metal surface free energy. Here, we have developed a unique two-step method for the synthesis of single Cu sites on ZSM-5 (termed CuS/ZSM-5) with high thermal stability. The atomic-level dispersion of single Cu sites was confirmed through scanning transmission electron microscopy, X-ray absorption fine structure (XAFS), and electron paramagnetic resonance spectroscopy. The CuS/ZSM-5 catalyst was compared to a CuO NP-based catalyst (termed CuN/ZSM-5) in the oxidation of NH3 to N2, with the former exhibiting superior activity and selectivity. Furthermore, operando XAFS and diffuse reflectance infrared Fourier transform spectroscopy studies were conducted to simultaneously assess the fate of the Cu and the surface adsorbates, providing a comprehensive understanding of the mechanism of the two catalysts. The study shows that the facile redox behavior exhibited by single Cu sites correlates with the enhanced activity observed for the CuS/ZSM-5 catalyst.

Ultra-Small High-Entropy Alloy as Multi-Functional Catalyst for Ammonia Based Fuel Cells.

Ammonia fuel cells using carbon-neutral ammonia as fuel are regarded as a fast, furious, and flexible next-generation carbon-free energy conversion technology, but it is limited by the kinetically sluggish ammonia oxidation reaction (AOR), oxygen reduction reaction (ORR), and hydrogen evolution reaction (HER). Platinum can efficiently drive these three types of reactions, but its scale-up application is limited by its susceptibility to poisoning and high cost. In order to reduce the cost and alleviate poisoning, incorporating Pt with various metals proves to be an efficient and feasible strategy. Herein, PtFeCoNiIr/C trifunctional high-entropy alloy (HEA) catalysts are prepared with uniform mixing and ultra-small size of 2 ± 0.5 nm by Joule heating method. PtFeCoNiIr/C exhibits efficient performance in AOR (Jpeak = 139.8 A g-1 PGM), ORR (E1/2 = 0.87 V), and HER (E10 = 20.3 mV), outperforming the benchmark Pt/C, and no loss in HER performance at 100 mA cm-2 for 200 h. The almost unchanged E1/2 in the anti-poisoning test indicates its promising application in real fuel cells powered by ammonia. This work opens up a new path for the development of multi-functional electrocatalysts and also makes a big leap toward the exploration of cost-effective device configurations for novel fuel cells.

Competition between ammonia-oxidizing archaea and complete ammonia oxidizers from freshwater environments.

Aerobic ammonia oxidizers (AOs) are prokaryotic microorganisms that contribute to the global nitrogen cycle by performing the first step of nitrification, the oxidation of ammonium to nitrite and nitrate. While aerobic AOs are found ubiquitously, their distribution is controlled by key environmental conditions such as substrate (ammonium) availability. Ammonia-oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) are generally found in oligotrophic environments with low ammonium availability. However, whether AOA and comammox share these habitats or outcompete each other is not well understood. We assessed the competition for ammonium between an AOA and comammox enriched from the freshwater Lake Burr Oak. The AOA enrichment culture (AOA-BO1) contained Nitrosarchaeum sp. BO1 as the ammonia oxidizer and Nitrospira sp. BO1 as the nitrite oxidizer. The comammox enrichment BO4 (cmx-BO4) contained the comammox strain Nitrospira sp. BO4. The competition experiments were performed either in continuous cultivation with ammonium as a growth-limiting substrate or in batch cultivation with initial ammonium concentrations of 50 and 500 µM. Regardless of the ammonium concentration, Nitrospira sp. BO4 outcompeted Nitrosarchaeum sp. BO1 under all tested conditions. The dominance of Nitrospira sp. BO4 could be explained by the ability of comammox to generate more energy through the complete oxidation of ammonia to nitrate and their more efficient carbon fixation pathway-the reductive tricarboxylic acid cycle. Our results are supported by the higher abundance of comammox compared to AOA in the sediment of Lake Burr Oak. IMPORTANCE: Nitrification is a key process in the global nitrogen cycle. Aerobic ammonia oxidizers play a central role in the nitrogen cycle by performing the first step of nitrification. Ammonia-oxidizing archaea (AOA) and complete ammonia oxidizers (comammox) are the dominant nitrifiers in environments with low ammonium availability. While AOA have been studied for almost 20 years, comammox were only discovered 8 years ago. Until now, there has been a gap in our understanding of whether AOA and comammox can co-exist or if one strain would be dominant under ammonium-limiting conditions. Here, we present the first study characterizing the competition between freshwater AOA and comammox under varying substrate concentrations. Our results will help in elucidating the niches of two key nitrifiers in freshwater lakes.

Comammox Nitrospira among dominant ammonia oxidizers within aquarium biofilter microbial communities.

Nitrification by aquarium biofilters transforms ammonia waste (NH3/NH4+) to less toxic nitrate (NO3-) via nitrite (NO2-). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) Nitrospira, previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 103 ± 1.5 × 103 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 103 ± 2.7 × 103 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 101 ± 1.1 × 102 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification. IMPORTANCE: Nitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira, and highlights the value of aquaria as microcosms for studying nitrifier ecology.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , first defined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Nitrosotalea devaniterrae gen. nov., sp. nov. and Nitrosotalea sinensis sp. nov., two acidophilic ammonia oxidising archaea isolated from acidic soil, and proposal of the new order Nitrosotaleales ord. nov. within the class Nitrososphaeria of the phylum Nitrososphaerota.

Two obligately acidophilic, mesophilic and aerobic soil ammonia-oxidising archaea were isolated from a pH 4.5 arable sandy loam (UK) and pH 4.7 acidic sulphate paddy soil (PR China) and designated strains Nd1T and Nd2T, respectively. The strains shared more than 99 % 16S rRNA gene sequence identity and their genomes were both less than 2 Mb in length, sharing 79 % average nucleotide identity, 81 % average amino acid identity and a DNA G+C content of approximately 37 mol%. Both strains were chemolithotrophs that fixed carbon dioxide and gained energy by oxidising ammonia to nitrite, with no evidence of mixotrophic growth. Neither strain was capable of using urea as a source of ammonia. Both strains were non-motile in culture, although Nd1T does possess genes encoding flagella components and therefore may be motile under certain conditions. Cells of Nd1T were small angular rods 0.5-1 µm in length and grew at pH 4.2-5.6 and at 20-30 °C. Cells of Nd1T were small angular rods 0.5-1 µm in length and grew at pH 4.0-6.1 and at 20-42 °C. Nd1T and Nd2T are distinct with respect to genomic and physiological features and are assigned as the type strains for the species Nitrosotalea devaniterrae sp. nov. (type strain, Nd1T=NCIMB 15248T=DSM 110862T) and Nitrosotalea sinensis sp. nov. (type strain, Nd2T=NCIMB 15249T=DSM 110863T), respectively, within the genus Nitrosotalea gen. nov. The family Nitrosotaleaceae fam. nov. and order Nitrosotaleales ord. nov. are also proposed officially.

Oxidation of Ammonia in Water Microdroplets Produces Nitrate and Molecular Hydrogen.

Water microdroplets containing dissolved ammonia (30-300 µM) are sprayed through a copper oxide mesh with a 200 µm average pore size, resulting in the formation of nitrate (NO3-) and the release of molecular hydrogen (H2). The products result from a redox process that takes place at the liquid-solid interface through contact electrification, where no external potential is applied. Oxidation is initiated by superoxide radical anions (O2-) that originate from the oxygen in the air surrounding the microdroplets and from the hydroxyl radicals (OH•) originating from the water-air interface. Two spin traps (TEMPO and DMPO) capture these radicals as well as NH2OH+•, HNO, NO•, NO2•, and NOOH, which are detected by mass spectrometry. We also directly observed N2O2-• by the same means. We found that the hydrogen atom from the ammonia molecule can be set free not only in the form of H• but also as H2, which is detected using a residue gas analyzer. The oxidation process can be significantly enhanced by a factor of 3 when the sprayed microdroplets are irradiated with ultraviolet light (265 nm, 5 W). 35% of 300 µM ammonia can be degraded within 20 µs, and the nitrate conversion rate is estimated to be 15 nmol·mg-1·h-1.

Physiological Influence of Fe and Cu Availability on Nitrogen Isotope Fractionation during Ammonia Oxidation.

Microbially mediated cycling processes play central roles in regulating the speciation and availability of nitrogen, a vital nutrient with wide implications for agriculture, water quality, wastewater treatment, ecosystem health, and climate change. Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by bacteria (AOB) and archaea (AOA) that require the trace metal micronutrients copper (Cu) and iron (Fe) for growth and metabolic catalysis. While stable isotope analyses for constraining nitrogen cycling are commonly used, it is unclear whether metal availability may modulate expression of stable isotope fractionation during ammonia oxidation, by varying growth or through regulation of metabolic metalloenzymes. We present the first study examining the influence of Fe and Cu availability on the kinetic nitrogen isotope effect in ammonia oxidation (15εAO). We report a general independence of 15εAO from the growth rate in AOB, except at a low temperature (10 °C). With AOA Nitrosopumilus maritimus SCM1, however, 15εAO decreases nonlinearly at lower oxidation rates. We examine assumptions involved in the interpretation of 15εAO values and suggest these dynamics may arise from physiological constraints that push the system toward isotopic equilibrium. These results suggest important links between isotope fractionation and environmental constraints on physiology in these key N cycling microorganisms.

Multidrug-resistant plasmid RP4 inhibits the nitrogen removal capacity of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox in activated sludge.

In wastewater treatment plants (WWTPs), ammonia oxidation is primarily carried out by three types of ammonia oxidation microorganisms (AOMs): ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox (CMX). Antibiotic resistance genes (ARGs), which pose an important public health concern, have been identified at every stage of wastewater treatment. However, few studies have focused on the impact of ARGs on ammonia removal performance. Therefore, our study sought to investigate the effect of the representative multidrug-resistant plasmid RP4 on the functional microorganisms involved in ammonia oxidation. Using an inhibitor-based method, we first evaluated the contributions of AOA, AOB, and CMX to ammonia oxidation in activated sludge, which were determined to be 13.7%, 41.1%, and 39.1%, respectively. The inhibitory effects of C2H2, C8H14, and 3,4-dimethylpyrazole phosphate (DMPP) were then validated by qPCR. After adding donor strains to the sludge, fluorescence in situ hybridization (FISH) imaging analysis demonstrated the co-localization of RP4 plasmids and all three AOMs, thus confirming the horizontal gene transfer (HGT) of the RP4 plasmid among these microorganisms. Significant inhibitory effects of the RP4 plasmid on the ammonia nitrogen consumption of AOA, AOB, and CMX were also observed, with inhibition rates of 39.7%, 36.2%, and 49.7%, respectively. Moreover, amoA expression in AOB and CMX was variably inhibited by the RP4 plasmid, whereas AOA amoA expression was not inhibited. These results demonstrate the adverse environmental effects of the RP4 plasmid and provide indirect evidence supporting plasmid-mediated conjugation transfer from bacteria to archaea.

Synthesis of amorphous-MnO2/Clinoptilolite and its utilization for NH4+-N oxidation in an anoxic environment.

Mediating the anoxic ammonia oxidation with manganese oxide (MnOx) can reduce the requirements of dissolved oxygen (DO) concentrations in constructed wetlands (CWs) and improve the removal of ammonium nitrogen (NH4+-N). Recent studies that employed natural manganese ore and/or mine waste as substrates in CWs may develop potentially negative environmental effects due to leachates. However, removing NH4+-N by anoxic ammonia oxidation is influenced by the crystal form of MnOx. In this study, a novel clinoptilolite-based amorphous-MnO2 (amorphous-MnO2/clinoptilolite) was synthesized by the sol-gel method as an alternative substrate to improve the efficiency of anoxic ammonia oxidation and reduce the impact of Mn ion leaching. According to the anoxic ammonia oxidation experiment of clinoptilolite, amorphous-MnO2/clinoptilolite, and manganese ore on NH4+-N, the amounts of NH4+-N removed were 24.55 mg/L/d, 44.55 mg/L/d, and 11.04 mg/L/d, respectively, and the initial NH4+-N concentration was 49.53 mg/L. These results indicated that the amorphous-MnO2/clinoptilolite had both the adsorption and the anoxic ammonia oxidation performance. The recycling experiment demonstrated that the effect of anoxic ammonia oxygen mediated by amorphous-MnO2 would not diminish with the gradual saturation of clinoptilolite for NH4+-N. Furthermore, the anoxic ammonia oxidation consumed NH4+-N in the clinoptilolite, which restored the adsorption capacity of the clinoptilolite and simultaneously decreased the leakage of manganese ions in the process, making it environmentally friendly. Therefore, the amorphous-MnO2/clinoptilolite provided an excellent substrate material for the constructed wetland under an anoxic environment, which greatly improved the nitrogen removal capacity compared to existing substrate materials.

Acid-tolerant Pseudomonas citronellolis YN-21 exhibits a high heterotrophic nitrification capacity independent of the amo and hao genes.

Heterotrophic nitrifying bacteria are found to be promising candidates for implementation in wastewater treatment systems due to their tolerance to extreme environments. A novel acid-resistant bacterium, Pseudomonas citronellolis YN-21, was isolated and reported to have exceptional heterotrophic nitrification capabilities in acidic condition. At pH 5, the highest NH4+ removal rate of 7.84 mg/L/h was displayed by YN-21, which was significantly higher than the NH4+ removal rates of other strains in neutral and alkaline environments. Remarkably, a distinct accumulation of NH2OH and NO3- was observed during NH4+ removal by strain YN-21, while traditional amo and hao genes were not detected in the genome, suggesting the possible presence of alternative nitrifying genes. Moreover, excellent nitrogen removal performance was displayed by YN-21 even under high concentrations of metal ion stress. Consequently, a broad application prospect in the treatment of leather wastewater and mine tailwater is offered by YN-21.

In situ electro-generated Ni(OH)2 synergistic with Cu cathode to promote direct ammonia oxidation to nitrogen.

To solve the problem of low removal rate and poor N2 selectivity in direct electrochemical ammonia oxidation (EAO), commercial Ni foam and Cu foam were used as anode and cathode of the EAO system, respectively. The coupling effect between the cathode and anode promoted nitrogen cycling during the reaction process, which improved N2 selectivity of the reaction system and promoted it to achieve a high ammonia removal rate. This study showed that the thin Ni(OH)2 with oxygen vacancy formed on the surface of Ni foam anode played an effective role in the dimerization of intermediate products in ammonia oxidation to form N2. This electrochemical system was used to treat real goose wastewater containing 422.5 mg/L NH4+-N and 94.5 mg/L total organic carbon (TOC). After treatment, this electrochemical system achieved good performance with an ammonia removal rate of 87%, N2 selectivity of 77%, and TOC removal rate of 72%. Therefore, this simple and efficient system with Ni foam anode and Cu foam cathode is a promising method for treating ammonia nitrogen wastewater.

Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700 days under anoxic condition. The synthetic wastewater containing 0.382 g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

Highly Selective Ammonia Oxidation on BiVO4 Photoanodes Co-catalyzed by Trace Amounts of Copper Ions.

High-efficient photoelectrocatalytic direct ammonia oxidation reaction (AOR) conducted on semiconductor photoanodes remains a substantial challenge. Herein, we develop a strategy of simply introducing ppm levels of Cu ions (0.5-10 mg/L) into NH3 solutions to significantly improve the AOR photocurrent of bare BiVO4 photoanodes from 3.4 to 6.3 mA cm-2 at 1.23 VRHE , being close to the theoretical maximum photocurrent of BiVO4 (7.5 mA cm-2 ). The surface charge-separation efficiency has reached 90 % under a low bias of 0.8 VRHE . This AOR exhibits a high Faradaic efficiency (FE) of 93.8 % with the water oxidation reaction (WOR) being greatly suppressed. N2 is the main AOR product with FEs of 71.1 % in aqueous solutions and FEs of 100 % in non-aqueous solutions. Through mechanistic studies, we find that the formation of Cu-NH3 complexes possesses preferential adsorption on BiVO4 surfaces and efficiently competes with WOR. Meanwhile, the cooperation of BiVO4 surface effect and Cu-induced coordination effect activates N-H bonds and accelerates the first rate-limiting proton-coupled electron transfer for AOR. This simple strategy is further extended to other photoanodes and electrocatalysts.

Subsurface Single-atom Catalyst Enabled by Mechanochemical Synthesis for Oxidation Chemistry.

Single-atom catalysts have garnered significant attention due to their exceptional atom utilization and unique properties. However, the practical application of these catalysts is often impeded by challenges such as sintering-induced instability and poisoning of isolated atoms due to strong gas adsorption. In this study, we employed the mechanochemical method to insert single Cu atoms into the subsurface of Fe2O3 support. By manipulating the location of single atoms at the surface or subsurface, catalysts with distinct adsorption properties and reaction mechanisms can be achieved. It was observed that the subsurface Cu single atoms in Fe2O3 remained isolated under both oxidation and reduction environments, whereas surface Cu single atoms on Fe2O3 experienced sintering under reduction conditions. The unique properties of these subsurface single-atom catalysts call for innovations and new understandings in catalyst design.

Gas Diffusion Electrodes for Electrocatalytic Oxidation of Gaseous Ammonia: Stepping Over the Nitrogen Energy Canyon.

As ammonia continues to gain more and more interest as a promising hydrogen carrier compound, so does the electrochemical ammonia oxidation reaction (AmOR). To avoid the liberation of H2 in a reverse Haber-Bosch reaction under release of the energetically more favorable N2, we propose the oxidation of ammonia to value-added nitrite (NO2 -), which is usually obtained during the Ostwald process. We investigated the anodic oxidation of gaseous ammonia directly supplied to a gas diffusion electrode (GDE) using a variety of compositionally different multi-metal catalysts coated on Ni foam under the simultaneous formation of H2 at the cathode. This will double the amount of H2 per ammonia molecule while applying a lower overpotential than that required for water electrolysis (1.4-1.8 V vs. RHE at 50 mA ⋅ cm-2). A selectivity study demonstrated that some of the catalyst compositions were able to produce significant amounts of NO2 -, and further investigations using the most promising catalyst composition Nif_AlCoCrCuFe integrated within a GDE demonstrated up to 88 % Faradaic efficiency for NO2 - at the anode coupled to close to 100 % Faradaic efficiency for the cathodic H2 production.

Oxidation of Ammonia Catalyzed by a Molecular Iron Complex: Translating Chemical Catalysis to Mediated Electrocatalysis.

Ammonia is a promising candidate in the quest for sustainable, clean energy. With its capacity to serve as an energy carrier, the oxidation of ammonia opens avenues for carbon-neutral approaches to address worldwide growing energy needs. We report the catalytic chemical oxidation of ammonia by an Earth-abundant transition metal complex, trans-[LFeII(MeCN)2][PF6]2, where L is a macrocyclic ligand bearing four N-heterocyclic carbene (NHC) donors. Using triarylaminium radical cations in MeCN, up to 182 turnovers of N2 per Fe were obtained from chemical catalysis with an extremely low loading of the Fe catalyst (0.043 mM, 0.004 mol % catalyst). This chemical catalysis was successfully transitioned to mediated electrocatalysis for the oxidation of ammonia. Molecular electrocatalysis by the Fe catalyst and the mediator (p-MeOC6H4)3N exhibited a catalytic half-wave potential (Ecat/2) of 0.18 V vs [Cp2Fe]+/0 in MeCN, and achieved 9.3 turnovers of N2 at an applied potential of 0.20 V vs [Cp2Fe]+/0 at -20 °C in controlled-potential electrolysis, with a Faradaic efficiency of 75 %. Based on computational results, the catalyst undergoes sequential oxidation and deprotonation steps to form [LFeIV(NH2)2]2+, and thereafter bimetallic coupling to form an N-N bond.

Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

Ecophysiological and Genomic Characterization of the Freshwater Complete Ammonia Oxidizer Nitrospira sp. Strain BO4.

Complete ammonia oxidizers (comammox) are a group of ubiquitous chemolithoautotrophic bacteria capable of deriving energy from the oxidation of ammonia to nitrate via nitrite. Here, we present a study characterizing the comammox strain Nitrospira sp. BO4 using a combination of cultivation-dependent and molecular methods. The enrichment culture BO4 was obtained from the sediment of Lake Burr Oak, a mesotrophic lake in eastern Ohio. The metagenome of the enrichment culture was sequenced, and a metagenome-assembled genome (MAG) was constructed for Nitrospira sp. BO4. The closest characterized relative of Nitrospira sp. BO4 was "Candidatus Nitrospira kreftii." All genes for ammonia and nitrite oxidation, reductive tricarboxylic acid (TCA) cycle, and other pathways of the central metabolism were detected. Nitrospira sp. BO4 used ammonia and oxidized it to nitrate with nitrite as the intermediate. The culture grew on initial ammonium concentrations between 0.01 and 3 mM with the highest rates observed at the lowest ammonium concentrations. Blue light completely inhibited the growth of Nitrospira sp. BO4, while white light reduced the growth and red light had no effect on the growth. Nitrospira sp. BO4 did not grow on nitrite as its sole substrate. When supplied with ammonium and nitrite, the culture utilized nitrite after most of the ammonium was consumed. In summary, the genomic information of Nitrospira sp. BO4 coupled with the growth experiments shows that Nitrospira sp. BO4 is a freshwater comammox species. Future research will focus on further characterization of the niches of comammox in freshwater environments. IMPORTANCE Nitrification is a key process in the global nitrogen cycle. Complete ammonia oxidizers (comammox) were discovered recently, and only three enrichment cultures and one pure culture have been characterized with respect to activity and growth under different conditions. The cultivated comammox strains were obtained from engineered systems such as a recirculating aquaculture system and hot water pipes. Here, we present the first study characterizing a comammox strain obtained from a mesotrophic freshwater lake. In freshwater environments, comammox coexist with ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our results will help elucidate physiological characteristics of comammox and the distribution and niche differentiation of different ammonia oxidizers in freshwater environments.

Comammox Nitrospira and Ammonia-Oxidizing Archaea Are Dominant Ammonia Oxidizers in Sediments of an Acid Mine Lake Containing High Ammonium Concentrations.

Exploring nitrifiers in extreme environments is vital to expanding our understanding of nitrogen cycle and microbial diversity. This study presents that complete ammonia oxidation (comammox) Nitrospira, together with acidophilic ammonia-oxidizing archaea (AOA), dominate in the nitrifying guild in sediments of an acid mine lake (AML). The lake water was characterized by acidic pH below 5 with a high ammonium concentration of 175 mg-N/liter, which is rare on the earth. Nitrification was active in sediments with a maximum nitrate production potential of 70.5 µg-N/(g-dry weight [dw] day) for mixed sediments. Quantitative PCR assays determined that in AML sediments, comammox Nitrospira and AOA amoA genes had relative abundances of 52% and 41%, respectively, among the total amoA genes. Further assays with 16S rRNA and amoA gene amplicon sequencing and metagenomics confirmed their dominance and revealed that the comammox Nitrospira found in sediments belonged to comammox Nitrospira clade A.2. Metagenomic binning retrieved a metagenome-assembled genome (MAG) of the comammox Nitrospira from sediments (completeness = 96.76%), and phylogenomic analysis suggested that it was a novel comammox Nitrospira. Comparative genomic investigation revealed that this comammox Nitrospira contained diverse metal resistance genes and an acidophile-affiliated F-type ATPase. Moreover, it had a more diverse genomic characteristic on nitrogen metabolism than the AOA in sediments and canonical AOB did. The results suggest that comammox Nitrospira is a versatile nitrifier that can adapt to acidic environments even with high ammonium concentrations. IMPORTANCE Ammonia-oxidizing archaea (AOA) was previously considered the sole dominant ammonia oxidizer in acidic environments. This study, however, found that complete ammonia oxidation (comammox) Nitrospira was also a dominant ammonia oxidizer in the sediments of an acidic mine lake, which had an acidic pH < 5 and a high ammonium concentration of 175 mg-N/liter. In combination with average nucleotide identity analysis, phylogenomic analysis suggested it is a novel strain of comammox Nitrospira. Moreover, the adaption of comammox Nitrospira to the acidic lake had been comprehensively investigated based on genome-centric metagenomic approaches. The outcomes of this study significantly expand our understanding of the diversity and adaptability of ammonia oxidizers in the acidic environments.