Complete Ammonia Oxidation Research Articles

Effect of long-term ammonia deficiency on nitrification performance and microbial dynamics in downflow sponge biofilm reactor

The downflow sponge biofilm (DSB) reactor has a promising nitrification capacity for ammonium (NH4+-N)-polluted raw water treatment. However, the highly fluctuating NH4+-N levels in raw water may pose a challenge to a stable nitrification process. It is unclear how the DSB reactor contends with NH4+-N deficiency. This study evaluated the robustness of a DSB reactor under long-term NH4+-N abundant and NH4+-N deficient conditions. Four NH4+-N deficient periods were investigated, comprising two 14-day partial NH4+-N deficient phases, one 56-day phase involving both partial and complete NH4+-N deficient, and one 90-day complete NH4+-N deficient phase. Repeated 14-day partial NH4+-N deficient phases had no impact on nitrification performance. However, shifting from partial to complete NH4+-N deficient condition resulted in a delay in NOx−-N production when NH4+-N was replenished. Following an additional 90 days of NH4+-N deficient phase, NH4+-N removal performance recovered faster than nitrite (NO2−-N) removal performance, which took 5 and 29 days, respectively. Furthermore, long-term NH4+-N deficiency resulted in a nominal loss of biomass in the DSB reactor. These findings suggested that the nitrification capacity of the DSB reactor is robust when subjected to long-term NH4+-N deficient conditions. The 16S rRNA amplicon sequencing results revealed that the growth of Nitrosomonas was suppressed, but Candidatus Nitrosotenuis predominated in NH4+-N deficient conditions, while Nitrospira persisted throughout the study. The observed persistence of Nitrospira may be attributed to their diverse metabolic capabilities, which include mixotrophy and complete ammonia oxidation metabolisms. Collectively, the co-existence of Ca. Nitrosotenius, Nitrosomonas, and Nitrospira contributed to stable NH4+-N removal performance in the robust DSB reactor. The DSB reactor shows promise for use in raw water treatment with highly fluctuating NH4+-N availability.

Shore-to-water spatial variations of complete ammonia oxidizers in a lake in Wuhan, China

Complete ammonia oxidizers (comammox bacteria) can convert ammonia into nitric acid through single-step nitrification. This study explored the spatial variations of comammox bacteria in the lakeshore area of the Houguan Lake in Wuhan, China. The abundance of the two comammox bacteria clades and two traditional ammonia-oxidizing microorganisms, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), generally showed a gradually decreasing trend from the shore to the water. Moreover, a similar decreasing trend was observed for the respective and total nitrification rate of three types of ammonia-oxidizing microorganisms. The average nitrification rate of AOA, AOB and comammox bacteria was 0.568, 0.718, and 0.935 mg N kg−1 d−1, respectively. Besides, comammox bacteria exhibited a high biological diversity, with clade A and clade B and three subclades of clade A all present. Among different clades, clade B played a dominant role in the ammonia oxidation process. Both the abundance and nitrification rate of comammox bacteria were significantly positively correlated with total carbon and total nitrogen, indicating that these two nutrient substances are important factors influencing this microorganism. Our results demonstrate that the spatial variations of environmental elements in the lakeshore area lead to gradual decreases of comammox bacteria from the shore to the water.

Comprehensive evaluation of primer pairs targeting the ammonia monooxygenase subunit A gene of complete ammonia-oxidizing Nitrospira.

Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.

Comammox rather than AOB dominated the efficient autotrophic nitrification-denitrification process in an extremely oxygen-limited environment

The discovery of complete ammonia oxidizer (comammox) has challenged the traditional understanding of the two-step nitrification process. However, their functions in the oxygen-limited autotrophic nitrification-denitrification (OLAND) process remain unclear. In this study, OLAND was achieved using comammox-dominated nitrifying bacteria in an extremely oxygen-limited environment with a dissolved oxygen concentrations of 0.05 mg/L. The ammonia removal efficiency exceeded 97 %, and the total nitrogen removal efficiency reached 71 % when sodium bicarbonate was used as the carbon source. The pseudo-first- and second-order models were found to best fit the ammonia removal processes under low and high loads, respectively, suggesting distinct ammonia removal pathways. Full-length 16S rRNA gene sequencing and metagenomic results revealed that comammox-dominated under different oxygen levels, in conjunction with anammox and heterotrophic denitrifiers. The abundance of enzymes involved in energy metabolism indicates the coexistence of anammox and autotrophic nitrification-heterotrophic denitrification pathways. The binning results showed that comammox bacteria engaged in horizontal gene transfer with nitrifiers, anammox bacteria, and denitrifiers to adapt to an obligate environments. Therefore, this study demonstrated that comammox, anammox, and heterotrophic denitrifiers play important roles in the OLAND process and provide a reference for further reducing aeration energy in the autotrophic nitrogen removal process.

Fabrication of Platinum-Decorated NiCo-Layered Double Hydroxide Nanoflowers for Electrocatalytic Ammonia Oxidation Reaction

The complete anodic oxidation of ammonia is an important part of direct ammonia fuel cells. Fabricating a high-performance electrocatalyst for ammonia oxidation reaction is meaningful for developing a direct ammonia fuel cell. Herein, we designed one platinum-decorated NiCo-layered double hydroxide nanoflower on Ni foam (Pt-NiCo-LDH-Ni foam) and measured the electrocatalytic performance via the cyclic voltammetry (CV) technique. The experimental results demonstrated that the optimized Pt-NiCo-LDH-Ni foam showed great electrocatalytic performance, with a low overpotential with a value of −0.573 V, a high current density of 17.75 mA cm−2 for the ammonia oxidation reaction, and good stability.

Growth of complete ammonia oxidizers on guanidine

Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1–3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.

Soil texture contributes to shaping comammox Nitrospira communities in rice-wheat rotation soils

Complete ammonia oxidizers (comammox Nitrospira) are intriguing discoveries that mark a significant milestone in the global nitrogen cycle. While numerous soil physiochemical variables have been identified as key influencers of comammox Nitrospira distribution, the role of soil texture in shaping these communities remains largely uncertain. Here, we explored the diversity, community structure of comammox Nitrospira, and their driving factors, including soil texture in 237 rice-wheat rotation soils. The results indicated that soil pH and texture were the primary factors influencing the Shannon diversity and richness of comammox Nitrospira. Comammox Nitrospira Shannon diversity and richness were positively associated with soil pH and silt content, but negatively correlated with clay content, suggesting that finer-textured soils harbored lower comammox Nitrospira diversity. Additionally, silt content emerged as the second most influential factor, after pH, shaping comammox Nitrospira community structure. Clade A.2 was found as the predominant comammox Nitrospira clade in rice-wheat rotation soils, representing 59.3 % of the total sequences. Clade A.2 exhibited a positive correlation with sand and clay contents but a negative association with silt content. Conversely, Clades A.3 and B demonstrated the opposite pattern. Overall, our study underscores the critical role of soil texture as a mediator of comammox Nitrospira diversity and community structure, emphasizing the need to consider soil texture in investigations of comammox Nitrospira.

Response of comammox Nitrospira clades A and B communities to long-term fertilization and rhizosphere effects and their relative contribution to nitrification in a subtropical paddy field of China

The recently discovered complete ammonia oxidation (comammox Nitrospira) containing clade A and clade B has further complemented our understanding of nitrification process. Nevertheless, understanding the community feature of comammox Nitrospira clades A and B and their relative contribution to nitrification in paddy rhizosphere are still in its infancy. In this study, we assessed the community diversity and structure of comammox Nitrospira clades A and B in paddy rhizosphere and bulk soils under thirty years of different fertilization strategies, i.e., non-fertilization control (CK), chemical fertilizers application (NPK), and NPK plus swine manure (NPKM), respectively. NPKM significantly increased the a-diversity (Chao1 and Shannon indices) of comammox Nitrospira clade A and altered the community structure (P < 0.05) but had little effect on clade B. A two-way analysis of variance (ANOVA) showed that the effect of long-term fertilization on soil comammox Nitrospira community and nitrification potential rate (PNR) was much greater than that of rhizosphere. Compared with NPK, soil PNR was greatly increased by 51.0% under the NPKM treatment in the rhizosphere (P < 0.05). Phylogenetic analysis showed that NPKM improved the relative abundances of sub-clade A.2.1 and sub-clade A.3.2 of the comammox clade A community, with an average increase of 212.2 and 210.4% in both rhizosphere and bulk soils relative to the NPK treatment. Soil organic matter, NH4+-N, and pH were significant soil drivers of comammox Nitrospira clades A and B community. Furthermore, linear regression and structural equation modeling clearly showed that comammox Nitrospira clade A a-diversity were significantly associated with soil PNR (P < 0.05). Our results suggest (i) that comammox Nitrospira clade A are sensitive to the organic fertilization; and (ii) that comammox Nitrospira clade A contribute more to nitrification than clade B under the long-term organic fertilized paddy soil.

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

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.

Long-term addition of organic manure stimulates the growth and activity of comammox in a subtropical Inceptisol

The discovery of complete ammonia oxidizers (comammox) has dramatically altered our perception of nitrogen (N) biogeochemistry. However, their functional importance vs. the canonical ammonia oxidizers (i.e., ammonia oxidizing-archaea (AOA) and bacteria (AOB)) in agroecosystems is still poorly understood. Accordingly, a new assay using acetylene, 3,4-dimethylpyrazole phosphate (DMPP), and 1-octyne was adopted to assess the ammonia (NH3) oxidation and nitrous oxide (N2O) production activity of these functional guilds in a subtropical Inceptisol under long-term different fertilization regimes. These regimes include CK (no fertilizer control), synthetic fertilizer only (NPK), organic manure only (M) and organic manure plus synthetic fertilizer (MNPK). AOA dominated NH3 oxidation in the M treatment, while AOB dominated both NH3 oxidation and N2O production in all treatments except M. Comammox always played a minor role in both NH3 oxidation and N2O production across all treatments. Both M and MNPK treatments significantly increased the activity and growth of comammox. Compared to NPK, comammox exhibited increases of 270 % and 326 % in the NH3 oxidation rates, and increases of 1472 % and 563 % in the N2O production rates in M and MNPK, respectively. Random forest model revealed that copper (Cu), comammox abundance, and dissolved organic nitrogen (DON) were the most important predictors for the NH3 oxidation rates of comammox. Redundancy analyses (RDA) showed that fertilizer treatments significantly altered the community composition of NH3 oxidizers, and pH was the overarching parameter underpinning the community shift of the NH3 oxidizers. Overall, this study provides evidence that comammox play a minor yet unneglectable role in the nitrification of agroecosystems, and the long-term addition of organic manure stimulates the growth and activity of comammox in a subtropical Inceptisol.

Evidence of comammox bacteria playing a dominant role in Lake Taihu sediments based on metagenomic analysis

The nitrification process plays an important role in the nitrogen cycle, in which the ammonia-oxidation process mediated by microorganisms is the rate-limiting step. Environmental factors can affect the distribution and activity of ammonia-oxidizing microorganisms (AOM), including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and complete ammonia oxidizer (comammox Nitrospira). At present, most studies have used amoA as a marker gene for the ammonia oxidation process to analyze the differences among AOM community composition and abundance in the environment. In this study, metagenomic sequencing was used to study the differences in community composition and functional gene distribution of nitrifying microorganisms in the sediments of Lake Taihu with different eutrophication levels. It was found that comammox Nitrospira and typical nitrite oxidizer NOB Nitrospira, which belong to Nitrospirota, had higher relative abundance at most sites compared to AOB and AOA. Furthermore, the network analysis of genes related to nitrogen cycle showed that the main survival mode of nitrogen metabolizing microorganisms was mutualism. Besides, the microbial genomes in the sediments of Lake Taihu were reconstructed by metagenomic binning, which showed that among the 167 bins obtained, 2 bins (bin 9 and bin 32) were annotated as comammox Nitrospira, and had high abundance in the macrophytes-dominated lakes (such as South Lake Taihu and West Coast). In addition, bin 9, which belongs to comammox Nitrospira, annotates amoA genes associated with ammonia oxidation, and other genes associated with urea decomposition and transport, suggesting functional diversity. Overall, these findings suggest that AOM have different distribution characteristics, among which comammox Nitrospira has high diversity and may be potentially dominant in macrophytes-dominated lakes.

High-rate pig manure substitution enhances comammox Nitrospira abundance and diversity in the Cinnamomum camphora coppice planting soils

Comammox Nitrospira represents a groundbreaking discovery in nitrogen cycle research, showcasing its remarking ability for complete ammonia oxidation, which challenges prior conceptions of nitrification. In this study, we examined the response of comammox Nitrospira gene abundance, diversity, and community structure to different rates of pig manure substitution (0 %, 25 %, 50 %, 75 %, and 100 %) in subtropical agroforestry soils. The abundance of ammonia-oxidizing microorganisms was assessed by qPCR, whereas the diversity and structure of comammox Nitrospira were determined by high-throughput sequencing. Our findings revealed that pig manure substitution led to an increase in soil pH, available phosphorus (AP), comammox Nitrospira abundance, and diversity within soils under Cinnamomum camphora coppice planting. Soil pH and AP were the primary factors influencing the diversity and community structure of comammox Nitrospira. Moreover, pig manure substitution significantly influenced the composition of comammox Nitrospira, notably by increasing the relative abundance of clade A.2.1 while reducing that of clade A.2.2. However, pig manure substitution did not exert a significant impact on net nitrification rates, suggesting bacterial relative abundances were more sensitive to manure substitution compared to the underlying biogeochemical processes. Overall, our results offer new insights into the response of comammox Nitrospira to different rates of pig manure substitution in Cinnamomum camphora coppice planting soils, highlighting the pivotal role of soil AP and pH as the key determinants shaping comammox Nitrospira diversity and community structure.

Electrochemically regulating COD/TN ratio of aged landfill leachate via rapid chlorine-mediated ammonia oxidation with RuO2-IrO2/Ti anode

Electrochemical oxidation process was employed to regulate the ratio of chemical oxygen demand to total nitrogen (COD/TN ratio) of aged landfill leachate, aiming to meet the requirement of biological treatment. The results indicate that the ammonia nitrogen can be rapidly removed through electrogenerated chlorine radicals over a typical chlorine evolution RuO2-IrO2/Ti anode. The increased input current and decreased flow rate are beneficial to COD/TN ratio improvement, and COD/TN ratio can be increased to more than 16 at 1000 mA (input current) and 0.3 L/h (flow rate). According to the obtained experimental data, we present a strategy for COD/TN ratio increase: (i) determining the COD/TN ratio balance coefficient (B) of aged landfill leachate, and selecting the anode with COD/TN ratio adjustment coefficient (TN) >B, where TN represents the ability to regulate COD/TN ratio, and B represents the specific TN to keep the same COD/TN ratio; (ii) performing the wastewater treatment experiments at different control parameters during electrochemical process; (iii) calculating the k of control parameter sensitivity curves to achieve different tolerance abilities, and further preferentially regulating the reaction process to high average current efficiency (ACE) of ammonia nitrogen and low ACE of COD. This work validates that the electrochemical process can increase the COD/TN ratio together with enhancing the biodegradability of such refractory wastewater, and also provides an effective tool for regulation of the COD/TN ratio via complete ammonia oxidation.

15N-DNA stable isotope probing reveals niche differentiation of ammonia oxidizers in paddy soils

Chemoautotrophic canonical ammonia oxidizers (ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB)) and complete ammonia oxidizers (comammox Nitrospira) are accountable for ammonia oxidation, which is a fundamental process of nitrification in terrestrial ecosystems. However, the relationship between autotrophic nitrification and the active nitrifying populations during 15N-urea incubation has not been totally clarified. The 15N-labeled DNA stable isotope probing (DNA-SIP) technique was utilized in order to study the response from the soil nitrification process and the active nitrifying populations, in both acidic and neutral paddy soils, to the application of urea. The presence of C2H2 almost completely inhibited NO3−-N production, indicating that autotrophic ammonia oxidation was dominant in both paddy soils. 15N-DNA-SIP technology could effectively distinguish active nitrifying populations in both soils. The active ammonia oxidation groups in both soils were significantly different, AOA (NS (Nitrososphaerales)-Alpha, NS-Gamma, NS-Beta, NS-Delta, NS-Zeta and NT (Ca. Nitrosotaleales)-Alpha), and AOB (Nitrosospira) were functionally active in the acidic paddy soil, whereas comammox Nitrospira clade A and Nitrosospira AOB were functionally active in the neutral paddy soil. This study highlights the effective discriminative effect of 15N-DNA-SIP and niche differentiation of nitrifying populations in these paddy soils.Key points• 15N-DNA-SIP technology could effectively distinguish active ammonia oxidizers.• Comammox Nitrospira clade A plays a lesser role than canonical ammonia oxidizers.• The active groups in the acidic and neutral paddy soils were significantly different.Graphical

The cooperative interaction of AOB and comammox clade A drives nitrification and N2O emissions in a long-term organic fertilized paddy soil

The newly discovered complete ammonia oxidizer (comammox Nitrospira) is able to single-step nitrification capability, and increased our understanding of soil nitrogen cycling. However, the response of comammox and ammonia-oxidizing bacteria (AOB) and archaea (AOA) to long-term fertilization and rhizosphere effects in paddy soils and their relative contribution to the nitrification-derived N2O emissions is still unclear. Here, we collected rhizosphere and bulk soils with thirty years of different fertilization strategies, i.e., non-fertilization (CK), chemical N, P, and K application (NPK), and NPK plus pig manure application (NPKM), respectively, to test changes in nitrification potential rate (PNR), N2O emission fluxes, abundance of ammonia oxidizers and their significant drivers. The result showed that NPKM significantly increased soil C and N levels, the proportion of soil middle-size particles (40.35–148.00 μm class), and soil PNR, but decreased soil N2O emissions, especially in the drying time of paddy (P < 0.05), compared to NPK fertilization. NPKM had the highest values of AOA, AOB, and comammox clade A amoA gene copy numbers (P < 0.05), but clade B was increased by the NPK in the rhizosphere soil. Furthermore, fertilization showed greater effects on ammonia oxidizers (except for clade B) than the rhizosphere effect. Mantel test showed that SOM, TP, pH, NH4+, and NO3− were main abiotic factors causing the niche separation among ammonia oxidizers. Linear regression analysis and structural equation model (SEM) showed that both PNR and N2O emission fluxes were significantly associated with the abundance of AOB and comammox clade A (P < 0.05). Therefore, our results underline the importance of AOB together with comammox clade A in nitrification and N2O production in long-term organic fertilized paddy fields, which could provide new ideas for the mitigation of N2O emission by adopting organic fertilization scenarios in Chinese paddy fields.

Lithology-driven soil properties control of N2O production by ammonia oxidizers in subtropical forest soils

Lithology can strongly influence soil's physical and chemical properties, significantly affecting soil nitrogen (N) transformation rates. Ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and complete ammonia oxidizers (comammox Nitrospira) constitute the major producers of soil nitrous oxide (N2O), but the importance of different lithology on their relative contributions still poorly understood, especially in terms of newly discovered and widespread comammox. To address this knowledge gap, three nitrification inhibitors, acetylene (ammonia oxidizers inhibitor), 1-octyne (AOB inhibitor) and 3,4-dimethylpyrazole phosphate (DMPP) (both AOB and comammox inhibitor), were used to selectively inhibit the activity of different ammonia oxidation groups in order to discriminate their respective roles in N2O production in forest soils developed on different lithologies (limestone and clastic rock). The results showed that AOB dominated N2O production (63.5 ± 3.3 %) in limestone soils with ammonium amendment, followed by AOA (34.5 ± 7.6 %), while comammox contributed 1 % of total N2O production with N2O yield of 0.03 ± 0.04 %. Higher N2O production by AOB in limestone soils is associated with higher soil pH, exchangeable calcium/magnesium and lower short-range order iron/aluminum minerals. However, the N2O yield by comammox (0.12 ± 0.12 %) in clastic rock soils was comparable to that by AOA (0.15 ± 0.14 %) and AOB (0.20 ± 0.13 %). Based on the results of the correlation analysis, comammox in the clastic rock soils produce greater amounts of N2O is closely related to the higher soil organic carbon and available phosphorus. The present study provided evidence that the lithology-driven soil properties markedly affect the N2O production from different ammonia oxidizers.

Complete ammonia oxidation (comammox) at pH 3–4 supports stable production of ammonium nitrate from urine

This study developed a new process that stably produced ammonium nitrate (NH4NO3), an important and commonly used fertilizer, from the source-separated urine by comammox Nitrospira. In the first stage, the complete conversion of ammonium to nitrate was achieved by comammox Nitrospira. In this scenario, the pH was maintained at 6 by adding external alkali, which also provided sufficient alkalinity for full nitrification. In the second stage, the NH4NO3 was produced directly by comammox Nitropsira by converting half of the ammonium in urine into nitrate. In this case, no alkali was added and pH automatically dropped and self-maintained at an extremely acidic level (pH 3–4). In both scenarios, negligible nitrite accumulation was observed, while the final product of the second stage contained ammonium and nitrate at the molar ratio of 1:1. The dominance of comammox Nitrospira over canonical ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was systematically proved by the combination of 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction, and metagenomics. Notably, metagenomic sequencing suggested that the relative abundance of comammox Nitrospira was over 20 % under the acidic condition at pH 3–4, while canonical AOB and NOB were undetectable. Batch experiments showed that the optimal pH for the enriched comammox Nitrospira was ∼7, which could sustain their activity in a wider pH range from 4 to 8 surprisingly but lost activity at pH 3 and 9. The findings not only present an application potential of comammox Nitrospira in nitrogen recovery from urine wastewater but also report the survivability of comammox bacteria in acidic environments.

Unraveling the important role of comammox Nitrospira to nitrification in the coastal aquaculture system.

Increasing nitrogen (N) input to coastal ecosystems poses a serious environmental threat. It is important to understand the responses and feedback of N removal microbial communities, particularly nitrifiers including the newly recognized complete ammonia-oxidizers (comammox), to improve aquaculture sustainability. In this study, we conducted a holistic evaluation of the functional communities responsible for nitrification by quantifying and sequencing the key functional genes of comammox Nitrospira-amoA, AOA-amoA, AOB-amoA and Nitrospira-nxrB in fish ponds with different fish feeding levels and evaluated the contribution of nitrifiers in the nitrification process through experiments of mixing pure cultures. We found that higher fish feeding dramatically increased N-related concentration, affecting the nitrifying communities. Compared to AOA and AOB, comammox Nitrospira and NOB were more sensitive to environmental changes. Unexpectedly, we detected an equivalent abundance of comammox Nitrospira and AOB and observed an increase in the proportion of clade A in comammox Nitrospira with the increase in fish feeding. Furthermore, a simplified network and shift of keystone species from NOB to comammox Nitrospira were observed in higher fish-feeding ponds. Random forest analysis suggested that the comammox Nitrospira community played a critical role in the nitrification of eutrophic aquaculture ponds (40-70 μM). Through the additional experiment of mixing nitrifying pure cultures, we found that comammox Nitrospira is the primary contributor to the nitrification process at 200 μM ammonium. These results advance our understanding of nitrifying communities and highlight the importance of comammox Nitrospira in driving nitrification in eutrophic aquaculture systems.

Nitrification inhibitors impose distinct effects on comammox bacteria and canonical ammonia oxidizers under high N fertilization regimes

The response of complete ammonia-oxidizing (comammox) bacteria to variable nitrogen (N) inputs and nitrification inhibitors (NIs) remains poorly understood. We conducted two experiments, in a soil where both clade A and B comammox Nitrospira were present, to identify the type (ammonium sulphate vs. urea) and the level of N-fertilization (0–500 mg-N-kg‐1d.w. soil) that are conducive to the growth of comammox, compared to canonical ammonia-oxidizing microorganisms (AOM), and subsequently to assess their response to three commercial (DCD, nitrapyrin, DMPP) and a new potent (ethoxyquin) NI under these favourable N fertilization regimes. The highest concentration of ammonium sulphate stimulated comammox compared to canonical AOM which showed a dose-dependent increase by both N sources. DMPP primarily, and DCD, imposed a more persistent inhibition on nitrification than nitrapyrin and ethoxyquin, a result which did not concur with the soil persistence of NIs (DMPP was the least persistent among the commercial NIs) but was mostly driven by their selective inhibitory activity on ammonia-oxidizing bacteria (AOB) that prevailed in the studied soil. The inhibition of AOB by DMPP and DCD was complemented by the proliferation of comammox and ammonia-oxidizing archaea (AOA). NIs induced significant changes on AOB and comammox communities. Comammox Nitrospira clade B were enriched by DCD and DMPP but depleted by nitrapyrin and ethoxyquin. We showed that (i) releasing comammox Nitrospira and AOA from AOB competition allows them to proliferate efficiently, even under high inorganic ammonium conditions; (ii) comammox clade B are more responsive than clade A to high inorganic N input and NIs.

Revolutionizing comammox enrichment: Novel approaches for comammox enrichment through reactor configuration and ammonia concentration mediation

The identification of comammox Nitrospira as a complete ammonia oxidizer marked a significant milestone in the field of ammonia oxidation. Nonetheless, the slow growth rate of comammox bacteria poses a challenge in differentiating them from conventional ammonia-oxidizers. In this study, different reactor configurations (SBR and SBBR) were used to explore the enrichment effect of comammox Nitrospira. The operation process of both reactors was organized into three distinct phases according to the concentration of NH4+ in the influent. With the increase in running time, the abundances of comammox bacteria in R1 (SBR) and R2 (SBBR) increased. The relative abundances of comammox amoA in Phase III of R2 were 27.9 times that of R1. After 240 days of enrichment, the abundances of Nitrospira in R1 and R2 increased from 0.03 % to 3.33 % and 22.79 %, respectively. The enrichment in R2 was about 6.8-fold of R1. The results showed that comammox Nitrospira is more competitive in an oligotrophic environment, and they are more favorable to grow in biofilms. This study shed new light on the impact of reactor configurations and provides a novel strategy for enriching comammox bacteria, highlighting the potential for optimizing wastewater treatment processes and advancing our understanding of microbial ecology.