Comammox Nitrospira Research Articles

Comammox Nitrospira was the dominant ammonia oxidizer in an acidic biofilm reactor at pH 5.5 and pH 5.

Nitrification is a vital process in the biological removal of inorganic nitrogen compounds. In order to ensure the stability and effectiveness of this process, buffer solutions should be added to the system to maintain neutral to slightly alkaline conditions. With a focus on the newly discovered comammox Nitrospira, this research investigates the transition of the nitrifying community within a biofilm reactor under different acidic levels (initiated at pH 6 and gradually decreased to pH 5). During the 305-day continuous operation experiment, it was observed that responsible ammonia oxidizers transitioned from ammonia-oxidizing bacteria (AOB) during the initial stages (setup stage and early stage of pH 6) to comammox Nitrospira under pH 5.5 and pH 5. Further analysis using next-generation sequencing targeting both the 16S rRNA region and amoA region revealed a shift in the dominant cluster of both Nitrospirae and comammox Nitrospira under varying pH conditions. Our study identified a distinct cluster of comammox Nitrospira that is phylogenetically closed to sequences found in acidic environments, but exhibits dissimilarity from known comammox Nitrospira isolates and the majority of environmental sequences. This cluster was found to be prevalent in the acidic biofilm reactor studied and thrived particularly well at pH 5. These findings underscore the potential significance of this distinct, uncultivated group of comammox Nitrospira in performing ammonia oxidation under acidic conditions. KEY POINTS: • Ammonia was effectively removed under pH 5.5 and 5 in the biofilm reactor • The dominant ammonia oxidizer was comammox Nitrospira when pH was 5.5 and 5 • A potential acidophilic cluster of comammox Nitrospira was identified in this acidic biofilm reactor.

Abundance, Diversity, and Community Composition of Comammox Nitrospira in Tea (Camellia Sinensis) Plantation Soils

Abundance, Diversity, and Community Composition of Comammox Nitrospira in Tea (Camellia Sinensis) Plantation Soils

Temporal enrichment of comammox Nitrospira and Ca. Nitrosocosmicus in a coastal plastisphere.

Plastic marine debris is known to harbor a unique microbiome (termed the "plastisphere") that can be important in marine biogeochemical cycles. However, the temporal dynamics in the plastisphere and their implications for marine biogeochemistry remain poorly understood. Here, we characterized the temporal dynamics of nitrifying communities in the plastisphere of plastic ropes exposed to a mangrove intertidal zone. The 39-month colonization experiment revealed that the relative abundances of Nitrospira and Candidatus Nitrosocosmicus representatives increased over time according to 16S rRNA gene amplicon sequencing analysis. The relative abundances of amoA genes in metagenomes implied that comammox Nitrospira were the dominant ammonia oxidizers in the plastisphere, and their dominance increased over time. The relative abundances of two metagenome-assembled genomes of comammox Nitrospira also increased with time and positively correlated with extracellular polymeric substances content of the plastisphere but negatively correlated with NH4+ concentration in seawater, indicating the long-term succession of these two parameters significantly influenced the ammonia-oxidizing community in the coastal plastisphere. At the end of the colonization experiment, the plastisphere exhibited high nitrification activity, leading to the release of N2O (2.52ng N2O N g-1) in a 3-day nitrification experiment. The predicted relative contribution of comammox Nitrospira to N2O production (17.9%) was higher than that of ammonia-oxidizing bacteria (4.8%) but lower than that of ammonia-oxidizing archaea (21.4%). These results provide evidence that from a long-term perspective, some coastal plastispheres will become dominated by comammox Nitrospira and thereby act as hotspots of ammonia oxidation and N2O production.

Lack of inhibitory effects of 1-Octyne and PTIO on ammonia oxidizers, nitrite oxidizers, and nitrate formation in acidic paddy soils

The feasibility of employing double inhibitors consisting of PTIO and 1-octyne with the 15N pool dilution technique to distinguish comammox process from archaeal and bacterial nitrification remains to be confirmed. As such, we carried out a nitrification inhibition experiment on acidic paddy soils at a pH of 5.0, 5.3, and 5.4 to investigate the effects of 1-octyne and PTIO on nitrate (NO3−) formation, ammonia (NH3)-oxidizing archaea (AOA) and bacteria (AOB), comammox Nitrospira, and nitrite (NO2−)-oxidizing bacteria (NOB) under urea fertilized- and unfertilized- conditions. We observed that 1-Octyne stimulated the growth of comammox Nitrospira clades A and B, and Nitrobacter-like NOB and Nitrospira-like NOB without inhibiting AOB growth. This observation indicates that 1-Octyne has the potential to promote comammox process and NO2− oxidization, and its usage may lead to an underestimation of AOB activity. Unexpectedly, we found that PTIO was incapable of causing a rapid inhibition of AOA growth and NO3− formation. In the short term, PTIO was usually not detrimental to the growth of comammox Nitrospira clades A and B, AOB, and Nitrobacter-like NOB. Instead, increased growth of AOB and Nitrospira-like NOB were observed following its application. Likely due to increased bacterial nitrification and NO2− oxidation and the reaction of PTIO with nitric oxide (NO) produced by non-nitrification processes, increased NO3− formation with PTIO was observable in acidic soils with varying N statuses. These findings highlight the need to develop novel and more efficient inhibition strategies to actualize rate distinction among AOA, AOB, comammox Nitrospira in acidic soils.

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.

Organic fertilizer significantly mitigates N2O emissions while increase contributed of comammox Nitrospira in paddy soils

Nitrification is the dominant process for nitrous oxide (N2O) production under aerobic conditions, but the relative contribution of the autotrophic nitrifiers (the ammonia-oxidising archaea (AOA), the ammonia-oxidising bacteria (AOB) and the comammox) to this process is still unclear in some soil types. This is particularly the case in paddy soils under different fertilization regimes. We investigated active nitrifiers and their contribution to nitrification and N2O production in a range of unfertilized and fertilized paddy soils, using 13CO2-DNA based stable isotope probing (SIP) technique combined with a series of specific nitrification inhibitors, including acetylene (C2H2), 3, 4-dimethylpyrazole phosphate (DMPP) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). The soils had a long-term history of fertilizer application, including chemical fertilizer only, a mixture of chemical fertilizers (70 %) and chicken manure (30 %) or a mixture of rice straw and chemical fertilizers. 13CO2-DNA-SIP and Illumina MiSeq sequencing demonstrated that comammox clades A.1 and B were active nitrifiers in all fertilized paddy soils. Inhibitor experiment showed that AOB largely contributed to nitrification activity and N2O emission in all paddy soils, while comammox contribution was more significant than AOA. Fertilization considerably altered nitrifiers' relative contribution to nitrification activity and N2O emissions. Applying organic fertilizers significantly decreased the N2O emissions but increased the contribution of comammox to the process. These findings expand the functional ecological niche of comammox, revealing their nitrification role and N2O production in other ecosystems than oligotrophic habitats.

Impact of variable hydrostatic pressure and intermittent operation on filtration performance and biofouling layer in gravity-driven membrane system for practical decentralized water supply

The gravity-driven membrane (GDM) systems offer a promising solution for decentralized drinking water supply due to its low maintenance and energy consumption. However, research on the practical application of GDM under variable hydrostatic pressure (variable load) and intermittent operation conditions, particularly in response to high-turbidity water, remains limited. This study investigates the long-term performance and characteristics of the biofouling layer of GDM under ultra-low variable hydrostatic pressure (20–60 mbar) and intermittent operation (8–12 h) conditions in practical decentralized water supply. The results indicate that the membrane flux (1.9–6.0 L∙m−2∙h−1, LMH) remained relatively stable despite variations in gravity-driven pressure (ΔP). Long-term operation of GDM can stably remove turbidity and potential pathogens from raw water. The total protein/polysaccharide ratios of extracellular polymeric substances (EPS) in the biofouling layer were below 0.50, reducing contaminant adhesion on the membrane surface. The 0.2–0.4 μm transparent exopolymer particles (TEP) fraction might be crucial for biofouling layer formation. The key species, belong to Proteobacteria and Patescibacteria, significantly alleviated membrane fouling by degrading polysaccharide and proteins in biofouling layer. Comammox Nitrospira were significantly enriched, contributing to efficient nitrification. GDM with variable load maintained a stable flux of 2.2–5.2 LMH under high-turbidity water conditions (300–1200 NTU), and simple forward flushing combined with sludge discharge can quickly restore ΔP. Overall, the variable load GDM system effectively manages membrane fouling and maintains stable filtration performance through the combined effects of variable load and intermittent operation, ensuring long-term and low-maintenance operation for decentralized water supply.

Analyzing performance and microbial mechanisms in an incineration leachate treatment after waste separation: Integrated metagenomic and metaproteomic analyses

The absence of food waste after separation poses a significant challenge to incineration leachate treatment, as it decreases the C/N ratio and COD of leachate, greatly impacting the biological treatment process. A one-year in-situ study was systematically conducted in an incineration leachate treatment plant that experienced waste separation, focusing on the variations in carbon and nitrogen removal performance as well as the involved microbial mechanism of the “anaerobic digestion (AD) + two-stage A/O” process. Results indicated that the biodegradability of leachate significantly decreased over time, with COD concentration decreasing by 10 times and the average C/N ratio decreasing from 12.3 to 1.4. The AD process was maintained stable, achieving a COD removal efficiency exceeding 92 %. The nitrification process also remained stable; while the denitrification process was significantly affected, and a nine-fold increase in external glucose addition was required to achieve a nitrogen removal efficiency of 85 %. Metagenomic analysis indicated that comammox Nitrospira (contributing 90 % to ammonia monooxygenase) occupied the dominant position over Nitrosomonas for nitrification due to the low NH4+-N concentration in A/O tanks (<35 mg/L), and Methanothrix was substituted by Methanosarcina for methanogenesis in AD unit. Metaproteomic results further elucidated that the expression of enzymes responsible for denitrification process, i.e., Nir, Nor, Nos (convert NO2− to N2), was decreased significantly, although the expression of enzymes related to glycolysis and TCA cycle were stimulated by glucose addition. The expression of Nar (convert NO3−-N to NO2−-N) remained stable, while the imbalance expression within denitrifying enzymes might have facilitated occurrence of partial denitrification, attributed to the low C/N ratio. The results prove that the function robustness and metabolic versatility were achieved in leachate treatment plant after waste separation but at the cost of the high external carbon resource addition, highlighting the urgent requirement for low-carbon nitrogen removal technologies.

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.

Distinct comammox Nitrospira in high-rate down-flow hanging sponge reactor treating municipal wastewater

A down-flow hanging sponge (DHS) reactor is a trickling filter system used for wastewater treatment, which employs sponges to retain biomass. This study assessed the process performance of a compact DHS combined with a sedimentation tank with seven phases at varying hydraulic retention times (HRT) over 500 days. The BOD of the DHS effluent was maintained at 4.0 ± 0.5 mg·L-1 for the shortest HRT 0.3 ± 0.1 h. The nitrification efficiency was considerably impacted by the reduced HRT, with NH4+-N and NO3–-N concentrations of 9.0 ± 1.2 mgN·L-1 and 2.2 ± 0.5 mgN·L-1, respectively. Nevertheless, the effluent complied with effluent discharge standards throughout the trial period. The number of comammox 16S rRNA gene copies ranged from 5.58 to 13.2 × 107 copies·mL−1, indicating that sponges biomass retained carrier can provide favorable conditions for comammox growth and could contribute to nitrification in the high-rate DHS reactor.

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.

Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Performances of full-scale biological nitrogen pre-treatment process for drinking water source: Seasonal variations and microbial community

This study investigated the performance of the full-scale unit over a two-year period to enhance nitrification efficiency and provide operational strategies. Results indicated that raw water quality from Donggan River was notably influenced by seasonal variations, particularly during dry and wet seasons, impacting the nitrification efficiency of the biological pretreatment process. Factors such as influent concentrations of ammonia and total Kjeldahl nitrogen were found to have significant effects on nitrification, with temperature and conductivity also showing correlations. The specific rate of ammonia removal was calculated to be approximately 0.1 kg-N/m3/d under the existing operational setup. Moreover, elevating dissolved oxygen levels above 4 mg/L was proposed to potentially boost ammonia oxidation based on findings from experiments conducted in lab-scale bioreactors. In times of increased influent ammonia levels, the elimination of about 1–3 mg-N/L of total nitrogen signified the activation of denitrification processes. This observation was corroborated by results from next-generation sequencing techniques, verifying the existence of denitrifying microorganisms. The real-time PCR analysis results indicated that the abundance of comammox amoA gene was comparable with the abundance of the AOB amoA gene, indicating the presence of comammox Nitrospira and their potential role on nitrification in the system.

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.

Comparative Analysis of Bacterial Information of Biofilms and Activated Sludge in Full-Scale MBBR-IFAS Systems.

This study extensively analyzed the bacterial information of biofilms and activated sludge in oxic reactors of full-scale moving bed biofilm reactor-integrated fixed-film activated sludge (MBBR-IFAS) systems. The bacterial communities of biofilms and activated sludge differed statistically (R = 0.624, p < 0.01). The denitrifying genera Ignavibacterium, Phaeodactylibacter, Terrimonas, and Arcobacter were more abundant in activated sludge (p < 0.05), while comammox Nitrospira was more abundant in biofilms (p < 0.05), with an average relative abundance of 8.13%. Nitrospira and Nitrosomonas had weak co-occurrence relationships with other genera in the MBBR-IFAS systems. Potential function analysis revealed no differences in pathways at levels 1 and 2 based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) between biofilms and activated sludge. However, in terms of pathways at level 3, biofilms had more potential in 26 pathways, including various organic biodegradation and membrane and signal transportation pathways. In comparison, activated sludge had more potential in only five pathways, including glycan biosynthesis and metabolism. With respect to nitrogen metabolism, biofilms had greater potential for nitrification (ammonia oxidation) (M00528), and complete nitrification (comammox) (M00804) concretely accounted for methane/ammonia monooxygenase (K10944, K10945, and K10946) and hydroxylamine dehydrogenase (K10535). This study provides a theoretical basis for MBBR-IFAS systems from the perspective of microorganisms.

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.

Nitrification inhibitor chlorate and nitrogen substrates differentially affect comammox Nitrospira in a grassland soil.

Through the combined use of two nitrification inhibitors, Dicyandiamide (DCD) and chlorate with nitrogen amendment, this study aimed to investigate the contribution of comammox Nitrospira clade B, ammonia oxidizing bacteria (AOB) and archaea (AOA) to nitrification in a high fertility grassland soil, in a 90-day incubation study. The soil was treated with nitrogen (N) at three levels: 0 mg-N kg-1 soil, 50 mg-N kg-1 soil, and 700 mg-N kg-1 soil, with or without the two nitrification inhibitors. The abundance of comammox Nitrospira, AOA, AOB, and nitrite oxidising bacteria (NOB) was measured using qPCR. The comammox Nitrospira community structure was assessed using Illumina sequencing. The results showed that the application of chlorate inhibited the oxidation of both NH4+ and NO2- in all three nitrogen treatments. The application of chlorate significantly reduced the abundance of comammox Nitrospira amoA and nxrB genes across the 90-day experimental period. Chlorate also had a significant effect on the beta diversity (Bray-Curtis dissimilarity) of the comammox Nitrospira clade B community. Whilst AOB grew in response to the N substrate additions and were inhibited by both inhibitors, AOA showed litle or no response to either the N substrate or inhibitor treatments. In contrast, comammox Nitrospira clade B were inhibited by the high ammonium concentrations released from the urine substrates. These results demonstrate the differential and niche responses of the three ammonia oxidising communities to N substrate additions and nitrification inhibitor treatments. Further research is needed to investigate the specificity of the two inhibitors on the different ammonia oxidising communities.