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.

Role of comammox bacteria in granular bioreactor for nitrogen removal via partial nitritation/anammox.

In this study, two bioprocess models were first constructed with the newly-discovered comammox process described as one-step and two-step nitrification and evaluated against relevant experimental data. The validated models were then applied to reveal the potential effect of comammox bacteria on the granular bioreactor particularly suitable for undertaking partial nitritation/anammox (PN/A) under different operating conditions of bulk dissolved oxygen (DO) and influent NH4+. The results showed although comammox bacteria-based PN/A could achieve > 80.0 % total nitrogen (TN) removal over a relatively wider range of bulk DO and influent NH4+ (i.e., 0.25-0.40 g-O2/m3 and 470-870 g-N/m3, respectively) without significant nitrous oxide (N2O) production (< 0.1 %), the bulk DO should be finely controlled based on the influent NH4+ to avoid the undesired full nitrification by comammox bacteria. Comparatively, conventional ammonium-oxidizing bacteria (AOB)-based PN/A not only required higher bulk DO to achieve > 80.0 % TN removal but also suffered from 1.7 %∼2.8 % N2O production.

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.

Biological treatment of N-methylpyrrolidone, cyclopentanone, and diethylene glycol monobutyl ether distilled residues and their effects on nitrogen removal in a full-scale wastewater treatment plant.

Manufacturing processes in semiconductor and photonics industries involve the use of a significant amount of organic solvents. Recycle and reuse of these solvents produce distillate residues and require treatment before being discharged. This study aimed to evaluate the performance of the biological treatment system in a full-scale wastewater treatment plant that treats wastewater containing distillate residues from the recycling of electronic chemicals. Batch experiments were conducted to investigate the optimal operational conditions for the full-scale wastewater treatment plant. To achieve good nitrogen removal efficiency with effluent ammonia and nitrate concentrations below 20 mg N/L and 50 mg N/L, respectively, it was suggested to control the ammonia concentration and pH of the influent below 500 mg N/L and 8.0, respectively. In addition, the biodegradability of N-methylpyrrolidone, diethylene glycol monobutyl ether, and cyclopentanone distillate residues from the electronic chemicals manufacturing process were evaluated under aerobic, anoxic, and anaerobic conditions. N-methylpyrrolidone and cyclopentanone distillate residues were suggested to be treated under anoxic condition. However, substrate inhibition occurred when using cyclopentanone distillate residue as a carbon source with chemical oxygen demand (COD) levels higher than 866 mg/L and nitrate levels higher than 415 mg N/L. Under aerobic condition, the COD from both N-methylpyrrolidone and cyclopentanone distillate residues could be easily degraded. Nevertheless, a negative effect on nitrification was observed, with a prolonged lag time for ammonia oxidation as the initial COD concentration increased. The specific ammonia oxidation rate and nitrate production rate decreased under high COD concentration contributed by N-methylpyrrolidone and cyclopentanone distillate residues. Furthermore, the biodegradability of diethylene glycol monobutyl ether distillate residue was found to be low under aerobic, anoxic, and anaerobic conditions. With respect to the abundance of nitrogen removal microorganisms in the wastewater treatment plant, results showed that Comammox may have an advantage over ammonia oxidizing bacteria under high pH conditions. In addition, Comammox may have higher resistance to environmental changes. Dominance of Comammox over ammonia oxidizing bacteria under high ammonia condition was first reported in this study.

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.

The superiority of hydrophilic polyurethane in comammox-dominant ammonia oxidation during low-strength wastewater treatment.

Carriers have been extensively employed to enhance nitrification performance during low-strength wastewater treatment by retaining slow-growing ammonia oxidizing microorganisms (AOMs). Still, there is a dearth of systematic understanding of biofilm properties and microbial community structure formed on different carriers. In this study, hydrophilic polyurethane foam (PUF) carriers were prepared and compared with five widely used commercial carriers, namely Kaldness 3, Biochip, activated carbon, volcanic rock, and zeolite. The results indicated that the biofilms formed on carriers enhanced microbial ammonia oxidation activity. Additionally, the biofilm developed on the PUF demonstrated the most superior performance among all selected carriers, not only exhibiting the highest abundant and the most active AOMs, with amoA gene abundance of 1.41 × 1013 copies/m3 and specific ammonia oxidation rate of 9.84 g NH4+-N/(m3 × h), but also possessing a compact structure, with 3.41 kg VSS/m3 and 46.83 mg extracellular polymeric substances/g VSS. The high-throughput sequencing analysis revealed that the comammox (CMX) Nitrospira dominated on biofilm due to the intrinsically low apparent half-saturation constant for substrate. A unique ecological community structure was established on PUF, characterized by low species diversity and high homogeneity in alignment with community characteristics of CMX. The biofilms on PUF contributed to the proliferation of CMX Nitrospira dominated by Nitrospira nitrosa, achieving the highest proportion among colonial three AOMs at 86.58 %. The appropriate average pore size, superior hydrophilicity, and large specific surface area of PUF carriers provided a robust foundation for the exceptional ammonia oxidation performance of the formed biofilms.

Comammox Nitrospira cooperate with anammox bacteria in a partial nitritation-anammox membrane bioreactor treating low-strength ammonium wastewater at high loadings.

Research has revealed that comammox Nitrospira and anammox bacteria engage in dynamic interactions in partial nitritation-anammox reactors, where they compete for ammonium and nitrite or comammox Nitrospria supply nitrite to anammox bacteria. However, two gaps in the literature are present: the know-how to manipulate the interactions to foster a stable and symbiotic relationship and the assessment of how effective this partnership is for treating low-strength ammonium wastewater at high hydraulic loads. In this study, we employed a membrane bioreactor designed to treat synthetic ammonium wastewater at a concentration of 60 mg N/L, reaching a peak loading of 0.36 g N/L/day by gradually reducing the hydraulic retention time to 4 hr. Throughout the experiment, the reactor achieved an approximately 80 % nitrogen removal rate through strategically adjusting intermittent aeration at every stage. Notably, the genera Ca. Kuenena, Nitrosomonas, and Nitrospira collectively constituted approximately 40 % of the microbial community. Under superior intermittent aeration conditions, the expression of comammox amoA was consistently higher than that of Nitrospira nxrB and AOB amoA in the biofilm, despite the higher abundance of Nitrosomonas than comammox Nitrospira, implying that the biofilm environment is favorable for fostering cooperation between comammox and anammox bacteria. We then assessed the in situ activity of comammox Nitrospira in the reactor by selectively suppressing Nitrosomonas using 1-octyne, thereby confirming that comammox Nitrospira played the primary role in facilitating the nitritation (33.1 % of input ammonium) rather than complete nitrification (7.3 % of input ammonium). Kinetic analysis revealed a specific ammonia-oxidizing rate 5.3 times higher than the nitrite-oxidizing rate in the genus Nitrospira, underscoring their critical role in supplying nitrite. These findings provide novel insights into the cooperative interplay between comammox Nitrospira and anammox bacteria, potentially reshaping the management of nitrogen cycling in engineered environments, and aiding the development of microbial ecology-driven wastewater treatment technologies.

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.

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.

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

Introduction: 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. Methods: 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. Results and Discussion: 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.

Irons differently modulate bacterial guilds for leading to varied efficiencies in simultaneous nitrification and denitrification (SND) within four aerobic bioreactors.

As a novel biological wastewater nitrogen removal technology, simultaneous nitrification and denitrification (SND) has gained increasing attention. Iron, serving as a viable material, has been shown to influence nitrogen removal. However, the precise impact of iron on the SND process and microbiome remains unclear. In this study, bioreactors amended with iron of varying valences were evaluated for total nitrogen (TN) removal efficiencies under aerobic conditions. The acclimated control reactor without iron addition (NCR) exhibited high ammonia nitrogen (AN) removal efficiency (98.9%), but relatively low TN removal (78.6%) due to limited denitrification. The reactor containing zero-valent iron (Fe0R) demonstrated the highest SND rate of 92.3% with enhanced aerobic denitrification, albeit with lower AN removal (84.1%). Significantly lower SND efficiencies were observed in reactors with ferrous (Fe2R, 66.3%) and ferric (Fe3R, 58.2%) iron. Distinct bacterial communities involved in nitrogen metabolisms were detected in these bioreactors. The presence of complete ammonium oxidation (comammox) genus Nitrospira and anammox bacteria Candidatus Brocadia characterized efficient AN removal in NCR. The relatively low abundance of aerobic denitrifiers in NCR hindered denitrification. Fe0R exhibited highly abundant but low-efficiency methanotrophic ammonium oxidizers, Methylomonas and Methyloparacoccus, along with diverse aerobic denitrifiers, resulting in lower AN removal but an efficient SND process. Conversely, the presence of Fe2+/Fe3+ constrained the denitrifying community, contributing to lower TN removal efficiency via inefficient denitrification. Therefore, different valent irons modulated the strength of nitrification and denitrification through the assembly of key microbial communities, providing insight for microbiome modulation in nitrogen-rich wastewater treatment.

Journey of the swift nitrogen transformation: Unveiling comammox from discovery to deep understanding.

COMplete AMMonia OXidizer (comammox) refers to microorganisms that have the function of oxidizing NH4+ to NO3- alone. The discovery of comammox overturned the two-step theory of nitrification in the past century and triggered many important scientific questions about the nitrogen cycle in nature. This comprehensive review delves into the origin and discovery of comammox, providing a detailed account of its detection primers, clades metabolic variations, and environmental factors. An in-depth analysis of the ecological niche differentiation among ammonia oxidizers was also discussed. The intricate role of comammox in anammox systems and the relationship between comammox and nitrogen compound emissions are also discussed. Finally, the relationship between comammox and anammox is displayed, and the future research direction of comammox is prospected. This review reveals the metabolic characteristics and distribution patterns of comammox in ecosystems, providing new perspectives for understanding nitrogen cycling and microbial ecology. Additionally, it offers insights into the potential application value and prospects of comammox.

Enhancing the relative abundance of comammox nitrospira in ammonia oxidizer community decreases N2O emission in nitrification exponentially.

Comammox Nitrospira and canonical ammonia-oxidizing bacteria (cAOB) generally coexist in activated sludge. In present study, the effect of comammox Nitrospira on N2O production during nitrification of activated sludge was investigated. Comammox Nitrospira and cAOB were separately enriched in two nitrifying reactors, with respective relative abundance of approximately 98% in ammonia oxidizer community. The N2O emission factor (EF) of nitrification in comammox Nitrospira dominated reactor was 0.35%, consistently lower than that (2.2%) in cAOB dominated reactor. When increasing the relative abundance of comammox Nitrospira in ammonia oxidizer community, the N2O EF of nitrification decreased exponentially, which suggested that comammox Nitrospira not only decreased N2O production directly but also might have reduced N2O yield by cAOB. When cAOB dominated the ammonia oxidizer community of sludge, decreasing pH to 6.3, lowering DO to less than 0.5 mg/L, and increasing nitrite concentration enhanced N2O EF dramatically. When comammox Nitrospira became the dominant ammonia oxidizer, however, the N2O EF correlated to nitrite insignificantly and a low DO of 0.2 mg/L and weakly acidic pH (6.3) decreased N2O EF by approximately 70% and 60%, respectively. These results imply that enhancing the relative abundance of comammox Nitrospira in sludge is an effective way to reducing N2O emissions and can also offset the promoting effects of acidic pH, low DO, and high nitrite concentration on N2O production during nitrification.

Different oxygen affinities of methanotrophs and Comammox Nitrospira inform an electrically induced symbiosis for nitrogen loss.

Aerobic methanotrophs establish a symbiotic association with denitrifiers to facilitate the process of aerobic methane oxidation coupled with denitrification (AME-D). However, the symbiosis has been frequently observed in hypoxic conditions continuing to pose an enigma. The present study has firstly characterized an electrically induced symbiosis primarily governed by Methylosarcina and Hyphomicrobium for the AME-D process in a hypoxic niche caused by Comammox Nitrospira. The kinetic analysis revealed that Comammox Nitrospira exhibited a higher apparent oxygen affinity compared to Methylosarcina. While the coexistence of comammox and AME-D resulted in an increase in methane oxidation and nitrogen loss rates, from 0.82 ± 0.10 to 1.72 ± 0.09 mmol CH4 d-1 and from 0.59 ± 0.04 to 1.30 ± 0.15 mmol N2 d-1, respectively. Furthermore, the constructed microbial fuel cells demonstrated a pronounced dependence of the biocurrents on AME-D due to oxygen competition, suggesting the involvement of direct interspecies electron transfer in the AME-D process under hypoxic conditions. Metagenomic and metatranscriptomic analysis revealed that Methylosarcina efficiently oxidized methane to formaldehyde, subsequently generating abundant NAD(P)H for nitrate reduction by Hyphomicrobium through the dissimilatory RuMP pathway, leading to CO2 production. This study challenges the conventional understanding of survival mechanism employed by AME-D symbionts, thereby contributing to the characterization responsible for limiting methane emissions and promoting nitrogen removal in hypoxic regions.

Downflow sponge biofilm reactors for polluted raw water treatment: Performance optimisation, kinetics, and microbial community.

Water outages caused by elevated ammonium (NH4+-N) levels are a prevalent problem faced by conventional raw water treatment plants in developing countries. A treatment solution requires a short hydraulic retention time (HRT) to overcome nitrification rate limitation in oligotrophic conditions. In this study, the performance of polluted raw water treatment using a green downflow sponge biofilm (DSB) technology was evaluated. We operated two DSB reactors, DSB-1 and DSB-2 under different NH4+-N concentration ranges (DSB-1: 3.2-5.0 mg L-1; DSB-2: 1.7-2.6 mg L-1) over 360 days and monitored their performance under short HRT (60 min, 30 min, 20 min, and 15 min). The experimental results revealed vertical segregation of organic removal in the upper reactor depths and nitrification in the lower depths. Under the shortest HRT of 15 min, both DSB reactors achieved stable NH4+-N and chemical oxygen demand removal (≥95%) and produced minimal effluent nitrite (NO2--N). DSB system could facilitate complete NH4+-N oxidation to nitrate (NO3--N) without external aeration energy requirement. The 16S rRNA sequencing data revealed that nitrifying bacteria Nitrosomonas and Nitrospira in the reactor were stratified. Putative comammox bacteria with high ammonia affinity was successfully enriched in DSB-2 operating at a lower NH4+-N loading rate, which is advantageous in oligotrophic treatment. This study suggests that a high hydraulic rate DSB system with efficient ammonia removal could incorporate ammonia treatment capability into polluted raw water treatment process and ensure safe water supply in many developing countries.

The significant contribution of comammox bacteria to nitrification in a constructed wetland revealed by DNA-based stable isotope probing.

The discovery of Comammox bacteria (CMX) has changed our traditional concept towards nitrification, yet its role in constructed wetlands (CWs) remains unclear. This study investigated the contributions of CMX and two canonical ammonia-oxidizing microorganisms, ammonia-oxidizing bacteria (AOB) and archaea to nitrification in four regions (sediment, shoreside, adjacent soil, and water) of a typical CW using DNA-based stable isotope probing. The results revealed that CMX not only widely occurred in sediment and shoreside zones with high abundance (5.08 × 104 and 6.57 × 104 copies g-1 soil, respectively), but also actively participated in ammonia oxidation, achieving ammonia oxidation rates of 1.43 and 2.00 times that of AOB in sediment and shoreside, respectively. Phylogenetic analysis indicated that N. nitrosa was the dominant and active CMX species. These findings uncovered the crucial role of CMX in nitrification of sediment and shoreside, providing a new insight into nitrogen cycle of constructed wetlands.

Urban and agricultural land use led to niche differentiation of AOA, AOB and comammox along the Beiyun River continuum.

River ecological health has been severely threatened by anthropogenic land-use pressures. Here, by combining remote sensing and molecular biology methods, we evaluated the impact of land-use activities on nitrification, a fundamental ecological process in rivers, which is conducted by ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB), or the newly discovered complete ammonia oxidisers (comammox). We explored the relationships of the abundance, activity, and diversity of AOA, AOB, and comammox in river sediments with land-use pressure by proposing a quantitative land use pattern index (LPI) over a 184 km continuum along the Beiyun River in North China. We found that comammox dominated nitrification in the forestry upstream (67.07 % in summer, 56.40 % in winter), while AOB became the major player in the urban middle (56.51 % in summer, 53.08 % in winter) and agricultural downstream reaches (62.98 % in summer, 50.74 % in winter). In addition, urban and agricultural land use lowered the α diversity of AOA and comammox, as well as simplified their co-occurrence networks, but promoted AOB diversity and complicated their networks. The structural equation model illustrated that the key drivers affecting the key taxa and activities were ammonia, and C/N for AOB, and total organic matter, and pH for comammox. We thus conclude that watershed urban and agricultural land use drive the niche differentiation of AOA, AOB, and comammox, specifically leading to a robust AOB community but weakened AOA and comammox communities. Our study connects the macro and micro worlds and provides a new paradigm for studying the variation in microbial communities as well as the potential ecological consequences under the increased anthropogenic land-use pressures in the Anthropocene.

Cooperation among nitrifying microorganisms promotes the irreversible biotransformation of sulfamonomethoxine.

Ammonia-oxidizing microorganisms, including AOA (ammonia-oxidizing archaea), AOB (ammonia-oxidizing bacteria), and Comammox (complete ammonia oxidization) Nitrospira, have been reported to possess the capability for the biotransformation of sulfonamide antibiotics. However, given that nitrifying microorganisms coexist and operate as communities in the nitrification process, it is surprising that there is a scarcity of studies investigating how their interactions would affect the biotransformation of sulfonamide antibiotics. This study aims to investigate the sulfamonomethoxine (SMM) removal efficiency and mechanisms among pure cultures of phylogenetically distinct nitrifiers and their combinations. Our findings revealed that AOA demonstrated the highest SMM removal efficiency and rate among the pure cultures, followed by Comammox Nitrospira, NOB, and AOB. However, the biotransformation of SMM by AOA N. gargensis is reversible, and the removal efficiency significantly decreased from 63.84 % at 167 h to 26.41 % at 807 h. On the contrary, the co-culture of AOA and NOB demonstrated enhanced and irreversible SMM removal efficiency compared to AOA alone. Furthermore, the presence of NOB altered the SMM biotransformation of AOA by metabolizing TP202 differently, possibly resulting from reduced nitrite accumulation. This study offers novel insights into the potential application of nitrifying communities for the removal of sulfonamide antibiotics (SAs) in engineered ecosystems.

Nitrogen source influences the interactions of comammox bacteria with aerobic nitrifiers.

While the co-existence of comammox Nitrospira with canonical nitrifiers is well documented in diverse ecosystems, there is still a dearth of knowledge about the mechanisms underpinning their interactions. Understanding these interaction mechanisms is important as they may play a critical role in governing nitrogen biotransformation in natural and engineered ecosystems. In this study, we tested the ability of two environmentally relevant factors (nitrogen source and availability) to shape interactions between strict ammonia and nitrite-oxidizing bacteria and comammox Nitrospira in continuous flow column reactors. The composition of inorganic nitrogen species in reactors fed either ammonia or urea was similar during the lowest input nitrogen concentration (1 mg-N/L), but higher concentrations (2 and 4 mg-N/L) promoted significant differences in nitrogen species composition and nitrifier abundances. The abundance and diversity of comammox Nitrospira were dependent on both nitrogen source and input concentrations as multiple comammox Nitrospira populations were preferentially enriched in the urea-fed system. In contrast, their abundance was reduced in response to higher nitrogen concentrations in the ammonia-fed system. The preferential enrichment of comammox Nitrospira in the urea-fed system could be associated with their ureolytic activity calibrated to their ammonia oxidation rates, thus minimizing ammonia accumulation, which may be partially inhibitory. However, an increased abundance of comammox Nitrospira was not associated with a reduced abundance of nitrite oxidizers in the urea-fed system while a negative correlation was found between them in the ammonia-fed system, the latter dynamic likely emerging from reduced availability of nitrite to strict nitrite oxidizers at low ammonia concentrations. IMPORTANCE: Nitrification is an essential biological process in drinking water and wastewater treatment systems for treating nitrogen pollution. The discovery of comammox Nitrospira and their detection alongside canonical nitrifiers in these engineered ecosystems have made it necessary to understand the environmental conditions that regulate their abundance and activity relative to other better-studied nitrifiers. This study aimed to evaluate two important factors that could potentially influence the behavior of nitrifying bacteria and, therefore, impact nitrification processes. Column reactors fed with either ammonia or urea were systematically monitored to capture changes in nitrogen biotransformation and the nitrifying community as a function of influent nitrogen concentration, nitrogen source, and reactor depth. Our findings show that with increased ammonia availability, comammox Nitrospira decreased in abundance while nitrite oxidizers abundance increased. Yet, in systems with increasing urea availability, comammox Nitrospira abundance and diversity increased without an associated reduction in the abundance of canonical nitrifiers.

Understanding vermicompost and organic manure interactions: impact on toxic elements, nitrification activity, comammox Nitrospira inopinata, and archaea/bacteria.

Vermicompost is a substantial source of nutrients, promotes soil fertility, and maintains or increases soil organic matter levels. Potentially toxic elements (PTEs) in vermicompost impact on nitrification activity. However, it is yet unknown how vermicompost affects nitrifying bacteria and archaea, comammox Nitrospira inopinata (complete ammonia oxidizers), net nitrification rates (NNRs), and PTEs. The effects of vermicompost application on NNRs, potential nitrification rates (NPs), PTEs, and the abundances of comammox N. inopinata bacteria, nitrite-oxidizing bacteria (NOB), and ammonia-oxidizing bacteria (AOB)/archaea (AOA) were studied. NNRs and NPs were significantly higher (p < 0.05) in fresh cow-dung vermicompost (stored for 40 days) as compared with other organic manure. The level of PTEs (Cu2+, Fe2+, Pb2+, Cd2+, and Zn2+) was significantly lower (p < 0.05) in vermicompost as compared with compost of waste material with Trichoderma and cow dung. Comammox N. inopinata, NOB, AOB, and AOA were significantly higher (p < 0.05) in stored cow-dung vermicompost (more than 1 year) as compared with other organic manure. The results of the scatterplot matrix analysis suggested that Fe2+, total nitrogen (TN), soil organic carbon (SOC), and total carbon (TC) were linearly correlated (p < 0.001) with NNRs and NPs in vermicompost and organic manure. Similarly, comammox N. inopinata bacteria, NOB, AOB, and AOA were linearly correlated (p < 0.001) with NNR and NP. These results indicated that vermicompost promoted nitrification activity by increasing microbial diversity and abundance, supplying nutrients and organic matter for microbial growth, and facilitating complex microbial interactions. It may be concluded that the influence of vermicompost, which played a great role in PTE concentration reduction, increased chemical, and biological properties, increased the growth rate of nitrifying bacteria/archaea and the nitrogen cycle.