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.

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.

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

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

Community assembly of comammox Nitrospira in coastal wetlands across southeastern China.

Complete ammonia oxidizers (comammox Nitrospira) are ubiquitous in coastal wetland sediments and play an important role in nitrification. Our study examined the impact of habitat modifications on comammox Nitrospira communities in coastal wetland sediments across tropical and subtropical regions of southeastern China. Samples were collected from 21 coastal wetlands in five provinces where native mudflats were invaded by Spartina alterniflora and subsequently converted to aquaculture ponds. The results showed that comammox Nitrospira abundances were mainly influenced by sediment grain size rather than by habitat modifications. Compared to S. alterniflora marshes and native mudflats, aquaculture pond sediments had lower comammox Nitrospira diversity, lower clade A.1 abundance, and higher clade A.2 abundance. Sulfate concentration was the most important factor controlling the diversity of comammox Nitrospira. The response of comammox Nitrospira community to habitat change varied significantly by location, and environmental variables accounted for only 11.2% of the variations in community structure across all sites. In all three habitat types, dispersal limitation largely controlled the comammox Nitrospira community assembly process, indicating the stochastic nature of these sediment communities in coastal wetlands. IMPORTANCE Comammox Nitrospira have recently gained attention for their potential role in nitrification and nitrous oxide (N2O) emissions in soil and sediment. However, their distribution and assembly in impacted coastal wetland are poorly understood, particularly on a large spatial scale. Our study provides novel evidence that the effects of habitat modification on comammox Nitrospira communities are dependent on the location of the wetland. We also found that the assembly of comammox Nitrospira communities in coastal wetlands was mainly governed by stochastic processes. Nevertheless, sediment grain size and sulfate concentration were identified as key variables affecting comammox Nitrospira abundance and diversity in coastal sediments. These findings are significant as they advance our understanding of the environmental adaptation of comammox Nitrospira and how future landscape modifications may impact their abundance and diversity in coastal wetlands.

Plant Species-Driven Distribution of Individual Clades of Comammox Nitrospira in a Subtropical Estuarine Wetland.

Plant species play a crucial role in mediating the activity and community structure of soil microbiomes through differential inputs of litter and rhizosphere exudates, but we have a poor understanding of how plant species influence comammox Nitrospira, a newly discovered ammonia oxidizer with pivotal functionality. Here, we investigate the abundance, diversity, and community structure of comammox Nitrospira underneath five plant species and a bare tidal flat at three soil depths in a subtropical estuarine wetland. Plant species played a critical role in driving the distribution of individual clades of comammox Nitrospira, explaining 59.3% of the variation of community structure. Clade A.1 was widely detected in all samples, while clades A.2.1, A.2.2, A.3 and B showed plant species-dependent distribution patterns. Compared with the native species Cyperus malaccensis, the invasion of Spartina alterniflora increased the network complexity and changed the community structure of comammox Nitrospira, while the invasive effects from Kandelia obovata and Phragmites australis were relatively weak. Soil depths significantly influenced the community structure of comammox Nitrospira, but the effect was much weaker than that from plant species. Altogether, our results highlight the previously unrecognized critical role of plant species in driving the distribution of comammox Nitrospira in a subtropical estuarine wetland.

Multi-scale Investigation of Ammonia-Oxidizing Microorganisms in Biofilters Used for Drinking Water Treatment.

Ammonia-oxidizing microorganisms (AOMs) include ammonia-oxidizing bacteria (AOB), archaea (AOA), and Nitrospira spp. sublineage II capable of complete ammonia oxidation (comammox). These organisms can affect water quality not only by oxidizing ammonia to nitrite (or nitrate) but also by cometabolically degrading trace organic contaminants. In this study, the abundance and composition of AOM communities were investigated in full-scale biofilters at 14 facilities across North America and in pilot-scale biofilters operated for 18 months at a full-scale water treatment plant. In general, the relative abundance of AOM in most full-scale biofilters and in the pilot-scale biofilters was as follows: AOB > comammox Nitrospira > AOA. The abundance of AOB in the pilot-scale biofilters increased with increasing influent ammonia concentration and decreasing temperature, whereas AOA and comammox Nitrospira exhibited no correlations with these parameters. The biofilters affected AOM abundance in the water passing through the filters via collecting and shedding but exhibited a minor influence on the composition of AOB and Nitrospira sublineage II communities in the filtrate. Overall, this study highlights the relative importance of AOB and comammox Nitrospira compared to AOA in biofilters and the influence of filter influent water quality on AOM in biofilters and their release into the filtrate.

Unraveling microbial characteristics of simultaneous nitrification, denitrification and phosphorus removal in a membrane-aerated biofilm reactor.

This study describes the simultaneous removal of carbon, ammonium, and phosphate from domestic wastewater by a membrane-aerated biofilm reactor (MABR) which was operated for 360 days. During the operation, the maximum removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) reached 93.1%, 83.98%, and 96.41%, respectively. Statistical analysis showed that the MABR could potentially treat wastewater with a high ammonium concentration and a relatively low C/N ratio. Dissolved oxygen and multiple pollutants, including ammonium, carbon, phosphate, and sulfate, shaped the structure of the microbial community in the MABR. High throughput sequencing uncovered the crucial microbiome in ammonium transformation in MABR. Phylogenetic analysis of the ammonia monooxygenase (amoA) genes revealed an important role for comammox Nitrospira in the nitrification process. Diverse novel phosphate-accumulating organisms (Thauera, Bacillus, and Pseudomonas) and sulfur-oxidizing bacteria (Thiobacillus, Thiothrix and Sulfurimonas) were potentially involved in denitrification in MABR. The results from this study suggested that MABR could be a feasible system for the simultaneous removal of nitrogen, carbon, phosphorus, and sulfur from sewage water.

Ecological distribution and function of comammox Nitrospira in the environment.

Complete ammonia oxidizers (Comammox) are of great significance for studying nitrification and expanding the understanding of the nitrogen cycle. Moreover, Comammox bacteria are also crucial in natural and engineered environments due to their role in wastewater treatment and maintaining the flux of greenhouse gases to the atmosphere. However, only few studies are there regarding the Comammox bacteria and their role in ammonia and nitrite oxidation in the environment. This review mainly focuses on summarizing the genomes of Nitrospira in the NCBI database. Ecological distribution of Nitrospira was also reviewed and the influence of environmental parameters on genus Nitrospira in different environments has been summarized. Furthermore, the role of Nitrospira in carbon cycle, nitrogen cycle, and sulfur cycle were discussed, especially the comammox Nitrospira. In addition, the overviews of current research and development regarding comammox Nitrospira, were summarized along with the scope of future research. KEY POINTS: • Most of Comammox Nitrospira are widely distributed in both aquatic and terrestrial ecosystems, but it has been studied less frequently in the extreme environments. • Comammox Nitrospira can be involved in different nitrogen transformation process, but rarely involved in nitrogen fixation. • The stable isotope and transcriptome techniques are important methods to study the metabolic function of comammox Nitrospira.

Comammox Nitrospira Bacteria Are Dominant Ammonia Oxidizers in Mainstream Nitrification Bioreactors Emended with Sponge Carriers.

Complete ammonia oxidation (i.e., comammox) is a newly discovered microbial process performed by a subset of the Nitrospira genus, and this unique microbial process has been ubiquitously detected in various wastewater treatment units. However, the operational conditions favoring comammox prevalence remain unclear. In this study, the dominance of comammox Nitrospira in four sponge biofilm reactors fed with low-strength ammonium (NH4+ = 23 ± 3 mg N/L) wastewater was proved by coupling 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. The results showed that comammox Nitrospira dominated in the nitrifying guild over canonical ammonia-oxidizing bacteria (AOB) constantly, despite the significant variation in the residual ammonium concentration (0.01-15 mg N/L) under different sets of operating conditions. This result indicates that sponge biofilms greatly favor retaining comammox Nitrospira in wastewater treatment and highlights an essential role of biomass retention in the comammox prevalence. Moreover, analyses of the assembled metagenomic sequences revealed that the retrieved amoA gene sequences affiliated with comammox Nitrospira (53.9-66.0% read counts of total amoA gene reads) were always higher than those (28.4-43.4%) related to ß-proteobacterial AOB taxa. The comammox Nitrospira bacteria detected in the present biofilm systems were close to clade A Candidatus Nitrospira nitrosa.

Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply.

The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02-0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR.

Integrating Genome-Resolved Metagenomics with Trait-Based Process Modeling to Determine Biokinetics of Distinct Nitrifying Communities within Activated Sludge.

Conventional bioprocess models for wastewater treatment are based on aggregated bulk biomass concentrations and do not incorporate microbial physiological diversity. Such a broad aggregation of microbial functional groups can fail to predict ecosystem dynamics when high levels of physiological diversity exist within trophic guilds. For instance, functional diversity among nitrite-oxidizing bacteria (NOB) can obfuscate engineering strategies for their out-selection in activated sludge (AS), which is desirable to promote energy-efficient nitrogen removal. Here, we hypothesized that different NOB populations within AS can have different physiological traits that drive process performance, which we tested by estimating biokinetic growth parameters using a combination of highly replicated respirometry, genome-resolved metagenomics, and process modeling. A lab-scale AS reactor subjected to a selective pressure for over 90 days experienced resilience of NOB activity. We recovered three coexisting Nitrospira population genomes belonging to two sublineages, which exhibited distinct growth strategies and underwent a compositional shift following the selective pressure. A trait-based process model calibrated at the NOB genus level better predicted nitrite accumulation than a conventional process model calibrated at the NOB guild level. This work demonstrates that trait-based modeling can be leveraged to improve our prediction, control, and design of functionally diverse microbiomes driving key environmental biotechnologies.

Rearrangements of the nitrifiers population in an activated sludge system under decreasing solids retention times.

Due to the key role of nitrite in novel nitrogen removal systems, nitrite oxidizing bacteria (NOB) have been receiving increasing attention. In this study, the coexistence and interactions of nitrifying bacteria were explored at decreasing solids retention times (SRTs). Four 5-week washout experiments were carried out in laboratory-scale (V = 10 L) sequencing batch reactors (SBRs) with mixed liquor from two full-scale activated sludge systems (continuous flow vs SBR). During the experiments, the SRT was gradually reduced from the initial value of 4.0 d to approximately 1.0 d. The reactors were operated under limited dissolved oxygen conditions (set point of 0.6 mg O2/L) and two process temperatures: 12 °C (winter) and 20 °C (summer). At both temperatures, the progressive SRT reduction was inefficient for the out-selection of both canonical NOB and comammox Nitrospira. However, the dominant NOB switched from Nitrospira to Ca. Nitrotoga, whereas the dominant AOB was always Nitrosomonas. The results of this study are important for optimizing NOB suppression strategies in the novel N removal processes, which are based on nitrite accumulation.

Evaluation of PCR primers for detecting the distribution of nitrifiers in mangrove sediments.

Ammonia-oxidizing archaea and ammonia-oxidizing bacteria (AOA and AOB), complete ammonia oxidizers (Comammox), and nitrite-oxidizing bacteria (NOB) play a crucial role in the nitrification process during the nitrogen cycle. However, their occurrence and diversity in mangrove ecosystems are still not fully understood. Here, a total of 11 pairs of PCR primers were evaluated to study the distribution and abundances of these nitrifiers in rhizosphere and non-rhizosphere sediments of a mangrove ecosystem. The amplification efficiency of these 11 pairs of primers was first evaluated and their performances were found to vary considerably. The CamoA-19F/CamoA-616R primer pair was suitable for the amplification of AOA in mangrove sediments, especially on the surface of rhizosphere sediments. Primer pair amoA1F/amoA2R was better for the characterization of novel AOB in the bacterial community of non-rhizosphere sediments of mangroves. In contrast, primer nxrB169F/nxrB638R showed a low abundance of NOB in mangrove sediments (except for R1). Comammox bacteria were abundant and diverse in mangrove sediments, as indicated by both the amoB gene for Comammox clade A and the amoA gene for Comammox Nitrospira clade B. However, the amoA gene for Comammox Nitrospira clade A was not successful in detecting them in the mangrove sediments. Furthermore, 568 operational taxonomic units (OTUs) were obtained by generating a clone library and a high abundance of OTUs was correlated with ammonium, pH, NO2-, and NO3-. Comammox and Comammox Nitrospira were identified by phylogenetic tree analysis, indicating that mangrove sediments harbor newly discovered nitrifiers. Additionally, many AOA and NOB were mainly distributed in the surface layer of the rhizosphere, whereas AOB and Comammox Nitrospira were in the subsurface of non-rhizosphere, as determined by qPCR analysis. Collectively, our findings highlight the limitations of some primers for the identification of specific nitrifying bacteria. Therefore, primers must be carefully selected to gain accurate insights into the ecological distribution of nitrifiers in mangroves. KEY POINTS: • Several sets of PCR primers perform well for the detection of nitrifiers in mangroves. • Mangroves are an important source of newly discovered nitrifiers. • Ammonium, pH, NO2-, and NO3- are important shapers of nitrifier communities in mangroves.

Response of substrate kinetics and biological mechanisms to various pH constrains for cultured Nitrobacter and Nitrospira in nitrifying bioreactor.

Nitrite (NO2-) oxidation is an essential step of biological nitrogen cycling in natural ecosystems, and is performed by chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Although Nitrobacter and Nitrospira are regarded as representative NOB in nitrification systems, little attention has focused on kinetic characterisation of the coexistence of Nitrobacter and Nitrospira at various pH values. Here, we evaluate the substrate kinetics, biological mechanism and microbial community dynamics of an enrichment culture including Nitrobacter (17.5 ± 0.9%) and Nitrospira (7.2 ± 0.6%) in response to various pH constrains. Evaluation of the Monod equation at pH 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 showed that the enrichment had maximum rate (rmax) and maximum substrate affinity (KS) for NO2- oxidation at pH 7.0, which was also supported by the largest absolute abundance of Nitrobacter nxrA (5.26 × 107 copies per g wet sludge) and Nitrospira nxrB (1.975 × 109 copies per g wet sludge) genes. Moreover, the predominant species for the Nitrobacter-like nxrA were N. vulgaris and N. winogradskyi, while for the Nitrospira-like nxrB, the predominant species were N. japonica, N. calida and Ca. N. bockiana. Furthermore, the rmax was strongly and positively correlated with the abundance of the Nitrobacter nxrA or Nitrospira nxrB genes, or N. winogradsk, whereas KS was positively correlated with the abundance of Nitrobacter nxrA or Nitrospira nxrB genes or Ca. N. bockiana. Overall, this study could improve basis kinetic parameters and biological mechanism of NO2- oxidation in WWTPs.

Different Engineering Designs Have Profoundly Different Impacts on the Microbiome and Nitrifying Bacterial Populations in Municipal Wastewater Treatment Bioreactors.

Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous "activated sludge" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.

Nitrospira as versatile nitrifiers: Taxonomy, ecophysiology, genome characteristics, growth, and metabolic diversity.

The global nitrogen cycle is of paramount significance as it affects important processes like primary productivity and decomposition. Nitrification, the oxidation of ammonia to nitrate via nitrite, is a key process in the nitrogen cycle. The knowledge about nitrification has been challenged during the last few decades with inventions like anaerobic ammonia oxidation, ammonia-oxidizing archaea, and recently the complete ammonia oxidation (comammox). The discovery of comammox Nitrospira has made a paradigm shift in nitrification, before which it was considered as a two-step process, mediated by chemolithoautotrophic ammonia oxidizers and nitrite oxidizers. The genome of comammox Nitrospira equipped with molecular machineries for both ammonia and nitrite oxidation. The genus Nitrospira is ubiquitous, comes under phylum Nitrospirae, which comprises six sublineages consisting of canonical nitrite oxidizers and comammox. The single-step nitrification is energetically more feasible; furthermore, the existence of diverse metabolic pathways in Nitrospira is critical for its establishment in various habitats. The present review discusses the taxonomy, ecophysiology, isolation, identification, growth, and metabolic diversity of the genus Nitrospira; compares the genomes of canonical nitrite-oxidizing Nitrospira and comammox Nitrospira, and analyses the differences of Nitrospira with other nitrifying bacteria.

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira.

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

Expanded Diversity and Metabolic Versatility of Marine Nitrite-Oxidizing Bacteria Revealed by Cultivation- and Genomics-Based Approaches.

Nitrite-oxidizing bacteria (NOB) are ubiquitous and abundant microorganisms that play key roles in global nitrogen and carbon biogeochemical cycling. Despite recent advances in understanding NOB physiology and taxonomy, currently very few cultured NOB or representative NOB genome sequences from marine environments exist. In this study, we employed enrichment culturing and genomic approaches to shed light on the phylogeny and metabolic capacity of marine NOB. We successfully enriched two marine NOB (designated MSP and DJ) and obtained a high-quality metagenome-assembled genome (MAG) from each organism. The maximum nitrite oxidation rates of the MSP and DJ enrichment cultures were 13.8 and 30.0 µM nitrite per day, respectively, with these optimum rates occurring at 0.1 mM and 0.3 mM nitrite, respectively. Each enrichment culture exhibited a different tolerance to various nitrite and salt concentrations. Based on phylogenomic position and overall genome relatedness indices, both NOB MAGs were proposed as novel taxa within the Nitrospinota and Nitrospirota phyla. Functional predictions indicated that both NOB MAGs shared many highly conserved metabolic features with other NOB. Both NOB MAGs encoded proteins for hydrogen and organic compound metabolism and defense mechanisms for oxidative stress. Additionally, these organisms may have the genetic potential to produce cobalamin (an essential enzyme cofactor that is limiting in many environments) and, thus, may play an important role in recycling cobalamin in marine sediment. Overall, this study appreciably expands our understanding of the Nitrospinota and Nitrospirota phyla and suggests that these NOB play important biogeochemical roles in marine habitats.IMPORTANCE Nitrification is a key process in the biogeochemical and global nitrogen cycle. Nitrite-oxidizing bacteria (NOB) perform the second step of aerobic nitrification (converting nitrite to nitrate), which is critical for transferring nitrogen to other organisms for assimilation or energy. Despite their ecological importance, there are few cultured or genomic representatives from marine systems. Here, we obtained two NOB (designated MSP and DJ) enriched from marine sediments and estimated the physiological and genomic traits of these marine microbes. Both NOB enrichment cultures exhibit distinct responses to various nitrite and salt concentrations. Genomic analyses suggest that these NOB are metabolically flexible (similar to other previously described NOB) yet also have individual genomic differences that likely support distinct niche distribution. In conclusion, this study provides more insights into the ecological roles of NOB in marine environments.

The effect of dissolved oxygen concentration on long-term stability of partial nitrification process.

Dissolved oxygen (DO) concentration is regarded as one of the crucial factors to influence partial nitrification process. However, achieving and keeping stable partial nitrification under different DO concentrations were widely reported. The mechanism of DO concentration influencing partial nitrification is still unclear. Therefore, in this study two same sequencing batch reactors (SBRs) cultivated same seeding sludge were built up with real-time control strategy. Different DO concentrations were controlled in SBRs to explore the effect of DO concentration on the long-term stability of partial nitrification process at room temperature. It was discovered that ammonium oxidation rate (AOR) was inhibited when DO concentration decreased from 2.5 to 0.5 mg/L. The abundance of Nitrospira increased from 1011.5 to 1013.7 copies/g DNA, and its relative percentage increased from 0.056% to 3.2% during 190 operational cycles, causing partial nitrification gradually turning into complete nitrification process. However, when DO was 2.5 mg/L the abundance of Nitrospira was stable and AOB was always kept at 1010.7 copies/g DNA. High AOR was maintained, and stable partial nitrification process was kept. Ammonia oxidizing bacteria (AOB) activity was significantly higher than nitrite oxidizing bacteria (NOB) activity at DO of 2.5 mg/L, which was crucial to maintain excellent nitrite accumulation performance.

Low Temperature and Neutral pH Define "Candidatus Nitrotoga sp." as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii.

Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides Nitrospira, "Candidatus Nitrotoga" has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of Nitrotoga in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent Nitrotoga sp. BS with regard to temperature and pH variations and evaluated its competitiveness against Nitrospira defluvii Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), and 16S amplicon sequencing, Nitrotoga sp. BS was clearly favored by its optimal growth parameters and dominated over Ns. defluvii at pH 7.4 and 17°C, whereas a pH of 6.4 was more selective for Ns. defluvii Our synthetic communities revealed that niche differentiation of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species.IMPORTANCE "Ca. Nitrotoga" is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between Nitrotoga and Nitrospira in bioreactors and identified conditions where the K strategist Ns. defluvii was almost replaced by Nitrotoga sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of Nitrotoga sp. BS versus Ns. defluvii at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel Nitrotoga cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.