硝化螺旋菌属的生态功能

Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo- and subsaline (1.3–12.8 g salt/l), highly alkaline (pH 8.9–10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members from  Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, “ Candidatus Nitrospira alkalitolerans”, revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira . Other functions aid in pH and cation homeostasis and osmotic stress defense. “ Ca . Nitrospira alkalitolerans” also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.

No abstract available

Abstract Nitrification is the two-step microbial oxidation of ammonia to nitrate via nitrite, and it can contribute to environmental problems in soils. Some nitrifiers have been cultivated from acidic soils at pH <5.5, allowing their metabolic potential and phylogeny to be investigated through genomic analyses. However, the genomic features of the genus Nitrospira remain poorly understood in the context of acid tolerance, despite its wide distribution in acidic environments. This study aimed to characterize the physiology and genomics of acid-tolerant Nitrospira enriched from an acidic soil. Using a metagenomic approach, two closed genomes of Nitrospira were reconstructed: a complete ammonia-oxidizing (comammox) bacterium and a nitrite-oxidizing bacterium (NOB). Both enriched Nitrospira survived at pH <5.5 in physiological tests, and the enriched comammox Nitrospira was phylogenetically close to clones derived from acidic soils. The active-site residues of hydroxylamine oxidase, a key nitrification enzyme, were conserved between the comammox Nitrospira characterized in this study and the previously reported betaproteobacterial ammonia oxidizers. This conservation suggests that existing nitrification inhibitors targeting this enzyme may also inhibit ammonia oxidation by comammox Nitrospira in acidic soils. Although the comammox and NOB Nitrospira in this study shared nearly all key metabolic pathways with Nitrospira species identified from neutral pH environments, both possessed passive urea transporters homologous to those found in acid-tolerant bacteria. These results revealed the acid tolerance of the enriched Nitrospira at pH <5.5, as well as their genomic features shared with acid-tolerant bacteria, rather than with previously reported Nitrospira species.

Microbially mediated nitrification plays an important role in the nitrogen (N) cycle, and rates of activity have been shown to change significantly with temperature. Despite this, the substrate affinities of nitrifying bacteria and archaea have not been comprehensively measured and are often assumed to be static in mathematical models of environmental systems. In this study, we measured the oxidation kinetics of ammonia- (NH3) oxidizing archaea (AOA), NH3-oxidizing bacteria (AOB), and two distinct groups of nitrite (NO2–)-oxidizing bacteria (NOB), of the genera Nitrobacter and Nitrospira, by measuring the maximum rates of apparent activity (Vmax(app)), the apparent half-saturation constant (Km(app)), and the overall catalytic efficiency (Vmax(app)/Km(app)) over a range of temperatures. Changes in Vmax(app) and Km(app) with temperature were different between groups, with Vmax(app) and catalytic efficiency increasing with temperature in AOA, while Vmax(app), Km(app), and catalytic efficiency increased in AOB. In Nitrobacter NOB, Vmax(app) and Km(app) increased, but catalytic efficiency decreased significantly with temperature. Nitrospira NOB were variable, but Vmax(app) increased while catalytic efficiency and Km(app) remained relatively unchanged. Michaelis–Menten (MM) and Haldane (H) kinetic models of NH3 oxidation and NO2– oxidation based on the collected data correctly predict nitrification potential in some soil incubation experiments, but not others. Despite previous observations of coupled nitrification in many natural systems, our results demonstrate significant differences in response to temperature strategies between the different groups of nitrifiers; and indicate the need to further investigate the response of nitrifiers to environmental changes.

Aerobic ammonia and nitrite oxidation reactions are fundamental biogeochemical reactions contributing to the global nitrogen cycle. Although aerobic nitrite oxidation yields 4.8-folds less Gibbs free energy (∆Gr ) than aerobic ammonia oxidation in the NH4 + -feeding marine recirculating trickling biofilter reactors operated in the present study, nitrite-oxidizing and not ammonia-oxidizing Nitrospira (sublineage IV) outnumbered ammonia-oxidizing Nitrosomonas (relative abundance; 53.8% and 7.59% respectively). CO2 assimilation efficiencies during ammonia or nitrite oxidation were 0.077 μmol-14 CO2 /μmol-NH3 and 0.053-0.054 μmol-14 CO2 /μmol-NO2 - respectively, and the difference between ammonia and nitrite oxidation was much smaller than the difference of ∆Gr . Free-energy efficiency of nitrite oxidation was higher than ammonia oxidation (31%-32% and 13% respectively), and high CO2 assimilation and free-energy efficiencies were a determinant for the dominance of Nitrospira over Nitrosomonas. Washout of Nitrospira and Nitrosomonas from the trickling biofilter reactors was also examined by quantitative PCR assay. Normalized copy numbers of Nitrosomonas amoA were 1.5- to 1.7-folds greater than Nitrospira nxrB and 16S rRNA gene in the reactor effluents. Nitrosomonas was more susceptible for washout than Nitrospira in the trickling biofilter reactors, which was another determinant for the dominance of Nitrospira in the trickling biofilter reactors.

In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7–78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.

No abstract available

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

No abstract available

Nitrospira has been revealed as a high versatile genus. Although previously considered only responsible for the conversion of nitrite to nitrate, now we know that Nitrospira can perform complete ammonia oxidation to nitrate too (comammox). Comammox activity was firstly reported as dominant in extremely limited oxygen environments, where anaerobic ammonia oxidation was also occurring (anammox). To explain the comammox selection, we developed an Individual-based Model able to describe Nitrospira and anammox growth in suspended flocs assembled in a dynamic nitrogen and oxygen-limiting environment. All known and hypothesized nitrogen transformations of Nitrospira were considered: ammonia and nitrite oxidation, comammox, nitrate-reducing ammonia oxidation, and anaerobic nitrite-reducing ammonia oxidation. Through bioenergetics analysis, the growth yield associated to each activity was estimated. The other kinetic parameters necessary to describe growth were calibrated according to the reported literature values. Our modeling results suggest that even extremely low oxygen concentrations (~1.0 µM) allow for a proportional growth of anammox versus Nitrospira similar to the one experimentally observed. The strong oxygen limitation was followed by a limitation of ammonia and nitrite, because anammox, without strong competitors, were able to grow faster than Nitrospira depleting the environment in nitrogen. These substrate limitations created an extremely competitive environment that proved to be decisive in the community assembly of Nitrospira and anammox. Additionally, a diversity of metabolic activities for Nitrospira was observed in all tested conditions, which in turn, explained the transient nitrite accumulation observed in aerobic environments with higher ammonia availability.

Abstract The genus Nitrospira, which includes canonical nitrite-oxidizing bacteria (NOB) and species capable of complete ammonia oxidation (comammox), plays an important role in the global biogeochemical nitrogen cycle. Typically, lineage IV Nitrospira predominate in marine environments, and other lineages are thought to be less abundant and remain poorly characterized in oceanic systems. Here, we recovered five novel metagenome-assembled genomes (MAGs) affiliated with Nitrospira lineage II–IV from deep-sea sediments. Notably, two of these MAGs represent members of lineage III and comammox Nitrospira, respectively, suggesting the presence of previously uncharacterized lineages in the deep sea. Phylogenetic and gene locus analyses indicated that deep-sea lineage III and comammox Nitrospira form distinct evolutionary clades that diverge from their terrestrial and coastal relatives, and we therefore designate these two marine-derived groups as “lineage III clade B” and “comammox clade A4”, respectively. Comparative read recruitment analyses revealed that these lineages exhibit potential pan-oceanic distribution in deep-sea sediments and waters, albeit at very low abundances. Furthermore, the identification of genes encoding amtB-type ammonium transporters (amtB), the ABC-type glycerol-3-phosphate transport system (ugpABCE), a multi-subunit Na+/H+ antiporter (mnh), and betaine transporters (BetT, opuABC) suggests that these newly discovered Nitrospira species possess adaptive capabilities to thrive in oligotrophic and saline marine environments. These findings provide novel insights into the occurrence, metabolic features, and adaptation strategies of lineage III and comammox Nitrospira, expand our understanding of Nitrospira diversity in the deep sea, and offer valuable perspectives on the evolutionary history of various Nitrospira lineages.

No abstract available

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. ABSTRACT 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.

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.

No abstract available

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (Km(app)_NO2- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.

The discovery of complete ammonia-oxidizing (comammox) Nitrospira has added an important new process to the microbial nitrogen cycle. While comammox Nitrospira have been detected in various ecosystems, only few studies have achieved their enrichment over other canonical nitrifiers. Here, we obtained a selective enrichment of comammox Nitrospira in a urine-fed membrane bioreactor in less than 200 days. By using 16S rRNA gene amplicon sequencing and quantitative PCR of the functional marker gene amoA, we observed a dominance (up to 30% relative abundance) of comammox Nitrospira over ammonia-oxidizing bacteria and archaea. Furthermore, the complete genomes of three new clade A comammox Nitrospira were recovered by metagenomics. These three strains were divergent from previously reported comammox species according to comparative genome and amoA-based analyses. In addition to the key genes for ammonia and nitrite oxidation, the three recovered genomes contained a complete urea utilization pathway. Our findings suggest that the urea present in the urine media played a significant role in the selective enrichment of these novel comammox Nitrospira, and support the diversity and versatility of their metabolism.

The recent discovery of comammox Nitrospira, a complete ammonia oxidizer, capable of completing the nitrification on their own has presented tremendous challenges to our understanding of the nitrification process. There are two divergent clades of comammox Nitrospira, Clade A and B. However, their population abundance, community structure and role in ammonia and nitrite oxidation are poorly understood. We conducted a 94-day microcosm study using a grazed dairy pasture soil amended with urea fertilizers, synthetic cow urine, and the nitrification inhibitor, dicyandiamide (DCD), to investigate the growth and community structure of comammox Nitrospira spp. We discovered that comammox Nitrospira Clade B was two orders of magnitude more abundant than Clade A in this fertile dairy pasture soil and the most abundant subcluster was a distinctive phylogenetic uncultured subcluster Clade B2. We found that comammox Nitrospira Clade B might not play a major role in nitrite oxidation compared to the role of canonical Nitrospira nitrite-oxidizers, however, comammox Nitrospira Clade B is active in nitrification and the growth of comammox Nitrospira Clade B was inhibited by a high ammonium concentration (700 kg synthetic urine-N ha–1) and the nitrification inhibitor DCD. We concluded that comammox Nitrospira Clade B: (1) was the most abundant comammox in the dairy pasture soil; (2) had a low tolerance to ammonium and can be inhibited by DCD; and (3) was not the dominant nitrite-oxidizer in the soil. This is the first study discovering a new subcluster of comammox Nitrospira Clade B2 from an agricultural soil.

No abstract available

Recent findings show that a subset of bacteria affiliated with Nitrospira, a genus known for its importance in nitrite oxidation for biological nutrient removal applications, are capable of complete ammonia oxidation (comammox) to nitrate. Early reports suggested that they were absent or present in low abundance in most activated sludge processes, and thus likely functionally irrelevant. Here we show the accumulation of comammox Nitrospira in a nitrifying sequencing batch reactor operated at low dissolved oxygen (DO) concentrations. Actual mainstream wastewater was used as influent after primary settling and an upstream pre-treatment process for carbon and phosphorus removal. The ammonia removal rate was stable and exceeded that of the treatment plant’s parallel full-scale high DO nitrifying activated sludge reactor. 16S rRNA sequencing showed a steady accumulation of Nitrospira to 53% total abundance and a decline in conventional ammonia oxidizing bacteria to <1% total abundance over 400+ days of operation. After ruling out other known ammonia oxidizers, qPCR confirmed the accumulation of comammox Nitrospira beginning around day 200, to eventually comprise 94% of all detected amoA and 4% of total bacteria by day 407. Quantitative fluorescence in-situ hybridization confirmed the increasing trend and high relative abundance of Nitrospira. These results demonstrate that comammox can be metabolically relevant to nitrogen transformation in wastewater treatment, and can even dominate the ammonia oxidizing community. Our results suggest that comammox may be an important functional group in energy efficient nitrification systems designed to operate at low DO levels.

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers (‘comammox’), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers will enable more encompassing studies of nitrifying microorganisms in diverse ecosystems.

Nitrous oxide (N2O) and NOy (nitrous acid (HONO) + nitric oxide (NO) + nitrogen dioxide (NO2)) are released as byproducts or obligate intermediates during aerobic ammonia oxidation, and further influence global warming and atmospheric chemistry. The ammonia oxidation process is catalyzed by groups of globally distributed ammonia-oxidizing microorganisms, which are playing a major role in atmospheric N2O and NOy emissions. Yet, little is known about HONO and NO2 production by the recently discovered, widely distributed complete ammonia oxidizers (comammox), able to individually perform the oxidation of ammonia to nitrate via nitrite. Here, we examined the N2O and NOy production patterns by comammox bacterium Nitrospira inopinata during aerobic ammonia oxidation, in comparison to its canonical ammonia-converting counterparts, representatives of the ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our findings, i) show low yield NOy production by the comammox bacterium compared to AOB; ii) highlight the role of the NO reductase in the biological formation of N2O based on results from NH2OH inhibition assays and its stimulation during archaeal and bacterial ammonia oxidations; iii) postulate that the lack of hydroxylamine (NH2OH) and NO transformation enzymatic activities may lead to a buildup of NH2OH/NO which can abiotically react to N2O ; iv) collectively confirm restrained N2O and NOy emission by comammox bacteria, an unneglectable consortium of microbes in global atmospheric emission of reactive nitrogen gases.

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.

The discovery of complete ammonia oxidation (comammox), oxidizing ammonia to nitrate via nitrite in a single organism, has redefined the traditional recognition of the two-step nitrification driven by two functional groups (ammonia-oxidizing and nitrite-oxidizing microorganisms). However, the understanding of the distribution and niche differentiation of comammox Nitrospira in the estuarine mudflats and their reclaimed agricultural soils is still limited. Here, we investigated the abundance, diversity and community structures of comammox Nitrospira in the mudflats and the reclaimed agricultural soils in the northern Yangtze River estuary. Quantitative PCR showed the abundances of amoA genes of comammox were lower than that of ammonia-oxidizing bacteria (AOB) in nearly all samples. Amplicon sequencing of amoA genes revealed that the community structures of comammox Nitrospira were significantly (P < 0.001) different between the original mudflats and the reclaimed agricultural soils, indicating niche differentiation among comammox Nitrospira clades (clade A.1, clade A.2, and clade B). The clade A.1 was the dominant group of comammox Nitrospira in the mudflats, while clade B predominated in the agricultural soils. However, the members of clade A.2 could be clearly divided into two groups, the mudflat-preferred and agricultural soil-preferred groups, suggesting more complicated ecological preferences within this sub-clade. Furthermore, it was demonstrated that salinity, organic matter (OM) and NO3–-N had a significantly influence on the distribution of comammox Nitrospira in the estuarine environment. Clade A.1 and nearly half members of clade A.2 were positively correlated with salinity, and negatively correlated with the concentrations of OM and NO3–-N. In contrast, the clade B and the other half members of clade A.2 showed the exact opposite pattern: a negative correlation with salinity and positive correlation with OM and NO3–-N. The co-occurrence network demonstrated that the operational taxonomic units (OTUs) within the same (sub-)clade were mostly positively correlated, indicating the similar niche preferences among the members from the same (sub-)clade of comammox Nitrospira. Taken together, our results revealed the niche differentiation of comammox Nitrospira in estuarine ecosystems where salinity and OM were the primary factors responsible for the distinct ecological distribution patterns.

Benzimidazole fungicides are frequently detected in aquatic environments and pose a serious health risk. Here, we investigated the metabolic capacity of the recently discovered complete ammonia-oxidizing (comammox) Nitrospira inopinata and kreftii to transform a representative set of benzimidazole fungicides (i.e., benzimidazole, albendazole, carbendazim, fuberidazole, and thiabendazole). Ammonia-oxidizing bacteria and archaea, as well as the canonical nitrite-oxidizing Nitrospira exhibited no or minor biotransformation activity towards all the five benzimidazole fungicides. In contrast, the investigated comammox bacteria actively transformed all the five benzimidazole fungicides, except for thiabendazole. The identified transformation products indicated hydroxylation, S-oxidation, and glycosylation as the major biotransformation pathways of benzimidazole fungicides. We speculated that these reactions were catalyzed by comammox-specific ammonia monooxygenase, cytochrome P450 monooxygenases, and glycosylases, respectively. Interestingly, the exposure to albendazole enhanced the expression of the antibiotic resistance gene acrB of Nitrospira inopinata, suggesting that some benzimidazole fungicides could act as environmental stressors that trigger cellular defense mechanisms. Altogether, this study demonstrated the distinct substrate specificity of comammox bacteria towards benzimidazole fungicides and implies their significant roles in the biotransformation of these fungicides in nitrifying environments.

A better understanding of r-/K-strategists nitrifiers will help to balance the design and operation of bioprocesses for efficient pollution removal from wastewater. The objectives of study were to investigate the nitrite oxidation biokinetics, biofilm property, microbial community and quorum sensing (QS) of nitrifying biofilm in a continuously flow reactor (CFR) and a sequencing batch reactor (SBR). Results showed that nitrite-oxidizing bacteria were estimated to have a nitrite half saturation constant of 76.23 and 224.73 μM in CFR and SBR, respectively. High-throughput and metagenomic sequencing results showed that Nitrospira and Candidatus Nitrospira defluvii were the dominated nitrite-oxidizing taxa performing nitrite oxidation in both reactors. Nitrifying biofilm developed in CFR and SBR showed obviously different properties. Biofilm in SBR had an obviously higher ratio of polysaccharide and protein in extracellular polymeric substances, and higher thickness than in CFR. Metagenomics and chemical analysis revealed various types of acyl-homoserine lactone (AHL) circuit genes (e.g., luxI, lasI, hdtS) and four types of AHL signaling substances (e.g., C6-HSL, C8-HSL, C10-HSL and 3-oxo-C10-HSL) in nitrifying biofilm. The concentrations of these AHLs in biomass and water phases were obviously higher in SBR than that in CFR. Together, AHLs-based QS might affect the formation of nitrifying biofilm and thus contribute to the different biokinetics of Nitrospira in CFR and SBR. Our insights may reveal the molecular mechanism of Nitrospira for different biokinetics, and indicate the AHL association with Nitrospira adaptation to various conditions.

Nitrospirales , including the genus Nitrospira , are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO_2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira ). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales . In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named “ Ca . Nitronereus thalassa”, represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, “ Ca . Nitronereus thalassa” harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, “ Ca . Nitronereus thalassa” appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

Nitrification, the oxidation of ammonia to nitrate via nitrite, is an important process in municipal wastewater treatment plants (WWTPs). Members of the Nitrospira genus that contribute to complete ammonia oxidation (comammox) have only recently been discovered and their relevance to engineered water treatment systems is poorly understood. This study investigated distributions of Nitrospira, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) in biofilm samples collected from tertiary rotating biological contactors (RBCs) of a municipal WWTP in Guelph, Ontario, Canada. Using quantitative PCR (qPCR), 16S rRNA gene sequencing, and metagenomics, our results demonstrate that Nitrospira species strongly dominate RBC biofilm samples and that comammox Nitrospira outnumber all other nitrifiers. Genome bins recovered from assembled metagenomes reveal multiple populations of comammox Nitrospira with distinct spatial and temporal distributions, including several taxa that are distinct from previously characterized Nitrospira members. Diverse functional profiles imply a high level of niche heterogeneity among comammox Nitrospira, in contrast to the sole detected AOA representative that was previously cultivated and characterized from the same RBC biofilm. Our metagenome bins also reveal two cyanase-encoding populations of comammox Nitrospira, suggesting an ability to degrade cyanate, which has only been shown previously for several Nitrospira representatives that are strict nitrite oxidizers. This study demonstrates the importance of RBCs as model systems for continued investigation of environmental factors that control the distributions and activities of AOB, AOA, comammox Nitrospira, and other nitrite oxidizers.

A bacterium capable of complete ammonia oxidation (comammox) has been widely found in various environments, whereas its industrial application is limited due to the difficulty of cultivation and/or enrichment. We developed a biological system to produce a high-quality nitrate solution for use in hydroponic fertilizer. The system was composed of two separate reactors for ammonification and nitrification and was found to have a stable and efficient performance in the conversion of organic nitrogen to nitrate. To determine the key microbes involved and better understand the system, the microbial communities in the reactors were analyzed by 16S rRNA gene sequencing in combination with a shotgun metagenomic analysis. Canonical ammonia-oxidizing bacteria, which can only catalyze the oxidation of ammonia to nitrite, were detected with negligible relative abundances, while a comammox Nitrospira-related operational taxonomic unit (OTU) dominated the nitrification reactor. Furthermore, the comammox-type ammonia monooxygenase was found to be 500 times more highly expressed than the canonical one by quantitative PCR, indicating that comammox was the main driver of the stable and efficient ammonia oxidation in the system. A microbial co-occurrence analysis revealed a strong positive correlation between Nitrospira and several OTUs, some of which, such as Anaerolinea OTU, have been found to co-exist with comammox Nitrospira in the biofilms of water treatment systems. Given that these OTUs were abundant only on microbe-attached carriers in the system, their co-existence within the biofilm could be beneficial to stabilize the Nitrospira abundance, possibly by physically preventing oxygen exposure as well as cell spillage.

The recent discovery of comammox Nitrospira as complete nitrifiers has significantly enriched our understanding on the nitrogen cycle, yet little is known about their metabolic transcripts in natural aquatic ecosystems. Using the genome-centric metatranscriptomics, we provided the first in-situ expression patterns of comammox Nitrospira along the Yangtze River. Our study confirmed widespread expressions of comammox Nitrospira, with the highest transcription accounting for 33.3% and 63.8% of amoA and nxrAB genes expressed in ammonia-oxidizing prokaryotes (AOPs) and Nitrospira sublineages I/II, respectively. Moreover, comammox two clades differed in nitrification, with clade A acting as the dominator to ammonia oxidation in comammox, and clade B contributing more transcripts to nitrite oxidation than to ammonia oxidation. Compared to canonical Nitrospira, comammox community had lower expressions of ammonia/nitrite transporters and nitrogen assimilatory genes, but far higher expressions in urea transport and hydrolysis, facilitating to derivation of ammonia and energy mainly through intracellular ureolytic metabolism. This suggests no need for "reciprocal-feeding" between canonical Nitrospira and AOPs in a natural river. Aerobic mixotrophy of comammox bacteria was suggested by expressions of genes coding for respiratory complexes I-V, oxidative/reductive TCA cycle, oxygen stress defenses, and transport/catabolism of simple carbohydrates and low-biosynthetic-cost amino acids. Intriguingly, significant positive correlations among expressions of ammonia monooxygenases, hydroxylamine dehydrogenase and copper-dependent nitrite reductase indicated that comammox Nitrospira had the potential of converting nitrite to nitric oxide accompanied by ammonia oxidation under low-C/N and aerobic conditions, while gene expressions in this pathway were significantly and positively associated with pH. Overall, this study illustrated novel transcriptional characteristics of comammox Nitrospira, and highlighted the necessity of reassessing their contributions to biogeochemical carbon and nitrogen cycling with perspective of in-situ meta-omics as well as culture experiments.

Summary The recently discovered comammox process encompasses both nitrification steps, the aerobic oxidation of ammonia and nitrite, in a single organism. All known comammox bacteria are affiliated with Nitrospira sublineage II and can be grouped into two distinct clades, referred to as A and B, based on ammonia monooxygenase phylogeny. In this study, we report high‐quality draft genomes of two novel comammox Nitrospira from the terrestrial subsurface, representing one clade A and one clade B comammox organism. The two metagenome‐assembled genomes were compared with other representatives of Nitrospira sublineage II, including both canonical and comammox Nitrospira. Phylogenomic analyses confirmed the affiliation of the two novel Nitrospira with comammox clades A and B respectively. Based on phylogenetic distance and pairwise average nucleotide identity values, both comammox Nitrospira were classified as novel species. Genomic comparison revealed high conservation of key metabolic features in sublineage II Nitrospira, including respiratory complexes I–V and the machineries for nitrite oxidation and carbon fixation via the reductive tricarboxylic acid cycle. In addition, the presence of the enzymatic repertoire for formate and hydrogen oxidation in the Rifle clades A and B comammox genomes, respectively, suggest a broader distribution of these metabolic features than previously anticipated.

Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. ABSTRACT Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [13C]bicarbonate and [13C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira’s in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.

Background The discovery of microorganisms capable of complete ammonia oxidation to nitrate (comammox) has prompted a paradigm shift in our understanding of nitrification, an essential process in N cycling, hitherto considered to require both ammonia oxidizing and nitrite oxidizing microorganisms. This intriguing metabolism is unique to the genus Nitrospira , a diverse taxon previously known to only contain canonical nitrite oxidizers. Comammox Nitrospira have been detected in diverse environments; however, a global view of the distribution, abundance, and diversity of Nitrospira species is still incomplete. Results In this study, we retrieved 55 metagenome-assembled Nitrospira genomes (MAGs) from newly obtained and publicly available metagenomes. Combined with publicly available MAGs, this constitutes the largest Nitrospira genome database to date with 205 MAGs, representing 132 putative species, most without cultivated representatives. Mapping of metagenomic sequencing reads from various environments against this database enabled an analysis of the distribution and habitat preferences of Nitrospira species. Comammox Nitrospira ’s ecological success is evident as they outnumber and present higher species-level richness than canonical Nitrospira in all environments examined, except for marine and wastewaters samples. The type of environment governs Nitrospira species distribution, without large-scale biogeographical signal. We found that closely related Nitrospira species tend to occupy the same habitats, and that this phylogenetic signal in habitat preference is stronger for canonical Nitrospira species . Comammox Nitrospira eco-evolutionary history is more complex, with subclades achieving rapid niche divergence via horizontal transfer of genes, including the gene encoding hydroxylamine oxidoreductase, a key enzyme in nitrification. Conclusions Our study expands the genomic inventory of the Nitrospira genus, exposes the ecological success of complete ammonia oxidizers within a wide range of habitats, identifies the habitat preferences of (sub)lineages of canonical and comammox Nitrospira species, and proposes that horizontal transfer of genes involved in nitrification is linked to niche separation within a sublineage of comammox Nitrospira. Video Abstract

In this study, we sought to investigate the impact of urbanization, the presence of concrete river bottoms, and nutrient pollution on microbial communities along the L.A. River. Six molecular markers were evaluated for the identification of bacteria, plants, fungi, fish, and invertebrates in 90 samples. PCA (principal components analysis) was used along with PAM (partitioning around medoids) clustering to reveal community structure, and an NB (negative binomial) model in DESeq2 was used for differential abundance analysis. PCA and factor analysis exposed the main axes of variation but were sensitive to outliers. The differential abundance of Proteobacteria was associated with soft-bottom sites, and there was an apparent balance in the abundance of bacteria responsible for nitrogen cycling. Nitrogen cycling was explained via ammonia-oxidizing archaea; the complete ammonia oxidizers, Nitrospira sp.; nitrate-reducing bacteria, Marmoricola sp.; and nitrogen-fixing bacteria Devosia sp., which were differentially abundant at soft-bottom sites (p adj < 0.002). In contrast, the differential abundance of several cyanobacteria and other anoxygenic phototrophs was associated with the impervious sites, which suggested the accumulation of excess nitrogen. The soft-bottom sites tended to be represented by a differential abundance of aerobes, whereas the concrete-associated species tended to be alkaliphilic, saliniphilic, calciphilic, sulfate dependent, and anaerobic. In the Glendale Narrows, downstream from multiple water reclamation plants, there was a differential abundance of cyanobacteria and algae; however, indicator species for low nutrient environments and ammonia-abundance were also present. There was a differential abundance of ascomycetes associated with Arroyo Seco and a differential abundance of Scenedesmaceae green algae and cyanobacteria in Maywood, as seen in the analysis that compared suburban with urban river communities. The proportion of Ascomycota to Basidiomycota within the L.A. River differed from the expected proportion based on published worldwide freshwater and river 18S data; the shift in community structure was most likely associated with the extremes of urbanization. This study indicates that extreme urbanization can result in the overrepresentation of cyanobacterial species that could cause reductions in water quality and safety.

Rice sheath blight pathogen, Rhizoctonia solani, produces numerous sclerotia to overwinter. As a rich source of nutrients in the soil, sclerotia may lead to the change of soil microbiota. For this purpose, we amended the sclerotia of R. solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points. At the phyla level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control. An increased abundance of ammonia-oxidizing bacterium (AOB) Nitrosospira and Nitrite oxidizing bacteria (NOB) i.e., Nitrospira was observed, where the latter is reportedly involved in the nitrifier denitrification. Moreover, Thiobacillus, Gemmatimonas, Anaeromyxobacter and Geobacter, the vital players in denitrification, N2O reduction and reductive nitrogen transformation, respectively, depicted enhanced abundance in R. solani sclerotia-amended samples. Furthermore, asymbiotic nitrogen-fixing bacteria, notably, Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control. Plant growth promoting bacteria, such as Kribbella, Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil. As per our knowledge, this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R. solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

The cyclic nature of specific conversions in the nitrogen cycle imposes strict limitations to the conversions observed in nature and explains for example why anaerobic ammonium oxidation (anammox) bacteria can only use nitrite – and not nitrate – as electron acceptor in catabolism, and why nitrite is required as additional electron donor for inorganic carbon fixation in anabolism. Furthermore, the biochemistry involved in nitrite-dependent anaerobic methane oxidation excludes the feasibility of using nitrate as electron acceptor. Based on the cyclic nature of these nitrogen conversions, we propose two scenarios that may explain the ecological role of recently discovered complete ammonia-oxidizing (comammox) Nitrospira spp., some of which were initially found in a strongly oxygen limited environment: (i) comammox Nitrospira spp. may actually catalyze an anammox-like metabolism using a biochemistry similar to intra-oxic nitrite-dependent methane oxidation, or (ii) scavenge all available oxygen for ammonia activation and use nitrate as terminal electron acceptor. Both scenarios require the presence of the biochemical machinery for ammonia oxidation to nitrate, potentially explaining a specific ecological niche for the occurrence of comammox bacteria in nature.

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The discovery of complete aerobic and anaerobic ammonia-oxidizing bacteria (Comammox and Anammox) significantly altered our understanding of the global nitrogen cycle. A high affinity for ammonia (Km(app),NH3 ≈ 63nM) and oxygen place the first described isolate, Comammox Nitrospira inopinata in the same trophic category as organisms such as some ammonia-oxidizing archaea. However, N. inopinata has a relatively low affinity for nitrite (Km,NO2 ≈ 449.2μM) suggesting it would be less competitive for nitrite than other nitrite-consuming aerobes and anaerobes. We examined the ecological relevance of the disparate substrate affinities by coupling it with Anammox (Nitrospira inopinata and Brocadia anammoxidans, respectively). Synthetic communities were established in hydrogel granules in which Comammox grew in the aerobic outer layer to provide Anammox with nitrite in the inner anoxic core to form dinitrogen gas. This spatial organization was confirmed with FISH imaging, supporting a mutualistic or commensal relationship. Successful co-habitation of Comammox N. inopinata and Anammox in synthetic granules broadens our limited understanding of the interplay between these two species and offers potential biotechnological applications to study any type of bacterial pairings in a systematic and reproducible manner.

ABSTRACT Nitrification is an essential process within the global nitrogen cycle and also occurs under extreme conditions, such as in geothermal environments. The nitrite‐oxidizing group Nitrospira represents key nitrifiers in these systems, as several species inhabit hot springs worldwide. Using different initial incubation temperatures, two novel moderately thermophilic Nitrospira enrichments, Nitrospira sp. Vd2 and Ca. N. neuquenensis E2OT, were obtained from sulfur‐rich mud pools in the geothermal field Las Máquinas (Neuquén Province, Argentina). Nitrospira sp. Vd2 belongs to the N. bockiana lineage V, whereas the second enrichment (E2OT) represents the novel taxonomic lineage VIII, together with cultures from Kamchatka (Kam‐Ns4a) and Garga hot springs (Ga3a). The vibrioid morphology of Ca. N. neuquenensis E2OT is strikingly different from all described, twisted rod‐shaped Nitrospira. Our study expands the knowledge of the taxonomic and genomic diversity of moderately thermophilic Nitrospira, by comparing the high‐quality draft genomes with those of previously described species. The recent discovery of quorum‐sensing genes outside the Nitrospira lineage II was confirmed for both Argentinian cultures. Notably, the genome GC contents of the enrichments Vd2 and E2OT are 60.6% and 69.4%, respectively. The latter is the highest observed for Nitrospira to date and might support thermotolerance up to 50°C.

Nitrous oxide (N2O) and nitric oxide (NO) are atmospheric trace gases that contribute to climate change and affect stratospheric and ground-level ozone concentrations. Ammonia oxidizing bacteria (AOB) and archaea (AOA) are key players in the nitrogen cycle and major producers of N2O and NO globally. However, nothing is known about N2O and NO production by the recently discovered and widely distributed complete ammonia oxidizers (comammox). Here, we show that the comammox bacterium Nitrospira inopinata is sensitive to inhibition by an NO scavenger, cannot denitrify to N2O, and emits N2O at levels that are comparable to AOA but much lower than AOB. Furthermore, we demonstrate that N2O formed by N. inopinata formed under varying oxygen regimes originates from abiotic conversion of hydroxylamine. Our findings indicate that comammox microbes may produce less N2O during nitrification than AOB. Ammonia-oxidizing bacteria and archaea are major producers of the gases nitrous oxide and nitric oxide. Here, Kits et al. show that a complete ammonia-oxidizing (comammox) bacterium emits nitrous oxide at levels that are comparable to those produced by ammonia-oxidizing archaea.

Due to global change, increasing nutrient input to ecosystems dramatically affects the nitrogen cycle, especially the nitrification process. Nitrifiers including ammonia-oxidizing archaea (AOAs), ammonia-oxidizing bacteria (AOBs), nitrite-oxidizing bacteria (NOBs), and recently discovered complete ammonia oxidizers (comammoxs) perform nitrification individually or in a community. However, much remains to be learned about their niche differentiation, coexistence, and interactions among those metabolically distinct nitrifiers. Here, we used synthetic microbial ecology approaches to construct synthetic nitrifying communities (SNCs) with different combinations of Nitrospira inopinata as comammox, Nitrososphaera gargensis as AOA, Nitrosomonas communis as AOB, and Nitrospira moscoviensis as NOB. Our results showed that niche differentiation and potential interactions among those metabolically distinct nitrifiers were determined by their kinetic characteristics. The dominant species shifted from N. inopinata to N. communis in the N4 community (with all four types of nitrifiers) as ammonium concentrations increased, which could be well explained by the kinetic difference in ammonia affinity, specific growth rate, and substrate tolerance of nitrifiers in the SNCs. In addition, a conceptual model was developed to infer niche differentiation and possible interactions among the four types of nitrifiers. This study advances our understanding of niche differentiation and provides new strategies to further study their interactions among the four types of nitrifiers.

Complete ammonia oxidizers (comammox), able to individually oxidize ammonia to nitrate, are considered to play a significant role in the global nitrogen cycle. However, the distribution of comammox Nitrospira in estuarine tidal flat wetland and the environmental drivers affecting their abundance and diversity remain unknown. Here, we present a large-scale investigation on the geographical distribution of comammox Nitrospira along the estuarine tidal flat wetlands of China, where comammox Nitrospira were successfully detected in 9 of the 16 sampling sites. The abundance of comammox Nitrospira ranged from 5.19 × 103 to 8.33 × 104 copies/g, 2.21 to 5.44-folds lower than canonical ammonia oxidizers: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Phylogenetic analysis based on the alpha subunit of the ammonia monooxygenase encoding gene (amoA) revealed that comammox Nitrospira Clade A, mainly originating from upstream river inputs, accounts for more than 80% of the detected comammox Nitrospira, whereas comammox Nitrospira clade B were rarely detected. Comammox Nitrospira abundance and dominant comammox Nitrospira OTUs varied within the estuarine samples, showing a geographical pattern. Salinity and pH were the most important environmental drivers affecting the distribution of comammox Nitrospira in estuarine tidal flat wetlands. The abundance of comammox Nitrospira was further negatively correlated with high ammonia and nitrite concentrations. Altogether, this study revealed the existence, abundance and distribution of comammox Nitrospira and the driving environmental factors in estuarine ecosystems, thus providing insights into the ecological niches of this recently discovered nitrifying consortium and their contributions to nitrification in global estuarine environments.

Water reservoir sediments represent a distinct habitat that harbors diverse microbial resources crucial for nitrogen cycling processes. The discovery of resuscitation promoting factor (Rpf) has been recognized as a crucial development in understanding the potential of microbial populations. However, our understanding of the relationship between microorganisms containing rpf-like genes and nitrogen-cycling functional populations remains limited. The present study explored the distribution patterns of rpf-like genes and nitrogen-cycling genes in various water reservoir sediments, along with their correlation with environmental factors. Additionally, the co-occurrence of rpf-like genes with genes associated with the nitrogen cycle and viable but non-culturable (VBNC) formation was investigated. The findings indicated the ubiquitous occurrence of Rpf-like domains and their related genes in the examined reservoir sediments. Notably, rpf-like genes were predominantly associated with Bradyrhizobium, Nitrospira, and Anaeromyxobacter, with pH emerging as the primary influencing factor for their distribution. Genera such as Nitrospira, Bradyrhizobium, Anaeromyxobacter, and Dechloromonas harbor the majority of nitrogen-cycling functional genes, particularly denitrification genes. The distribution of nitrogen-cycling microbial communities in the reservoir sediments was mainly influenced by pH and NH4+. Notably, correlation network analysis revealed close connections between microorganisms containing rpf-like genes and nitrogen-cycling functional populations, as well as VBNC bacteria. These findings offer new insights into the prevalence of rpf-like genes in the water reservoir sediments and their correlation with nitrogen-cycling microbial communities, enhancing our understanding of the significant potential of microbial nitrogen cycling.

Apparent contribution of complete ammonia-oxidizing organisms (comammox) to the global nitrogen cycle highlights the necessity for understanding niche differentiation of comammox bacteria among other ammonia oxidizers. While the high affinity for ammonia of the comammox species Nitrospira inopinata suggests their niche partitioning is expected to be centered in oligotrophic environments, their absence in nutrient-depleted environments (such as the oceans) suggests that other (abiotic) factors might control their distribution and spatial localization within microbial communities. Many ammonia- and nitrite-oxidizing organisms are sensitive to light; however, the photosensitivity of comammox has not been explored. Since comammox bacteria encode enzymatic machinery homologous to canonical ammonia-and nitrite-oxidizers, we hypothesized that comammox N. inopinata, the only available pure culture of this group of microorganisms, may be inhibited by illumination in a similar manner. We evaluated the impact of light intensity, wavelength, and duration on the degree of photoinhibition for cultures of the comammox species N. inopinata and the soil ammonia-oxidizing archaea Nitrososphaera viennensis. Both species were highly sensitive to light. Interestingly, mimicking diurnal light exposure caused an uncoupling of ammonia and nitrite oxidation in N. inopinata, indicating nitrite oxidation might be more sensitive to light exposure than ammonia oxidation. It is likely that light influences comammox spatial distribution in natural environments such as surface fresh waters according to diurnal cycles, light attenuation coefficients, and the light penetration depths. Our findings therefore provide ecophysiological insights for further studies on comammox both in field and laboratory settings.

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Nitrite-oxidizing bacteria (NOB) are vital players in the global nitrogen cycle that convert nitrite to nitrate during the second step of nitrification. Within this functional guild, members of the genus Nitrospira are most widespread, phylogenetically diverse, and physiologically versatile, and they drive nitrite oxidation in many natural and engineered ecosystems. Despite their ecological and biotechnological importance, our understanding of their energy metabolism is still limited. A major bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing and fastidious microorganisms. In this study, we cultivated Nitrospira moscoviensis under nitrite-oxidizing conditions in a continuous stirred tank reactor (CSTR) system. This cultivation setup enabled accurate control of physicochemical parameters and avoided fluctuating levels of their energy substrate nitrite, thus ensuring constant growth conditions and furthermore allowing continuous biomass harvesting. Transcriptomic analyses under these conditions supported the predicted core metabolism of N. moscoviensis, including expression of all proteins required for carbon fixation via the reductive tricarboxylic acid cycle, assimilatory nitrite reduction, and the complete respiratory chain. Here, simultaneous expression of multiple copies of respiratory complexes I and III suggested functional differentiation. The transcriptome also indicated that the previously assumed membrane-bound nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation, is formed by three soluble subunits. Overall, the transcriptomic data greatly refined our understanding of the metabolism of Nitrospira. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of these fascinating microorganisms.

With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3− uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments. ABSTRACT Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira. Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2− in the presence of 13C-HCO3− (labeled) or 12C-HCO3− (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3− uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome. IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3− uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

Nitrification directly contributes to the ammonia removal in sponges and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei.

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Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

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Marine microorganisms are important for the global nitrogen cycle, but marine nitrifiers, especially aerobic nitrite oxidizers, remain largely unexplored. To increase the number of cultured representatives of marine nitrite-oxidizing bacteria (NOB), a bioreactor cultivation approach was adopted to first enrich nitrifiers and ultimately nitrite oxidizers from Dutch coastal North Sea water. With solely ammonia as the substrate an active nitrifying community consisting of novel marine Nitrosomonas aerobic ammonia oxidizers (ammonia-oxidizing bacteria) and Nitrospina and Nitrospira NOB was obtained which converted a maximum of 2 mmol of ammonia per liter per day. Switching the feed of the culture to nitrite as a sole substrate resulted in a Nitrospira NOB dominated community (approximately 80% of the total microbial community based on fluorescence in situ hybridization and metagenomic data) converting a maximum of 3 mmol of nitrite per liter per day. Phylogenetic analyses based on the 16S rRNA gene indicated that the Nitrospira enriched from the North Sea is a novel Nitrospira species with Nitrospira marina as the next taxonomically described relative (94% 16S rRNA sequence identity). Transmission electron microscopy analysis revealed a cell plan typical for Nitrospira species. The cytoplasm contained electron light particles that might represent glycogen storage. A large periplasmic space was present which was filled with electron dense particles. Nitrospira-targeted polymerase chain reaction analyses demonstrated the presence of the enriched Nitrospira species in a time series of North Sea genomic DNA samples. The availability of this new Nitrospira species enrichment culture facilitates further in-depth studies such as determination of physiological constraints, and comparison to other NOB species.

Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2−) to nitrate (NO3−), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.

Nitrification is a central process in the global nitrogen cycle, yet the ecological niches and growth strategies of ammonia-oxidizing microorganisms in oligotrophic environments remain poorly understood. To investigate the ecological responses of complete ammonia oxidizers (Comammox) and ammonia-oxidizing archaea (AOA), a membrane bioreactor (MBR) system with two distinct ammonia loading rates (ALRs) was employed in this study. Metagenomic and meta-transcriptomic analyses showed that Comammox species-including Candidatus Nitrospira nitrosa and Candidatus Nitrospira inopinata-underwent a pronounced "bloom" only at high ALR, where their DNA and mRNA relative abundances reached 4.7 % and 5.63 %, respectively. Meanwhile, AOA steadily increased under both high and low ALR in oligotrophic environments. Network analysis further indicated stronger cooperative interactions between Comammox and AOA in higher ALR, highlighting distinct ecological strategies that underpin ammonia oxidation in oligotrophic environments. These findings not only support the development of low-carbon nitrogen removal processes in wastewater treatment but also clarify the impact of nitrogen loading on the distribution of ammonia-oxidizing microorganisms in natural ecosystems and provide insights into the origin and evolutionary pathways of these essential microbes.

Aquaponic systems are sustainable solutions for food production combining fish growth (aquaculture) and production of vegetables (hydroponic) in one recirculating system. In aquaponics, nitrogen-enriched wastewater from fish in the aquaculture serves as fertilizer for the plants in the hydroponics, while the nitrogen-depleted and detoxified water flows back to the aquaculture. To investigate bacterial nitrogen-cycling in such an aquaponic system, measurements of nitrogen species were coupled with time-resolved 16S rRNA gene profiling and the functional capacity of organisms was studied using metagenomics. The aquaponic system was consistently removing ammonia and nitrite below 23 µM and 19 µM, and nitrate to steady-state concentrations of about 0.5 mM. 16S rRNA gene amplicon sequencing of sediments exposed in the pump sump revealed that typical signatures of canonical ammonia-oxidising microorganisms were below detection limit. However, one of the most abundant operational taxonomic units (OTU) was classified as a member of the genus Nitrospira with a relative abundance of 3.8%. For this genus, also genome scaffolds were recovered encoding the only ammonia monooxygenase genes identified in the metagenome. This study indicates that even in highly efficient aquaponic systems, comammox Nitrospira were found to participate in ammonium removal at low steady-state ammonia concentrations.

Chemoautotrophic ammonia-oxidizers and nitrite-oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long-term fertilization regimes remain largely under-investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira-like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long-term fertilization shifted the composition of active Nitrospira-like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.

An anoxic-aerobic membrane bioreactor (AO-MBR), an anaerobic side-stream reactor (ASSR) coupled MBR (A-MBR), and an MBR with ASSR packed with carriers (AP-MBR) were operated parallelly to investigate biological nutrient removal, microbial community structure and mass balance of nutrients in sludge reduction systems. Compared to AO-MBR, A-MBR and AP-MBR were both efficient in COD and NH4+-N removal, had significantly higher nitrogen removal, reduced sludge production by 35.0% and 45.9%, but deteriorated biological phosphorus removal. Nitrosomonadaceae and Nitrospira were major bacteria responsible for ammonium and nitrite oxidation in the three systems. Inserting ASSR and packing carriers both favored denitrifying bacteria enrichment and organic substances release, and thus resulted in higher nitrate uptake rate (NUR) in the anoxic tank. Higher endogenous NUR in ASSR than in anoxic tank also indicated that ASSR and carriers both accelerated sludge decay. Denitrification and sludge reduction occurred in ASSR played important roles in biological nutrient removal.

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Nitrate production during anammox can decrease total nitrogen removal efficiency, which will negatively impact its usefulness for the removal of nitrogen from waste streams. However, neither the performance characteristics nor physiological shifts associated with nitrate accumulation in anammox reactors under different nitrogen loading rates (NLRs) is well understood. Consequently, these parameters were studied in a lower NLR anammox reactor, termed R1, producing higher than expected levels of nitrate and compared with a higher NLR reactor, termed R2, showing no excess nitrate production. While both reactors showed high NH4+-N removal efficiencies (>90%), the total nitrogen removal efficiency (<60%) was much lower in R1 due to higher nitrate production. Metagenomic analysis found that the number of reads derived from anammox bacteria were significantly higher in R2. Another notable trend in reads occurrence was the relatively higher levels of reads from genes predicted to be nitrite oxidoreductases (nxr) in R1. Binning yielded 27 high quality draft genomes from the two reactors. Analysis of bin occurrence found that R1 showing both a decrease in anammox bacteria and an unexpected increase in nxr. In-situ assays confirmed that R1 had higher rates of nitrite oxidation to nitrate and suggested that it was not solely due to obligate NOB, but other nxr-containing bacteria are important contributors as well. Our results demonstrate that nitrate accumulation can be a serious operational concern for the application of anammox technology to low-strength wastewater treatment and provide insight into the community changes leading to this outcome.

The recent discovery of Nitrospira species capable of complete ammonia oxidation (comammox) in non-marine natural and engineered ecosystems under mesothermal conditions has changed our understanding of microbial nitrification. However, little is known about the occurrence of comammox bacteria or their ability to survive in moderately thermal and/or hyperthermal habitats. Here, we report the wide distribution of comammox Nitrospira in five terrestrial hot springs at temperatures ranging from 36 to 80°C and provide metagenome-assembled genomes of 11 new comammox strains. Interestingly, the identification of dissimilatory nitrate reduction to ammonium (DNRA) in thermophilic comammox Nitrospira lineages suggests that they have versatile ecological functions as both sinks and sources of ammonia, in contrast to the described mesophilic comammox lineages, which lack the DNRA pathway. Furthermore, the in situ expression of key genes associated with nitrogen metabolism, thermal adaptation, and oxidative stress confirmed their ability to survive in the studied hot springs and their contribution to nitrification in these environments. Additionally, the smaller genome size and higher GC content, less polar and more charged amino acids in usage profiles, and the expression of a large number of heat shock proteins compared to mesophilic comammox strains presumably confer tolerance to thermal stress. These novel insights into the occurrence, metabolic activity, and adaptation of comammox Nitrospira in thermal habitats further expand our understanding of the global distribution of comammox Nitrospira and have significant implications for how these unique microorganisms have evolved thermal tolerance strategies.

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The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Abstract Nitrite‐oxidizing bacteria (NOB) catalyse the second nitrification step and are the main biological source of nitrate. The most diverse and widespread NOB genus is Nitrospira, which also contains complete ammonia oxidizers (comammox) that oxidize ammonia to nitrate. To date, little is known about the occurrence and biology of comammox and canonical nitrite oxidizing Nitrospira in extremely alkaline environments. Here, we studied the seasonal distribution and diversity, and the effect of short‐term pH changes on comammox and canonical Nitrospira in sediments of two saline, highly alkaline lakes. We identified diverse canonical and comammox Nitrospira clade A‐like phylotypes as the only detectable NOB during more than a year, suggesting their major importance for nitrification in these habitats. Gross nitrification rates measured in microcosm incubations were highest at pH 10 and considerably faster than reported for other natural, aquatic environments. Nitrification could be attributed to canonical and comammox Nitrospira and to Nitrososphaerales ammonia‐oxidizing archaea. Furthermore, our data suggested that comammox Nitrospira contributed to ammonia oxidation at an extremely alkaline pH of 11. These results identify saline, highly alkaline lake sediments as environments of uniquely strong nitrification with novel comammox Nitrospira as key microbial players.

Nitrospira are the most widespread and diverse known nitrite-oxidizing bacteria and key nitrifiers in natural and engineered ecosystems. Nevertheless, their ecophysiology and environmental distribution are understudied because of the recalcitrance of Nitrospira to cultivation and the lack of a molecular functional marker, which would allow the detection of Nitrospira in the environment. Here we introduce nxrB, the gene encoding subunit beta of nitrite oxidoreductase, as a functional and phylogenetic marker for Nitrospira. Phylogenetic trees based on nxrB of Nitrospira were largely congruent to 16S ribosomal RNA-based phylogenies. By using new nxrB-selective polymerase chain reaction primers, we obtained almost full-length nxrB sequences from Nitrospira cultures, two activated sludge samples, and several geographically and climatically distinct soils. Amplicon pyrosequencing of nxrB fragments from 16 soils revealed a previously unrecognized diversity of terrestrial Nitrospira with 1801 detected species-level operational taxonomic units (OTUs) (using an inferred species threshold of 95% nxrB identity). Richness estimates ranged from 10 to 946 coexisting Nitrospira species per soil. Comparison with an archaeal amoA dataset obtained from the same soils [Environ. Microbiol. 14: 525-539 (2012)] uncovered that ammonia-oxidizing archaea and Nitrospira communities were highly correlated across the soil samples, possibly indicating shared habitat preferences or specific biological interactions among members of these nitrifier groups.

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO2, and requires exogenous vitamin B12. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O2 concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb3-type terminal oxidase increased under O2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

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Nitrification is defined as conversion of the ammonium(NH4+-N) to nitrate and it is generally divided into two categories: autotrophic nitrification and heterotrophic nitrification. With the metagenomes retrieved from the Nitrospira highly enriched biosamples, both the ammonium oxidation gens (full set of AMO and hydroxylamine dehydrogenase (HAO) genes) and nitrite oxidoreductase (NXR) coding genes necessary for nitrite oxidation were found in a completely nitrifying bacterium from the genus Nitrospira. Various species of Nitrospira (containing phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes) had been found in many engineered systems and were proved to be comammox bacterium. Some anammox bacteria were found to use mono or dimethylamine and even methanol as electron donors, but the enzymes the employed in the special metabolism were not known. Many heterotrophic nitrifying bacteria have been isolated and characterized from soil, activated sludge, wastewater treatment system. A denitrification process was developed with poly(3-hydroxybutyrate) (PHB) granulesas source of carbon for denitrification. It is possible from metabolic foundations that the heterotrophic nitrifier can consume intracellular stored PHB as the alternative carbon source. This direction still waiting for further exploration.

ABSTRACT Nitrite-oxidizing bacteria (NOB) play a crucial role in global nitrogen cycling, yet their presence and adaptations in acidic environments remain poorly understood. This study unveils the cultivation and characterization of a novel acid-tolerant NOB, NS4 culture, affiliated with lineage II (Nitrospira_D) within the genus Nitrospira. Enriched and isolated from a freshwater pond sediment, NS4 culture exhibits remarkable physiological and genomic traits that shed light on NOB survival strategies in low pH conditions. NS4 culture demonstrates the optimal growth at pH 6 and 0.5 mM nitrite concentration, with a maximum growth rate of 0.62 day⁻¹. Kinetic analyses reveal a high affinity for nitrite (Km(app) = 4.02 µM), suggesting adaptation to oligotrophic environments. Phylogenomic and genomic-relatedness analyses position NS4 culture as a novel member within the genus Nitrospira, for which we propose as “Candidatus Nitrospira acidotolerans.” Genomic investigations indicate the presence of a complete reductive tricarboxylic acid cycle and genes for nitrite oxidation, confirming its chemolithoautotrophic lifestyle. Intriguingly, NS4 genome lacks complete pathways for cobalamin biosynthesis, implying a potential dependence on symbiotic partners for this essential cofactor. The NS4 genome harbors genes associated with acid resistance, including chaperones, transporters, and amino acid metabolism, suggesting a genetic potential for adaptation or resistance to low pH conditions. This discovery expands our understanding of NOB diversity and adaptability, offering insights into nitrogen cycling in acid-impacted ecosystems. The physiological and genomic traits of this acid-tolerant NOB open new insights for exploring the ecological significance of NOB in previously overlooked acidic habitats. IMPORTANCE Nitrite-oxidizing bacteria (NOB) are integral to the global nitrogen cycle, yet their adaptations to acidic environments remain poorly understood. This study introduces Candidatus Nitrospira acidotolerans, an acid-tolerant NOB highly enriched from freshwater pond sediment. By combining physiological and genomic analyses, this work reveals unique adaptations that enable survival and nitrite oxidation under low pH conditions. Notably, the NS4 culture demonstrates high nitrite affinity and resistance to acidic stress, suggesting its ecological significance in acid-impacted ecosystems. Additionally, NS4 genomic traits reveal genetic potential of metabolic dependencies, including reliance on symbiotic partners for cobalamin synthesis. These findings expand our understanding of NOB diversity and their role in nitrogen cycling under extreme conditions, offering novel insights into microbial ecology and potential applications in managing nitrogen processes in acidic environments. Nitrite-oxidizing bacteria (NOB) are integral to the global nitrogen cycle, yet their adaptations to acidic environments remain poorly understood. This study introduces Candidatus Nitrospira acidotolerans, an acid-tolerant NOB highly enriched from freshwater pond sediment. By combining physiological and genomic analyses, this work reveals unique adaptations that enable survival and nitrite oxidation under low pH conditions. Notably, the NS4 culture demonstrates high nitrite affinity and resistance to acidic stress, suggesting its ecological significance in acid-impacted ecosystems. Additionally, NS4 genomic traits reveal genetic potential of metabolic dependencies, including reliance on symbiotic partners for cobalamin synthesis. These findings expand our understanding of NOB diversity and their role in nitrogen cycling under extreme conditions, offering novel insights into microbial ecology and potential applications in managing nitrogen processes in acidic environments.

Background In the late stages of landfill operation, leachate becomes brackish and contains high concentrations of ammonia with limited organic carbon. At leachate treatment facilities, it is typically subjected to nitrification followed by denitrification, with methanol supplied as an external electron donor. This unique environment may harbor novel microorganisms, including nitrifiers. Although a variety of microorganisms are involved in nitrification, their substrate specificity and salinity tolerance remain insufficiently understood. In this study, a genome-centric metagenome analysis was conducted on the microbiome from a leachate treatment facility at a closed landfill. Results A total of 68 metagenome-assembled genomes (MAGs) were reconstructed, including 64 putative novel species. Among these, two Nitrospira MAGs were recovered: a novel complete ammonia-oxidizing bacterium (comammox), Nitrospira LAS72 (88.72% completeness, 2.10% contamination), and canonical nitrite-oxidizing Nitrospira LAS18 (99.98% completeness, 2.29% contamination). Comparative genomic analysis with 260 publicly available Nitrospira genomes revealed that LAS18 represents a new sub-lineage within lineage VII of the Nitrospira genus. Two ammonia-oxidizing archaea (AOA), Candidatus Nitrosocosmicus LAS21 and Nitrosarchaeum LAS73, were also identified, while canonical ammonia-oxidizing bacteria were not detected. Given the brackish conditions (1.23% salinity) and the methanol-fed operation of the treatment facility, the genomic potential for osmotic stress adaptation and methanol metabolism was investigated. Comammox Nitrospira LAS72 harbors biosynthetic pathways for several compatible solutes (osmoprotectants), including glycine betaine, proline, trehalose, and L-glutamate. Moreover, comammox Nitrospira LAS72 possesses genetic potential for oxidizing formaldehyde, suggesting that it may exploit these methanol-derived intermediates as energy sources. These features indicate that LAS72 may withstand osmotic fluctuations through the production of various osmoprotectants and thrive under the unique conditions of a methanol-fed environment. Conclusions The discovery of novel comammox Nitrospira and canonical Nitrospira forming a new sub-lineage within lineage VII of the Nitrospira genus in an ammonia-rich brackish environment provides the first genomic evidence for evolutionary adaptation among nitrifiers to saline, methanol-fed environments. These findings enhance our understanding of the ecological and evolutionary dynamics shaping nitrifier communities in complex treatment ecosystems. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-026-02351-7.

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Abstract Nitrification in aquarium biofilters transforms toxic ammonia (NH₃/NH₄+) into less toxic nitrate (NO₃-) via nitrite (NO₂-). Known freshwater aquarium nitrifiers include ammonia- and nitrite-oxidizing bacteria, ammonia-oxidizing archaea (AOA), and complete ammonia-oxidizing Nitrospira (CMX), with CMX recently shown to dominate most freshwater aquarium biofilters. However, little is known about nitrifier succession during aquarium establishment in home settings. Based on CMX prevalence in mature aquariums and the rapid growth of ammonia-oxidizing bacteria (AOB), we hypothesized that AOB initially dominate before CMX establish. To test this, we monitored microbial succession and water chemistry in three home aquariums over 12 weeks, collecting weekly samples from aquarium water, biofilter beads, and sponge filters. Biofilter DNA was analyzed via 16S rRNA gene sequencing and quantitative PCR (qPCR) targeting amoA genes. Nitrification reduced ammonia and nitrite to undetectable levels by week 3 in two aquariums and by week 8 in the third. Ammonia oxidizer detection by qPCR coincided with the onset of ammonia oxidation, with AOA preferentially colonizing biofilter beads. Metagenomic profiling of week 12 biofilter samples confirmed AOA and comammox Nitrospira amoA genes in all aquariums, along with nxrB genes from both comammox and canonical Nitrospira nitrite oxidizers. These results provide insight into the establishment of ammonia oxidizers in residential aquariums. Future work should explore factors influencing nitrifier community assembly, including inoculation sources (e.g. live plants, biological supplements), fish load, and water chemistry.

“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. ABSTRACT 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.

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Microbial community structures in mesophilic and low-temperature anammox and partial nitrification–anammox reactors were examined by a 16S rRNA–gene amplicon sequencing analysis. The anammox bacterium, Jettenia sp., was dominant, and nitrifying bacteria, including Nitrosomonas sp. (aerobic ammonia–oxidizing bacterium) and Nitrospira sp., (nitrite–oxidizing bacterium) coexisted in the bioreactors. Core coexisting bacteria, such as Sulfurisoma sp. and Zeimonas sp., showed oxygen-scavenging and NO3– reduction potentials. Sulfurisoma-related bacteria are distributed across wastewater treatment plants worldwide, particularly in denitrification systems. These results underscore the ecological and functional importance of microbial consortia in enhancing nitrogen removal efficiency.

Chlorella sp. and Navicula sp. were separately used to construct an algal-bacterial symbiotic system in two identical sequencing batch reactors (R1 and R2) to explore the influence of algal species differences on nitrite accumulation. The Navicula-bacterial symbiotic system showed a higher nitrite accumulation efficiency of 85% and a stronger resistance to ammonia load. It secreted twice as many extracellular polymeric substances than the Chlorella-bacterial symbiotic system. Nitrospira and SM1A02 were the dominant functional genera of nitrite-oxidizing bacteria in R1. The dominant functional genus of ammonium-oxidizing bacteria and the dominant functional genus of denitrifying bacteria were Ellin6067 and unclassified_Saprospiraceae in R2, respectively. In general, this research provided some reference for the construction of an algal-bacterial symbiotic system and achieving nitrite accumulation through an algal-bacterial symbiotic system.

The integration of powdered activated carbon and biofilm carriers in a membrane bioreactor (MBR) presents a promising approach to address the challenge of long hydraulic retention time (HRT) for nitrification of hydrolysed urine. This study investigated the effect of the incorporation in the MBR on microbial dynamics, focusing on dominant nitrifying bacteria. The results showed that significant shifts in microbial compositions were observed with the feed transition to full-strength urine and across different sludge growth forms. Remarkably, the nitrite-oxidizing bacteria Nitrospira were highly enriched in the suspended sludge. Simultaneously, ammonia-oxidizing bacteria, Nitrosococcaceae thrived in the attached biomass, showing a significant seven-fold increase in relative abundance compared to its suspended counterpart. Consequently, the incorporated MBR displayed 36% higher nitrification rate and 40% HRT reduction compared to the conventional MBR. This study provides valuable insights on the potential development of household or building scale on-site nutrient recovery from urine to fertiliser.

The aim of this study was to evaluate two moving bed biofilm reactors (MBBR) without nitrifying bacteria inoculation. Biofilms and viable bacterial colonies were evaluated after 124 days. MBBR bioreactors received water from Oreochromis niloticus fish farming and water quality parameters were monitored daily. Four distinct phases with different fish stocking density were established.: phase 1 (2.40 kg m-3), phase 2 (4.95 kg m-3), phase 3 (8.71 kg m-3) and phase 4 (12.23 kg m-3). The successful maturation of the bioreactors occurred around on the 100th experimental day when the nitration rate increased to 57 % in MBBR1 and 38 % in MBBR2. 105 species were identified in the biofilms, which were grouped into 65 genera, three of which were essential: Pseudomonas (21.7 %), Nitrospira (15.1 %) and Gemmobacter (11.2 %). MBBR start-up without bacterial inoculation is time-consuming, however, strengthened by important nitrifying groups.

The conventional functional microorganisms for nitrogen and phosphorus removal, such as Nitrosomonas, Nitrobacter, Nitrospira and Candidatus Accumulibacter, were hotspots in past research. However, the role of diverse unconventional functional microorganisms was neglected. In this study, a biofilm system was developed to explore the potential role of unconventional functional microorganisms in nutrients removal. According to the results of microbial community dynamics and metagenomics, complete ammonia oxidizing (comammox) bacteria was 20 times more abundant than ammonia-oxidizing bacteria (AOB) at day 121 and its abundance of amoA gene was almost the same as AOB. Although Nitrospira dominated the nitrite-oxidizing bacteria (NOB), diverse unconventional nxrB-containing microorganisms, particularly Chloroflexi, also significantly contributed to the nitrite oxidation. Binning analysis showed that Myxococcota-affiliated Haliangium had the necessary genes owns by phosphorus-accumulating organisms (PAO) and was likely to be the primary PAO since its abundance (6.38 %) was much higher than other conventional PAO (0.70 %). Comparing metagenome-assembled genomes of comammox bacteria with AOB and ammonia-oxidizing archaea (AOA), it possessed potential metabolic versatility in hydrogen and phosphorus, which may be the primary reason for the positive effect of the alternating anaerobic and aerobic conditions on the enrichment of comammox bacteria. Collectively, our findings broaden the understanding on the microbial mechanism of nitrogen and phosphorus removal in biofilm system.

Microplastics (MPs) and nanoplastics (NPs), as emerging pollutants, are frequently detected in wastewater treatment plants. However, studies comparing the effects of MPs versus NPs on nitrogen removal by activated sludge are rarely reported. Here, the responses of nitrogen removal performance, microbial community and functional genes to MPs and NPs in sequencing batch reactors were investigated. Results revealed that MPs (10 and 1000 μg/L) had no effects on nitrogen removal. While upon exposure to NPs, although low concentration (10 μg/L) of NPs showed no remarkable influence on nitrogen removal, high level (1000 μg/L) of NPs decreased NH4+-N removal efficiency by 24.48% and caused accumulation of NO3--N and NO2--N. These inhibitory probably due to the acute toxicity of NPs to activated sludge, which was reflected by the increasing reactive oxygen species generation and lactate dehydrogenase release. The toxic effects of NPs further declined the relative abundance of nitrifiers (e.g., Nitrospira) and denitrifiers (e.g., Dechloromonas). These negative effects, accompanied by a decrease in abundance of amoA and nxrA genes related to nitrification (30.01% and 65.24% of control) and narG, nirK and nirS genes associated with denitrification (78.59%, 61.39%, and 86.17% of control), directly illustrated the attenuate phenomenon observed in nitrogen removal.

A sequencing batch reactor (SBR) was operated to investigate variations of extracellular polymeric substances (EPS) and microbial community during the start‐up of the single‐stage partial nitritation/anammox (SPN/A) process at intermittent aeration mode. The SPN/A system was successfully started on day 34, and the nitrogen removal efficiency and total nitrogen loading rate were 82.29% and 0.31 kg N/(m3·day), respectively. Furthermore, the relationship between the protein secondary structures and microbial aggregation was strongly related. The α‐helix/ (β‐sheet + random coil) ratios increased obviously from 0.20 ± 0.03 to 0.23 ± 0.01, with the sludge aggregation mean size increased from 56 to 107 μm during the start‐up of SPN/A. During the start‐up of SPN/A, Candidatus Kuenenia was the primary anammox bacteria, whereas Nitrospira was the main functional bacteria of nitrite‐oxidizing bacteria. Correlation between the microbial community and EPS components was performed. The EPS and microbial community played important roles in keeping stable nitrogen removal and the formation of sludge granules.

To address ammonium nitrogen (NH4+-N) and nitrite accumulation in intensive marine shrimp aquaculture, a marine recirculating aquaculture system (RAS) for Penaeus vannamei centered on a moving bed biofilm reactor (MBBR) was constructed to investigate the microbial basis of nitrogen removal. The results showed that the MBBR contributed most to NH4+-N removal, demonstrating favorable nitrification potential under marine conditions (0.513 mg·L−1·h−1). The biofilm carrier formed a complete attached layer and developed a mature biofilm structure. Microbial community analysis revealed clear differentiation between the biofilm and sediment. The biofilm community was dominated by norank_f__Caldilineaceae (9.89%). Linear discriminant analysis effect size identified the nitrifying genus Nitrospira to be significantly enriched on the biofilm side (α = 0.05, linear discriminant analysis > 2.0). In addition, PICRUSt2-based functional prediction suggested a higher potential in biofilm than in sediment for ammonia oxidation and downstream nitrogen transformation, involving ammonia monooxygenase (EC:1.14.99.39), hydroxylamine dehydrogenase (EC:1.7.2.6), nitrate reductase (EC:1.7.99.4), and nitrite reductase (EC:1.7.2.1). Thus, this study provides a microbial basis and process strategy for P. vannamei RAS.

A lab-scale anaerobic-anoxic-oxic system was used to investigate the nitrogen removal mechanism under low dissolved oxygen (DO) conditions. When DO was decreased from 2 to 0.5 mg L-1, chemical oxygen demand (COD) and NH4+ removals were not influenced, while total nitrogen removal increased from 69% to 79%. Further batch tests indicated that both the specific nitrification rate and denitrification rate greatly increased under low DO conditions. When DO was decreased from 2 to 0.5 mg L-1, the oxygen half saturation constant value for ammonia oxidizing bacteria (AOB) decreased from 0.39 to 0.29 mg-O2 L-1, and for nitrite oxidizing bacteria (NOB), it reduced from 0.29 to 0.09 mg-O2 L-1. Correspondingly, the observed yield coefficients increased from 0.05 to 0.10 mg-cell mg-1-N for AOB, and from 0.02 to 0.06 mg-cell mg-1-N for NOB. High-throughput sequencing revealed that the relative abundances of AOB increased from 6.13% to 6.54%, Nitrospira-like NOB increased from 3.67% to 6.50%, and denitrifiers increased from 2.84% to 7.04%. Improved simultaneous nitrification and denitrification under low DO conditions contributed to the enhanced nitrogen removal.

This study aims to offer insights into how ciprofloxacin (CIP) impact bacterial community structures in the Sponge-MBR process when CIP is spiked into hospital wastewater. We found that the CIP toxicity decreased richness critical phylotypes such as phylum class ẟ-, β-, ɣ-proteobacteria, and Flavobacteria that co-respond to suppress denitrification and cake fouling to 37% and 28% respectively. Cluster analysis shows that the different community structures were formed under the influence of CIP toxicity. CIP decreased attached growth biomass by 2.3 times while increasing the concentration of permeate nitrate by 3.8 times, greatly affecting TN removal by up to 26%. Ammonia removal was kept stable by inflating the ammonia removal rate (p < 0.003), with the wealthy Nitrospira genus guaranteeing the nitrification activity. In addition, we observed an increasing richness of Chloroflexi and Planctomycetes, which may play a role in fouling reduction in the Sponge-MBR. Therefore, if the amount of antibiotics in hospital wastewater continues to increase, it is so important to extend biomass retention for denitrification recovery.

In this study, a novel sponge biocarriers (SB) in moving bed bioreactor (MBBR) treating recirculating aquaculture systems wastewater was evaluated for the first time. Two lab-scale MBBRs were operated simultaneously for 116 days under various hydraulic retention times (HRTs). The reactors R1 and R2 were filled with K5 plastic carriers and SB, respectively. From the results, at an optimum HRT of 6 h, ammonia removal efficiency and nitrification rate were 86.67 ± 2.4% and 1.43 mg/L.h for the R1 and, 91.65 ± 1.3% and 1.52 mg/L.h for the R2, respectively. The microbial community analysis showed that the predominant genera in the nitrifying community were Nitrosomonas (AOB) and Nitrospira (NOB) in co-existence with heterotrophic genera Hyphomicrobium, Mesorhizobium, Zhizhongheella, and Klebsiella spp. Modified Stover-Kincannon model examined the ammonia removal kinetics, and the values of kinetic parameters obtained were Umax: 0.909 and 1.111 g/L.d and KB: 0.929 and, 1.108 g/L.d for the R1 and R2, respectively. The correlation coefficients (R2) of the MBBRs were higher than 0.98, indicating that the model adequately described the experimental data. Overall, MBBR, filled with the proposed novel SB operated at 6 h HRT, can achieve the highest nitrification performance and increase the diversity of the functional microbial communities.

Understanding the ecological relationship of microbial community under external stimulation is crucial for environmental restoration. Black carbon (e.g., biochar) have been widely deemed as a strategy to enhance pollutants removal because of its structure and redox-active property. However, the underlying ecological mechanism of microbial community under black carbon addition is poorly understood. The major purposes of this study were to determine the microbial assemblage and functional patterns via null model test, network analysis, and function prediction. The results showed that the nitrate removal efficiency of modified black carbon system achieved 46.44%. Both deterministic and stochastic processes were significant for mediating the microbial assemblage and the deterministic process dominated (>95%) the assemblage of community. Keystone taxa in the black carbon systems, involving Sulfuricella, Allorhizobium, and Nitrospira, stimulated the shift of community composition regarding the nitrogen removal. The existence of black carbon and the biotic interactions increased biological nitrate utilization and promoted nitrogen removal. Overall, this study presents the mechanism of bacterial community assembly and provides insight into biological nitrogen removal from microbial ecological perspective.

The novel immobilized microbial granules (IMG) shows a significant effect of nitrification for freshwater aquaculture. However, there is lack of evaluation study on the performance of nitrification at high salinity due to the concentration of recycled water or seawater utilization. A laboratory scale moving bed bioreactor (MBBR) with IMG was tested on recycled synthetic aquaculture wastewater for the nitrification at 2.5 mg/L NH3-N daily. The results indicated that IMG showed a high salinity tolerance and effectively converted ammonia to nitrate up to 92% at high salinity of 35.0 g/L NaCl. As salinity increased from near zero to 35.0 g/L, the microbial activity of nitrite oxidation bacteria (NOB) in the IMG decreased by 86.32%. The microbial community analysis indicated that salinity significantly influenced the community structure. It was found that Nitrosomonas sp. and Nitrospira sp. were the dominant genera for ammonia oxidation bacteria (AOB) and NOB respectively at different salinity levels.

The application of MBR in high saline wastewater treatment is mainly constrained by poor nitrogen removal and severe membrane fouling caused by high salinity stress. A novel carriers-enhanced MBR system was successfully developed for treating saline mariculture wastewater, which showed efficient TN removal (93.2%) and fouling control. High-throughput sequencing revealed the enhancement mechanism of bio-carriers under high saline condition. Bio-carriers substantially improved the community structure, representatively, nitrifiers abundance (Nitrosomonas, Nitrospira) increased from 2.18% to 9.57%, abundance of denitrifiers (Sulfurimonas, Thermogutta, etc.) also rose from 3.81% to 14.82%. Thereby, the nitrogen removal process was enhanced. Noteworthy, ammonia oxidizer (Nitrosomonas, 8.26%) was the absolute dominant nitrifiers compared with nitrite oxidizer (Nitrospira, 1.13%). This supported the finding of shortcut nitrification-denitrification process in hybrid system. Moreover, a series of biomacromolecule degraders (Lutibacterium, Cycloclasticus, etc.) were detected in bio-carriers, which could account for the mitigation of membrane fouling as result of EPS and SMP degradation.

Linear alkylbenzene sulfonates (LAS) are widely detected in wastewater, and pose potential risks to environment. The influences of LAS on the typical pollutants removal in sequencing batch reactors (SBRs) were evaluated. The results indicated that the removal efficiency of COD, NH4+ and PO43- was respectively reduced by 10.5-27.5%, 5.4-7.3% and11.6-28.9% with the exposure of 10-50 mg/L LAS. Mechanisms investigation found that LAS disrupted the sludge structure and reduced the biomass in reactors due to the saponification effects. Also, the presence of LAS altered the microbial community of activated sludge, and reduced the abundances of functional bacterial responsible for pollutants removal (i.e.Candidatus Accumulibacter, Nitrospira, Denitratisoma and etc.). Moreover, the LAS exhibited negative impacts on the microbial activity with increased LDH release but decreased ATP concentration. The genes expressions for microbial metabolism (i.e. carbohydrate metabolisms, energy metabolism) and typical pollutants removal (i.e. electron transport, phosphonate transport) were all downregulated in LAS-exposed SBRs.

Achievement of nitrite accumulation is critical for the application of advanced nitrogen removal processes. Two lab-scale sequencing batch biofilm reactors (SBBRs) and two sequencing batch reactors (SBRs) were operated under intermittent aeration with different feeding patterns. The nitrite accumulation ratio was 56.6% (pulse feeding) and 68.9% (constant feeding) in SBBRs with nitritation. Nitrate accounted for 98% of the effluent nitrogen in SBRs with complete nitrification. The dominant nitrifier was Nitrosomonas in SBBRs and Nitrospira in SBRs. Four types of acyl homoserine lactones (AHLs) were detected. N‑[(RS)‑3‑Hydroxybutyryl]‑l‑homoserine lactone and N‑octanoyl‑l‑homoserine lactone had a high concentration in the extracellular polymeric substance phase, and had an obvious relationship with nitrite accumulation and ammonia removal. Various microbial communities coexisted in nitrifying systems, with diverse microbial interactions. Microorganisms harboring AHLs-related genes had more interactions with each other, suggesting that nitritation could be regulated by AHLs based quorum sensing.

Simultaneous nitrification and denitrification under low dissolved oxygen conditions is an energy-saving modification of the activated sludge process to achieve efficient nitrogen removal. Geographically distinct full-scale treatment plants are excellent platforms to address the links of microbial community with operating parameters. Mixed liquor samples were collected from a sequencing batch reactor plant, oxidation ditch plant, and step-feed activated sludge plant. Next-Generation Sequencing of the samples showed that the microbial communities were similar at the phylum level among the plants, being dominated by Proteobacteria. Microbial composition of functional groups was similar between the react fill and react phases of the sequencing batch reactors, among four sequencing batch reactors, and among four oxidation ditches. Nitrospira was the only identified genus of autotropic nitrifying bacteria with a relative abundance of 2.2-2.5% in the oxidation ditches and 0.4-0.7% at the other plants. Heterotrophic nitrifying-aerobic denitrifying bacteria were dominated by Dechloromonas with a relative abundance of 0.4-1.0%. Microbial community composition and nitrogen removal mechanisms were related to overall level and local zonation of dissolved oxygen, mixed liquor suspended solids concentration, nitrogen and organic loadings, and solids retention time. Low dissolved oxygen and low organic and nitrogen loadings favored growth of Nitrospira.

The rapid growth of nitrite-oxidizing bacteria (NOB) in reactor prevents the application of anaerobic ammonium oxidation (anammox) technology to main-stream wastewater treatment. How to eliminate NOB and reserve anaerobic ammonium oxidation bacteria (AnAOB) simultaneously becomes the biggest challenge. In this study two coupled biological aeration filters (BAFs) were built up to treat domestic sewage. In BAF1 nitrogen removal concentration was 21.4 mg/L via heterotrophic denitrification pathway. Backwash was conducted to BAF2 to improve nitrogen removal performance. After backwash Nitrospira proportion declined from 10.8% to 2.1%, while Candidatus Kuenenia percentage increased from 5.6% to 10.2%. Nitrogen removal concentration improved from 8.6 mg/L to 22.8 mg/L via anammox pathway in BAF2, and total nitrogen removal concentration reached to 44.2 mg/L in two coupled BAFs during aeration process. These findings could provide a new strategy for the application of anammox technology to main-stream wastewater treatment.

Abstract The microbial community diversity in anaerobic-, anoxic- and oxic-biological zones of a conventional Carrousel oxidation ditch system for domestic wastewater treatment was systematically investigated. The monitored results of the activated sludge sampled from six full-scale WWTPs indicated that Proteobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Verrucomicrobia, Acidobacteria and Nitrospirae were dominant phyla, and Nitrospira was the most abundant and ubiquitous genus across the three biological zones. The anaerobic-, anoxic- and oxic-zones shared approximately similar percentages across the 50 most abundant genera, and three genera (i.e. uncultured bacterium PeM15, Methanosaeta and Bellilinea) presented statistically significantly differential abundance in the anoxic-zone. Illumina high-throughput sequences related to ammonium oxidizer organisms and denitrifiers with top50 abundance in all samples were Nitrospira, uncultured Nitrosomonadaceae, Dechloromonas, Thauera, Denitratisoma, Rhodocyclaceae (norank) and Comamonadaceae (norank). Moreover, environmental variables such as water temperature, water volume, influent ammonium nitrogen, influent chemical oxygen demand (COD) and effluent COD exhibited significant correlation to the microbial community according to the Monte Carlo permutation test analysis (p < 0.05). The abundance of Nitrospira, uncultured Nitrosomonadaceae and Denitratisoma presented strong positive correlations with the influent/effluent concentration of COD and ammonium nitrogen, while Dechloromonas, Thauera, Rhodocyclaceae (norank) and Comamonadaceae (norank) showed positive correlations with water volume and temperature. The established relationship between microbial community and environmental variables in different biologically functional zones of the six representative WWTPs at different geographical locations made the present work of potential use for evaluation of practical wastewater treatment processes.

Bioturbation by the razor clam, Sinonovacula constricta, influenced the redox conditions and changed the sedimentary environment, providing a suitable micro-environment for microbial growth. However, the mechanism of the integrated razor clam-microbial community in organic matter mineralization remains elusive. To study this mechanism, an in situ experiment was conducted to investigate the bioturbation effects of S. constricta on the microbial community and enzyme activities in the sediment of an ecological aquaculture wastewater treatment system. According to principal coordinate analysis (PCoA) and PERMANOVA, we found that the microbial community was significantly (P < 0.05) influenced by the bioturbation effect of S. constricta. Linear discriminant analysis effect size (LEfSe) showed that species involved in reduced effluent concentrations of TN and TP, such as Prolixibacteraceae, Nitrospira and Actinobacteria, were increased significantly (P < 0.05) by S. constricta. Molecular ecological network (MENs) analysis indicated that the bioturbation effect of S. constricta increased the complexity of interspecies interaction and changed the topological properties of individual OTUs. The results elucidated that S. constricta increased the microbial community network, as substantiated by a higher total number of nodes and a shorter geodesic distance. Zi < 2.5 and Pi <0.62 in MENs showed that the S. constricta treatment significantly increased (P < 0.05) the potential microbial community, with the keystone (OTU747049) Proteobacteria and (OTU74388) Bacteroidetes family Prolixibacteraceae, which connected different co-expressed OTUs. Furthermore, S. constricta significantly increased (P < 0.05) the enzymatic activities (alkaline phosphatase (APA), dehydrogenase and urease) of the substrate at different sampling depths. Overall, this study provides evidence that the bioturbation effect of S. constricta changes the microbial community structure, increases enzymatic activities and accelerates the degradation of organic matter in an aquaculture wastewater environment.

The impact of hydraulic retention time (HRT) on the performance and microbial community structure of control and electro‐bioreactors was investigated. Control bioreactors and electro‐bioreactors were operated at HRT ranging between 6 and 75 hr. The total bacterial counts in addition to the removal efficiency of NH4+–N, sCOD, and PO43−–P was assessed in all the reactors tested. In addition, Illumina sequencing was performed to determine the microbial communities that developed in these reactors under each HRT condition. Phylogenetic analysis showed that Proteobacteria and Bacteroidetes were the dominant phyla in those reactors. In addition, Nitrospira sp. and Pseudomonas sp. were found to be present in electro‐bioreactors with higher relative abundance than in control bioreactors. The results presented here are the first to determine what different microbial communities in wastewater electro‐bioreactors due to the application of an electric current under different HRTs.

The aim of this study was to investigate the microbial community of three tannery wastewater treatment plants (WWTPs) involved in nitrification by Illumina MiSeq sequencing. The results showed that highly diverse communities were present in tannery wastewater. A total of six phyla, including Proteobacteria (37-41%), Bacteroidetes (6.04-16.80), Planctomycetes (3.65-16.55), Chloroflexi (2.51-11.48), Actinobacteria (1.91-9.21), and Acidobacteria (3.04-6.20), were identified as the main phyla, and Proteobacteria dominated in all the samples. Within Proteobacteria, Beta-proteobacteria was the most abundant class, with the sequence percentages ranging from 9.66% to 17.44%. Analysis of the community at the genus level suggested that Thauera, Gp4, Ignavibacterium, Phycisphaera, and Arenimonas were the core genera shared by at least two tannery WWTPs. A detailed analysis of the abundance of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) indicated that Nitrosospira, Nitrosomonas, and Nitrospira were the main AOB and NOB in tannery wastewater, respectively, which exhibited relatively high abundance in all samples. In addition, real-time quantitative PCR was conducted to validate the results by quantifying the abundance of the AOB and total bacteria, and similar results were obtained. Overall, the results presented in this study may provide new insights into our understanding of key microorganisms and the entire community of tannery wastewater and contribute to improving the nitrogen removal efficiency.

A metagenomic approach was used to investigate how the microbial community composition changes when an anammox-based granular sludge reactor is seeded with nitritation-anammox biomass from a wastewater treatment plant. In the seed sample, the abundance of Candidatus Kuenenia stuttgartiensis was similar to Candidatus Jettenia caeni (12.63 vs. 11.68%). This biomass was typical in terms of microbial nitrogen conversion; both ammonia (Nitrosomonas sp.) and nitrite (Nitrospira sp.) oxidizing bacteria were detected. In the lab-scale reactor, Candidatus Kuenenia stuttgartiensis and Candidatus Jettenia caeni bacteria were also present in equal proportions (18.57 vs. 20.89%). On the contrary, Candidatus Nitrospira defluvii bacteria were highly abundant in this reactor, but no known ammonia-oxidizing bacteria were detected. In light of recent studies showing that Nitrospira sp. are capable of complete nitrification, the results presented here may well indicate that both stages of nitrification in the anammox-based granular sludge reactor were performed by this bacteria.

The Sundarbans mangrove, located at the mouth of the Ganges and Brahmaputra Rivers, is the world’s largest tidal mangrove forest. These mangroves are also one of the most striking sources of microbial diversity, essential in productivity, conservation, nutrient cycling, and rehabilitation. Hence, the main objective of this study was to use metagenome analysis and provide detailed insight into microbial communities and their functional roles in the Sundarbans mangrove ecosystem. A comparative analysis was also done with a non-mangrove region of the Sundarbans ecosystem to assess the capability of the environmental parameters to explain the variation in microbial community composition. The study found several dominant bacteria, viz., Alphaproteobacteria, Actinomycetota, Bacilli, Clostridia, Desulfobacterota, Gammaproteobacteria, and Nitrospira, from the mangrove region. The mangrove sampling site reports several salt-tolerant bacteria like Alkalibacillus haloalkaliphilus, Halomonas anticariensis, and Salinivibrio socompensis. We found some probiotic species, viz., Bacillus clausii, Lactobacillus curvatus, Vibrio mediterranei and Vibrio fluvialis, from the Sundarbans mangrove. Nitrifying bacteria in Sundarbans soils were Nitrococcus mobilis, Nitrosococcus oceani, Nitrosomonas halophila, Nitrospirade fluvii, and others. Methanogenic archaea, viz., Methanoculleus marisnigri, Methanobrevibacter gottschalkii, and Methanolacinia petrolearia, were highly abundant in the mangroves as compared to the non-mangrove soils. The identified methanotrophic bacterial species, viz., Methylobacter tundripaludum, Methylococcus capsulatus, Methylophaga thiooxydans, and Methylosarcina lacus are expected to play a significant role in the degradation of methane in mangrove soil. Among the bioremediation bacterial species identified, Pseudomonas alcaligenes, Pseudomonas mendocina, Paracoccus denitrificans, and Shewanella putrefaciens play a significant role in the remediation of environmental pollution. Overall, our study shows for the first time that the Sundarbans, the largest mangrove ecosystem in the world, has a wide range of methanogenic archaea, methanotrophs, pathogenic, salt-tolerant, probiotic, nitrifying, and bioremediation bacteria.

Quorum sensing (QS) can function to shape the microbial community interactions, composition, and function. In wastewater treatment systems, acylated homoserine lactone (AHL)-based QS has been correlated with the conversion of floccular biomass into microbial granules, as well as EPS production and the nitrogen removal process. However, the role of QS in such complex communities is still not fully understood, including the QS-proficient taxa and the functional QS genes involved. To address these questions, we performed a metagenomic screen for AHL genes in an activated sludge microbial community from the Ulu Pandan wastewater treatment plant (WWTP) in Singapore followed by functional validation of luxI activity using AHL biosensors and LC–MSMS profiling. We identified 13 luxI and 30 luxR homologs from the activated sludge metagenome. Of those genes, two represented a cognate pair of luxIR genes belonging to a Nitrospira spp. and those genes were demonstrated to be functionally active. The LuxI homolog synthesized AHLs that were consistent with the dominant AHLs in the activated sludge system. Furthermore, the LuxR homolog was shown to bind to and induce expression of the luxI promoter, suggesting this represents an autoinduction feedback system, characteristic of QS circuits. Additionally, a second, active promoter was upstream of a gene encoding a protein with a GGDEF/EAL domain, commonly associated with modulating the intracellular concentration of the secondary messenger, c-di-GMP. Thus, the metagenomic approach used here was demonstrated to effectively identify functional QS genes and suggests that Nitrospira spp. maybe QS is active in the activated sludge community.

ABSTRACT Microbes commonly exist in diverse and complex communities where species interact, and their genomic repertoires evolve over time. Our understanding of species interaction and evolution has increased during the last decades, but most studies of evolutionary dynamics are based on single species in isolation or in experimental systems composed of few interacting species. Here, we use the microbial ecosystem found in groundwater-fed sand filter as a model to avoid this limitation. In these open systems, diverse microbial communities experience relatively stable conditions, and the coupling between chemical and biological processes is generally well defined. Metagenomic analysis of 12 sand filters communities revealed systematic co-occurrence of at least five comammox Nitrospira species, likely promoted by low ammonium concentrations. These Nitrospira species showed intrapopulation sequence diversity, although possible clonal expansion was detected in a few abundant local comammox populations. Nitrospira species showed low homologous recombination and strong purifying selection, the latter process being especially strong in genes essential in energy metabolism. Positive selection was detected for genes related to resistance to foreign DNA and phages. We found that, compared to other habitats, groundwater-fed sand filters impose strong purifying selection and low recombination on comammox Nitrospira populations. These results suggest that evolutionary processes are more affected by habitat type than by species identity. Together, this study improves our understanding of species interaction and evolution in complex microbial communities and sheds light on the environmental dependency of evolutionary processes. IMPORTANCE Microbial species interact with each other and their environment (ecological processes) and undergo changes in their genomic repertoire over time (evolutionary processes). How these two classes of processes interact is largely unknown, especially for complex communities, as most studies of microbial evolutionary dynamics consider single species in isolation or a few interacting species in simplified experimental systems. In this study, these limitations are circumvented by examining the microbial communities found in stable and well-described groundwater-fed sand filters. Combining metagenomics and strain-level analyses, we identified the microbial interactions and evolutionary processes affecting comammox Nitrospira, a recently discovered bacterial type capable of performing the whole nitrification process. We found that abundant and co-occurrent Nitrospira populations in groundwater-fed sand filters are characterized by low recombination and strong purifying selection. In addition, by comparing these observations with those obtained from Nitrospira species inhabiting other environments, we revealed that evolutionary processes are more affected by habitat type than by species identity.

The abundance and phylogenetic diversity of functional genes involved in nitrification were assessed in Rothamsted field plots under contrasting management regimes—permanent bare fallow, grassland, and arable (wheat) cultivation maintained for more than 50 years. Metagenome and metatranscriptome analysis indicated nitrite oxidizing bacteria (NOB) were more abundant than ammonia oxidizing archaea (AOA) and bacteria (AOB) in all soils. The most abundant AOA and AOB in the metagenomes were, respectively, Nitrososphaera and Ca. Nitrososcosmicus (family Nitrososphaeraceae) and Nitrosospira and Nitrosomonas (family Nitrosomonadaceae). The most abundant NOB were Nitrospira including the comammox species Nitrospira inopinata, Ca. N. nitrificans and Ca. N. nitrosa. Anammox bacteria were also detected. Nitrospira and the AOA Nitrososphaeraceae showed most transcriptional activity in arable soil. Similar numbers of sequences were assigned to the amoA genes of AOA and AOB, highest in the arable soil metagenome and metatranscriptome; AOB amoA reads included those from comammox Nitrospira clades A and B, in addition to Nitrosomonadaceae. Nitrification potential assessed in soil from the experimental sites (microcosms amended or not with DCD at concentrations inhibitory to AOB but not AOA), was highest in arable samples and lower in all assays containing DCD, indicating AOB were responsible for oxidizing ammonium fertilizer added to these soils.

Freshwater mussel assemblages of the Upper Mississippi River (UMR) sequester tons of ammonia- and urea-based biodeposits each day and aerate sediment through burrowing activities, thus creating a unique niche for nitrogen (N) cycling microorganisms. This study explored how mussels impact the abundance of N-cycling species with an emphasis on Candidatus Nitrospira inopinata, the first microorganism known to completely oxidize ammonia (comammox) to nitrate. This study used metagenomic shotgun sequencing of genomic DNA to compare nitrogen cycling species in sediment under a well-established mussel assemblage and in nearby sediment without mussels. Metagenomic reads were aligned to the prokaryotic RefSeq non-redundant protein database using BLASTx, taxonomic binning was performed using the weighted lowest common ancestor algorithm, and protein-coding genes were categorized by metabolic function using the SEED subsystem. Linear discriminant analysis (LDA) effect sizes were used to determine which metagenomes and metabolic features explained the most differences between the mussel habitat sediment and sediment without mussels. Of the N-cycling species deemed differentially abundant, Nitrospira moscoviensis and “Candidatus Nitrospira inopinata” were responsible for creating a distinctive N-cycling microbiome in the mussel habitat sediment. Further investigation revealed that comammox Nitrospira had a large metabolic potential to degrade mussel biodeposits, as evidenced the top ten percent of protein-coding genes including the cytochrome c-type biogenesis protein required for hydroxylamine oxidation, ammonia monooxygenase, and urea decomposition SEED subsystems. Genetic marker analysis of these two Nitrospira taxons suggested that N. moscoviensis was most impacted by diverse carbon metabolic processes while “Candidatus Nitrospira inopinata” was most distinguished by multidrug efflux proteins (AcrB), NiFe hydrogenase (HypF) used in hydrogen oxidation and sulfur reduction coupled reactions, and a heme chaperone (CcmE). Furthermore, our research suggests that comammox and NOB Nitrospira likely coexisted by utilizing mixotrophic metabolisms. For example, “Candidatus Nitrospira inopinata” had the largest potentials for ammonia oxidation, nitrite reduction with NirK, and hydrogen oxidation, while NOB Nitrospira had the greatest potential for nitrite oxidation, and nitrate reduction possibly coupled with formate oxidation. Overall, our results suggest that this mussel habitat sediment harbors a niche for NOB and comammox Nitrospira, and ultimately impacts N-cycling in backwaters of the UMR.

No abstract available

Ammonia-oxidizing activity of different ammonia-oxidizing microorganisms (AOMs), such as ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and complete ammonia oxidizers (comammoxs), were investigated by adding the inhibitors such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, octyne, and KCLO3 in biofilm systems with different salinities. It was found that the ammonia-oxidizing activity of all AOMs gradually decreased with increasing salinity. The ammonia-oxidizing activity of AOB was consistently higher than those of AOA and comammox at different salinities. Moreover, nitrite-oxidizing bacteria (NOB) were more sensitive to changes in salinity than AOMs. Metagenomic analysis revealed that nitrifiers were detected at high level, with the AOB Nitrosomonas sp. comprising 24.9 % and the NOB Nitrospira sp. comprising 47.2% of all nitrifiers. The main functional genes involved in the nitrification reaction were amoABC, hao, and nxrAB. This study demonstrates that higher abundance of functional microorganisms and genes is related to the ammonia-oxidizing activity and ammonia removal contribution rate.

Recently, floating membrane filter cultivation was adopted to simulate solid surface and enrich surface-adapted soil ammonia-oxidizing archaea (AOA) communities from agricultural soil, as opposed to the conventional liquid medium. Here, we conducted metagenomic sequencing to recover nitrifier bins from the floating membrane filter cultures and reveal their genomic properties. Phylogenomic analysis showed that AOA bins recovered from this study, designated FF_bin01 and FF_bin02, are affiliated with the Nitrososphaeraceae family, while the third bin, FF_bin03, is a nitrite-oxidizing bacterium affiliated with the Nitrospiraceae family. Based on the ANI/AAI analysis, FF_bin01 and FF_bin02 are identified as novel species within the genera "Candidatus Nitrosocosmicus" and Nitrososphaera, respectively, while FF_bin03 represents a novel species within the genus Nitrospira. The pan and core genome analysis for the 29 AOA genomes considered in this study revealed 5,784 orthologous clusters, out of which 653 were core orthologous clusters. Additionally, 90 unique orthologous clusters were conserved among the Nitrososphaeraceae family, suggesting their potential role in enhancing culturability and adaptation to diverse environmental conditions. Intriguingly, FF_bin01 and FF_bin02 harbor a gene encoding manganese catalase and FF_bin03 also possesses a heme catalase gene, which might enhance their growth on the floating membrane filter. Overall, the floating membrane filter cultivation has proven to be a promising approach for isolating distinct soil AOA, and further modifications to this technique could stimulate the growth of a broader range of uncultivated nitrifiers from diverse soil environments.

Efficient removal of ammonia nitrogen and sulfamethoxazole (SMX) from wastewater has become increasingly critical due to their detrimental effects on aquatic ecosystems and public health. This study aimed to investigate the nitrogen transformation and SMX removal in a membrane aerated biofilm reactor (MABR) under different SMX concentrations (0 ∼ 200 μg·L-1) with a nitrifying membrane bioreactor (MBR) as a control. Results suggested that SMX removal in MABR was better than that of MBR with SMX addition (50 ∼ 200 μg·L-1). Membrane aerated biofilms tended to secrete more extracellular polymeric substances (EPS) and generate less antioxidant enzymes in response to SMX stress when compared with nitrifying sludge in MBR. Metagenomic analysis indicated that distinct succession of microbial community was observed in both systems after SMX addition, and the relative abundance of nitrifying bacteria (Nitrosomonas, Nitrospira, and Nitrobacter) evidently decreased under SMX concentration of 200 μg·L-1. The proliferation of predominant antibiotic resistance gene (ARG) sul2 was suppressed more obviously in MABR than that in MBR. Thus, this study provided extensive insights into the advantages of nitrifying MABR in simultaneous removal of ammonium and antibiotics with less risk of associated ARGs spread.

Synchronous sludge reduction and nitrogen removal have attracted increasing attention, while the underlying mechanisms of diverse nitrogen metabolism within the complicated processes remain unclear. Four anoxic/oxic membrane bioreactors, three of which were upgraded by anaerobic side-stream reactors (ASSR) and carriers (APSSR-MBRs), were operated to determine effects of hydraulic retention time of ASSRs. APSSR-MBRs achieved more significant nitrogen removal and higher nitrate uptake rate because of more denitrifying bacteria and the supernumerary release of secondary substrates. Ammonia uptake rate showed the diverse Nitrospira preceded over anaerobic decay and sulfide inhibition in the ASSR, and made the reactor exhibit higher nitrification capacity. Metagenomic analysis indicated that APSSR-MBRs showed higher abundances of genes related to nitrogen consumption processes, and higher abundances on the carriers, confirming their pivotal roles in nitrogen metabolism. This study provided novel perspectives to build a bridge between process model and nitrogen metabolism in the sludge reduction system..

Currently, little is known about the microbial diversity in the sediments of Pacmanus and Desmos hydrothermal fields in Manus Basin. In this study, Illumina-based sequencing of 16S rRNA gene amplicons and metagenomic analysis were conducted to investigate the microbial populations and metabolic profiles in the sediments from four different regions in Pacmanus and Desmos hydrothermal fields. It was found that Gammaproteobacteria and Thaumarchaeota were the most abundant bacterial and archaeal populations, respectively. The autotrophic prokaryotes in the four communities probably fixed CO2 via four major pathways, i.e. Calvin-Benson-Bassham cycle, reductive acetyl-CoA cycle, rTCA cycle, and 3-hydroxypropionate/4-hydroxybutyrate cycle. Ammonia-oxidizing Thaumarchaeota, nitrifiers, denitrifiers, and sulfur oxidizers belonging to the subgroups of Proteobacteria (e.g., alpha, beta, gamma, and epsilon), Nitrospira, and Nitrospina, and sulfate-reducing Desulfobacterales likely played critical roles in nitrogen and sulfur cycling, in which ammonia, sulfur compounds, and hydrogen could be utilized as potential energy sources. These findings revealed new insights into the operational mechanism of the microbial communities associated with Pacmanus and Desmos hydrothermal fields.

This study used two laboratory-scale sequencing batch reactors (SBRs) to evaluate the shifts in nitrification kinetics and microbial communities of an activated sludge sewage treatment system (main stream) during bioaugmentation with nitrifiers cultivated on real sludge reject water (side stream). Although bioaugmentation exerted a strong influence on the microbial community and the nitrification kinetics in the main stream, there was 58% of maximum ammonia uptake rate (AUR) and 80% of maximum nitrite uptake rate (NUR) loss of the seed source after bioaugmentation. In addition, nitrite accumulation occurred during bioaugmentation due to the unequal and asynchronous increase of the AUR (from 2.88 to 13.36 mg N/L·h) and NUR (from 0.76 to 4.34 mg N/L·h). FISH results showed that ammonia oxidizing bacteria (AOB) was inclined to be washed out with effluent in contrast to nitrite oxidizing bacteria (NOB), and Nitrosococcus mobilis lineage was the dominant AOB, while the dominant NOB in the main stream gradually transferred from Nitrospira to Nitrobacter. Nitrospina and Nitrococcus which existed in the seed source could not be detected in the main stream. It can be inferred that nitrite accumulation occurred due to the mismatch of NOB structure but washed out with effluent.

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

Amid growing concerns over water pollution, the secondary effluent from wastewater treatment plants poses significant threats to aquatic ecosystems with limited self-purification capacity. Secondary effluent-constructed wetlands (SECWs) offer a sustainable solution for advanced nitrogen removal from this low-ammonia effluent, yet the functional role of comammox bacteria remains largely unexplored in such habitats. This study investigated the abundance, activity, kinetics, and ecological adaptations of comammox in typical SECWs. Quantitative PCR and amplicon sequencing revealed that comammox Nitrospira ubiquitously presented across all samples, even numerically dominated over ammonia-oxidizing bacteria/archaea (AOB/AOA). Consistent results from double-inhibition assays and DNA-stable isotope probing microcosm experiments indicated that comammox actively participated in nitrification, contributing 2.03-3.89 times those of canonical nitrifiers. Substrate kinetic and metagenomic analyses identified the Nitrospira nitrosa cluster as the sole active comammox population in SECWs, which exhibited relatively lower ammonia affinity (Km(app) = 0.055 ± 0.007 mg N/L) than other comammox species and distinct genomic adaptations to SECW-specific stressors, potentially explaining its dominance. Compared to AOB, comammox combines high substrate affinity with environmental resilience, aligning with K-strategist traits that enable it to outperform r-strategist AOB in SECWs. Overall, it is within this niche differentiation among comammox species and nitrifiers that the N. nitrosa cluster numerically and functionally dominated the nitrification process in SECWs, positioning comammox Nitrospira as pivotal biocatalysts for advanced nitrogen removal in engineered ecosystems.

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A lab-scale pure moving bed sequencing batch biofilm reactor (MBSBBR) was employed to investigate changes in nitrification kinetics and microbial diversity. The MBSBBR operated under different aeration strategies (defined by the ratio of the duration of the subphases with (t1) and without (t2) aeration (R = t2/t1)) − continuous (R = 0) and intermittent (with constant time of non-aerated subphases (t2 = 10 min) and variable duration of subphases with aeration (t1 = 40 min–R = 1/4, t1 = 30 min–R = 1/3, t1 = 20 min–R = 1/2) and dissolved oxygen (DO) concentrations (6 mg/L; 3.5 mg/L). Moreover, the reactor’s organic (OLR) and nitrogen (NLR) loading rates were changed in the following ranges: OLR—537–402 gCOD/m3·d, NLR—64–48 gN/m3·d. The obtained results showed that, irrespective of changes introduced in particular series, a highly effective nitrification process (93.36 ± 2.13%) was achieved. The activity of bacteria capable of oxidizing ammonia nitrogen changed differently from that of bacteria capable of oxidizing nitrites (NOB). An increase in R was the primary factor changing the activity of ammonia-oxidizing microorganisms. NOB activity was affected only by the reduction of OLR and NLR. NOB were the predominant bacterial group, consistent with the kinetics studies. A DO decrease caused an increase in the abundance of AOB, NOB, and Comammox bacteria. Comammox bacteria were the most abundant at R = 1/2 and DO = 3.5 mg/L.

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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.

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Nitrification in terrestrial ecosystems is mediated by NH 4 + ‐oxidizing bacteria (AOB) and archaea (AOA). Presently, we understand relatively little about how physiological capacities and environmental tolerances influence their community composition, and how compositional differences may, in turn, mediate nitrification. The relatively recent discovery of comammox Nitrospira , which can completely oxidize NH 4 + to NO 3 − unlike their AOA and AOB counterparts, adds further complexity to deriving this understanding. Using a well‐studied series of northern temperate forests, we explored how the abundance of NH 4 + ‐oxidizing microorganisms responded to co‐occurring gradients of NH 4 + availability and soil pH. Here, we used quantitative polymerase chain reaction to assess the abundance of comammox Nitrospira and compared it to that of AOA and AOB. Importantly, comammox Nitrospira were absent in most sites, except those with the greatest supply of NH 4 + ; AOA (10 6 copies/g) and AOB (10 7 copies/g) were less abundant and were more equally distributed. Soil pH was significantly related to comammox Nitrospira abundance, displaying a unimodal distribution with a maximum at ~pH 5.0. In forests we studied, the biogeochemical process of nitrification is mediated by changes in the relative abundance of AOA, AOB, and comammox Nitrospira , thereby linking composition to function in soil microbial communities.

Abstract The discovery of comammox Nitrospira in low pH environments has reshaped the ammonia oxidation process in acidic settings, providing a plausible explanation for the higher nitrification rates observed in weakly acidic soils. However, the response of comammox Nitrospira to varying pH levels and its ecological role in these environments remains unclear. Here, a survey across soils with varying pH values (ranging from 4.4 to 9.7) was conducted to assess how comammox Nitrospira perform under different pH conditions. Results showed that comammox Nitrospira dominate ammonia oxidation in weakly acidic soils, functioning as a K‐strategy species characterized by slow growth and stress tolerance. As a key species in this environment, comammox Nitrospira may promote bacterial cooperation under low pH conditions. Genomic evidence suggested that cobalamin sharing is a potential mechanism, as comammox Nitrospira uniquely encode a metabolic pathway that compensates for cobalamin imbalance in weakly acidic soils, where 86.8% of metagenome‐assembled genomes (MAGs) encode cobalamin‐dependent genes. Additionally, we used DNA stable‐isotope probing (DNA‐SIP) to demonstrate its response to pH fluctuations to reflect how it responds to the decrease in pH. Results confirmed that comammox Nitrospira became dominant ammonia oxidizers in the soil after the decrease in pH. We suggested that comammox Nitrospira will become increasingly important in global soils, under the trend of soil acidification. Overall, our work provides insights that how comammox Nitrospira perform in weakly acidic soil and its response to pH changes.

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The discovery of complete ammonium oxidation (comammox) has subverted the traditional perception of two-step nitrification, which plays a key role in achieving biological nitrogen removal from wastewater. Floccular sludge-based treatment technologies are being applied at the majority of wastewater treatment plants in service where detection of various abundances and activities of comammox bacteria have been reported. However, limited efforts have been made to enrich and subsequently characterize comammox bacteria in floccular sludge. To this end, a lab-scale sequencing batch reactor (SBR) in the step-feeding mode was applied in this work to enrich comammox bacteria through controlling appropriate operational conditions (dissolved oxygen of 0.5 ± 0.1 g-O2/m3, influent ammonium of 40 g-N/m3 and uncontrolled longer sludge retention time). After 215-d operation, comammox bacteria gradually gained competitive advantages over counterparts in the SBR with a stable nitrification efficiency of 92.2 ± 2.2 %: the relative abundance of Nitrospira reached 42.9 ± 1.3 %, which was 13 times higher than that of Nitrosomonas, and the amoA gene level of comammox bacteria increased to 7.7 ± 2.1 × 106 copies/g-biomass, nearly 50 times higher than that of conventional ammonium-oxidizing bacteria. The enrichment of comammox bacteria, especially Clade A Candidatus Nitrospira nitrosa, in the floccular sludge led to (i) apparent affinity constants for ammonium and oxygen of 3.296 ± 0.989 g-N/m3 and 0.110 ± 0.004 g-O2/m3, respectively, and (ii) significantly low N2O and NO production, with emission factors being 0.136 ± 0.026 % and 0.023 ± 0.013 %, respectively.

This study aimed to investigate the effect of soil pH change, and nitrogen amendment on ammonia oxidiser abundance and comammox Nitrospira community composition. The experimental design used soil mesocosms placed in a temperature-controlled incubator for 90 days. A Templeton silt loam was used as its physiochemical properties are typical of the region's dairy farms. The results showed that comammox Nitrospira clade B preferred the natural (pH 6.1-6.2) soil pH with no applied nitrogen. Furthermore, synthetic urine (N700) decreased the abundance of comammox Nitrospira clade B. This may have been because the large amounts of available ammonia in the N700 treatments inhibited the growth of comammox Nitrospira. These results suggest that while comammox Nitrospira clade B are present in New Zealand dairy farm soils, but their role in nitrification in the very high nitrogen environment under a urine patch in grazed pastures may be limited. Further research is needed to confirm this. In contrast to comammox, the AOB community (dominated by Nitrosospira) responded positively to the application of synthetic urine. The response was greatest in the high pH soil (7.1), followed by the natural and then the low pH (4.9) soils. This may be due to the difference in ammonia availability. At high pH, the ammonia/ammonium equilibrium favours ammonia production. Calculated ammonia availability in the N700 treatments accurately predicted the AOB amoA gene abundance. Interestingly, the AOA community abundance (which was predominantly made up of Thaumarchaeota group I.1b clade E) seemed to prefer the natural and high pH soils over the low pH. This may be due to the specific lineage of AOA present. AOA did not respond to the application of nitrogen.

Nitrification plays a crucial role in aquatic ecosystems and in the biofilters used in fish farms. Despite their importance, the role of canonical nitrifiers, comammox bacteria, and archaea has not yet been sufficiently investigated. The aim of this study was to characterize the microbiome of the external canister biofilter in a freshwater fish aquarium, with particular focus on the role of comammox Nitrospira and their competition with other nitrifiers. To achieve this, a comprehensive approach combining metagenome sequencing and co-occurrence network analysis was used to study the interactions between microorganisms in portable biofilter. The fish were subjected to a changing feeding regime that affected the ecological relationships and abundance of different microbial taxa. The results showed the presence of two types of nitrifiers in the biofilter: comammox Nitrospira and ammonia-oxidizing archaea (AOA). Five comammox Nitrospira genomes were reconstructed, with comammox clade B being the most abundant with an average abundance of 7.8 ± 0.4%. In addition, two families of archaea were identified: Nitrosopumilaceae and Nitrososphaeraceae, with an average abundance of 4.3 ± 0.4%. Heterotrophs were also abundant in the bacterial community, particularly in the genera Actinomycetota, Planctomycetota, and Pseudomonadota. Network analysis indicated competitive interactions between comammox and heterotrophs, whereas no competition was observed between comammox and AOA. The predominance of comammox Nitrospira, and AOA over canonical nitrifiers emphasizes their better adaptation to oligotrophic environments. This study highlights the importance of competition within the biofilter microbiome and the role of ecological interaction networks, which can contribute to the optimization of water purification systems in RASs.

Despite their widespread presence in acidic environments, the stability and adaptative mechanisms of complete ammonia oxidization (comammox) bacteria remain poorly understood. In this three-year study, comammox Nitrospira consistently dominated both abundance and activity in an acidic nitrifying reactor (pH = 6.3-6.8), as revealed by metagenomic and cDNA-based 16S rRNA sequencing. Batch tests demonstrated their decent nitrification down to pH 4.7, while ceasing at pH 4.2. Genome-centric metatranscriptomics revealed that comammox Nitrospira upregulated a Rh-type ammonium transporter to enhance substrate uptake under acidic conditions. Active proton transport, mediated by NADH dehydrogenases and F-type ATPase, was identified as a primary strategy for maintaining pH homeostasis in comammox Nitrospira. Genes associated with carbon acquisition, chemotaxis, and DNA repair were upregulated at low pH, suggesting these processes play roles in acid adaptation. These findings enhance the understanding of ecological roles and adaptive mechanisms of comammox bacteria in acidic environments.

Intermittent and continuous aeration strategies in combination with solids retention time (SRT) were investigated in terms of their impact on the performance and ecology of mainstream nitrification. Two lab-scale reactors (R1 and R2) were seeded with the same inoculum and subjected to intermittent aeration and continuous low aeration, respectively at statistically similar air-supply rates. For both reactors, SRT was progressively decreased from 8 d during Phase I to 4 and 2.5 d during Phases II and III, respectively. Compared to R2 (93 ± 4.8 %), R1 achieved more stable and higher ammonia oxidation (99 ± 0.13 %) averaged across SRTs. In R1, Nitrosospira were the dominant AOB, while in R2, AOB were a combination of Nitrosospira and Nitrosomonas. Among NOB, Nitrospira were more abundant than Nitrobacter for both R1 and R2 across SRTs. Intermittent aeration in R1 supported higher relative abundance of Comammox Nitrospira than R2. Notably, the enrichment patterns for nitrifying bacteria in the two reactors reflected distinct drivers (beyond microbial kinetics), including inoculum composition, extant oxygen or nitrogen concentrations or both. Overall, intermittent aeration strategies are integral to the design of biological nitrogen removal processes, although other considerations such as operational feasibility and process emissions might ultimately influence operating strategies.

Abstract Chemolithoautotrophic nitrifiers are model groups for linking phylogeny, evolution, and ecophysiology. Ammonia-oxidizing bacteria (AOB) typically dominate the first step of ammonia oxidation at high ammonium supply rates, ammonia-oxidizing archaea (AOA) and complete ammonia-oxidizing Nitrospira (comammox) are often active at lower supply rates or during AOB inactivity, and nitrite-oxidizing bacteria (NOB) complete canonical nitrification. Soil virus communities are dynamic but contributions to functional processes are largely undetermined. In addition, characterizing viruses infecting hosts with low relative abundance, such as nitrifiers, may be constrained by vast viral diversity, partial genome recovery, and difficulties in host linkage. Here, we describe a targeted incubation study that aimed to determine whether growth of different nitrifier groups in soil is associated with active virus populations and if process-focused analyses facilitate characterization of high-quality virus genomes. dsDNA viruses infecting different nitrifier groups were enriched in situ via differential host inhibition. Growth of each nitrifier group was consistent with predicted inhibition profiles and concomitant with the abundance of their viruses. These included 61 high-quality/complete virus genomes 35–173 kb in length with minimal similarity to validated families. AOA viruses lacked ammonia monooxygenase sub-unit C (amoC) genes found in marine AOA viruses but some encoded AOA-specific multicopper oxidase type 1 (MCO1), previously implicated in copper acquisition, and suggesting a role in supporting energy metabolism of soil AOA. Findings demonstrate focused incubation studies facilitate characterization of active host-virus interactions associated with specific processes and viruses of soil AOA, AOB, and NOB are dynamic and potentially influence nitrogen cycling processes.

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The ecophysiology of complete ammonia-oxidizing bacteria (CMX) of the genus Nitrospira and their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the specific contribution of their activity to nitrification processes has remained unclear. We aimed to disentangle the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen in oligotrophic carbonate rock aquifers. CMX amoA genes accounted on average for 16 to 75% of the total groundwater amoA genes detected. Nitrification rates were positively correlated to CMX clade A associated phylotypes and AOB affiliated with Nitrosomonas ureae. Short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed a large fraction to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOB and AOA was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater appears to be primarily governed by AOB. Higher growth yields at lower ammonia turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations.

Interrelated successive transformation steps of nitrification are performed by distinct microbial groups – the ammonia-oxidizers, comprising ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nitrite-oxidizers such as Nitrobacter and Nitrospira, which are the dominant genera in the investigated soils. Hence, not only their presence and activity in the investigated habitat is required for nitrification, but also their temporal and spatial interactions. To demonstrate the interdependence of both groups and to address factors promoting putative niche differentiation within each group, temporal and spatial changes in nitrifying organisms were monitored in an unfertilized grassland site over an entire vegetation period at the plot scale of 10 m2. Nitrifying organisms were assessed by measuring the abundance of marker genes (amoA for AOA and AOB, nxrA for Nitrobacter, 16S rRNA gene for Nitrospira) selected for the respective sub-processes. A positive correlation between numerically dominant AOA and Nitrospira, and their co-occurrence at the same spatial scale in August and October, suggests that the nitrification process is predominantly performed by these groups and is restricted to a limited timeframe. Amongst nitrite-oxidizers, niche differentiation was evident in observed seasonally varying patterns of co-occurrence and spatial separation. While their distributions were most likely driven by substrate concentrations, oxygen availability may also have played a role under substrate-limited conditions. Phylogenetic analysis revealed temporal shifts in Nitrospira community composition with an increasing relative abundance of OTU03 assigned to sublineage V from August onward, indicating its important role in nitrite oxidation.

ABSTRACT Understanding the changes in bacterial community structure in different microenvironments of Camellia oleifera is essential to better explore the benign interaction between beneficial microorganisms and plants. Using Camellia oleifera trees, a Chinese wooden oil plant as a model ecosystem, we characterized the archaeal and bacterial microbiome across five different tissue-level niches using 16S rRNA gene analyses. Our research indicates that the diversity of Camellia oleifera endophytic bacterial communities is highly dependent on the plant compartment. The species replacement process (69.90%) is the dominant factor in the differences in bacterial community structure. The dominant bacteria phyla (Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Chloroflexi, and Verrucomicrobia) of Camellia oleifera show a significant plant compartment (roots, stems, leaves, fruits) enrichment effects. A variety of bacteria (Hymenobacter, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Mesorhizobium, Bradyrhizobium, Bacillus, Ochrobactrum, Pantoea, Pseudomonas, etc.) with nitrogen-fixed potentials are enriched in Camellia oleifera tissue. In addition, the hub bacterial groups of Camellia oleifera are Nitrospira, Haemophilus, Staphylococcus, Ruminiclostridium, and Ochrobactrum. They are widespread colonization in various tissues with a low relative abundance and may play an important role in the nitrogen cycle, host life promotion, and plant defense. This study provides a holistic understanding of the endosphere bacterial community structure, which is one of the most complete ecological niche-level analyses of Camellia oleifera. These results provide a scientific theoretical basis for an in-depth discussion of plant-endosphere microbial interaction and better exploration of benign interaction of beneficial microorganisms and plants. IMPORTANCE Microorganisms inhabited various tissues of plants and play a key role in promoting plant growth, nutritional absorption, and resistance. Our research indicates that the diversity of Camellia oleifera endophytic bacterial communities is highly dependent on the plant compartment. Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Chloroflexi, and Verrucomicrobia are dominant bacteria phyla. The tissues of Camellia oleifera contain various bacteria with nitrogen fixation potential, host life promotion, and plant defense. This study provides a scientific theoretical basis for an in-depth discussion of plant-endosphere microbial interaction and better exploration of benign interaction of beneficial microorganisms and plants. Microorganisms inhabited various tissues of plants and play a key role in promoting plant growth, nutritional absorption, and resistance. Our research indicates that the diversity of Camellia oleifera endophytic bacterial communities is highly dependent on the plant compartment. Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Chloroflexi, and Verrucomicrobia are dominant bacteria phyla. The tissues of Camellia oleifera contain various bacteria with nitrogen fixation potential, host life promotion, and plant defense. This study provides a scientific theoretical basis for an in-depth discussion of plant-endosphere microbial interaction and better exploration of benign interaction of beneficial microorganisms and plants.

Nitrite is a pivotal component of the marine nitrogen cycle. The fate of nitrite determines the loss or retention of fixed nitrogen, an essential nutrient for all organisms. Loss occurs via anaerobic nitrite reduction to gases during denitrification and anammox, while retention occurs via nitrite oxidation to nitrate. Nitrite oxidation is usually represented in biogeochemical models by one kinetic parameter and one oxygen threshold, below which nitrite oxidation is set to zero. Here we find that the responses of nitrite oxidation to nitrite and oxygen concentrations vary along a redox gradient in a Pacific Ocean oxygen minimum zone, indicating niche differentiation of nitrite-oxidizing assemblages. Notably, we observe the full inhibition of nitrite oxidation by oxygen addition and nitrite oxidation coupled with nitrogen loss in the absence of oxygen consumption in samples collected from anoxic waters. Nitrite-oxidizing bacteria, including novel clades with high relative abundance in anoxic depths, were also detected in the same samples. Mechanisms corresponding to niche differentiation of nitrite-oxidizing bacteria across the redox gradient are considered. Implementing these mechanisms in biogeochemical models has a significant effect on the estimated fixed nitrogen budget.

Although the distribution of ammonia/nitrite oxidizers had been profiled in different habitats, current understanding is still limited regarding their niche differentiation in the integrated biofilm reactors, the symbiotic associations of ammonia/nitrite oxidizers, as well as the parasitic interaction between viruses and those functional organisms involved in the nitrogen cycle. Here, the integrated metagenomics and metatranscriptomics are applied to profile the ammonia/nitrite oxidizers communities and transcriptional activities changes along the flowpath of a concatenated full-scale rotating biological contactor (RBC) (frontend Stage-A and backend Stage-B). 19 metagenome-assembled genomes (MAGs) of ammonia/nitrite oxidizers were recovered by using a hybrid assembly approach, including four ammonia-oxidizing bacteria (AOB), two ammonia-oxidizing archaea (AOA), two complete ammonia oxidation bacteria (comammox), eight nitrite-oxidizing bacteria (NOB), and three anaerobic ammonium oxidation bacteria (anammox). Diverse AOB and anammox dominated Stage-A and collectively contributed to nitrogen conversion. With the decline of ammonia concentration along the flowpath, comammox and AOA appeared and increased in relative abundance in Stage-B, accounting for 8.8% of the entire community at the end of this reactor, and their dominating role in nitrogen turnover was indicated by the high transcription activity of their corresponding function genes. Moreover, the variation in the abundance of viruses infecting ammonia and nitrite oxidizers suggests that viruses likely act as a biotic factor mediating ammonia/nitrite oxidizer populations. This study demonstrates that complex factors shaped niche differentiation and symbiotic associations of ammonia/nitrite oxidizers in the RBC and highlights the importance of RBCs as model systems for the investigation of biotic and abiotic factors affecting the composition of microbiomes.