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Nitrification, the sequential aerobic oxidation of ammonia via nitrite to nitrate, is a key process of the biogeochemical nitrogen cycle and catalyzed by two aerobic microbial guilds (nitrifiers): ammonia oxidizers and nitrite-oxidizing bacteria (NOB). NOB are generally considered as metabolically restricted and dependent on ammonia oxidizers. Here, we report that, surprisingly, key NOB of many ecosystems ( Nitrospira ) convert urea, an important ammonia source in nature, to ammonia and CO 2 . Thus, Nitrospira supply urease-negative ammonia oxidizers with ammonia and receive nitrite produced by ammonia oxidation in return, leading to a reciprocal feeding interaction of nitrifiers. Moreover, Nitrospira couple formate oxidation with nitrate reduction to remain active in anoxia. Accordingly, Nitrospira are unexpectedly flexible and contribute to nitrogen cycling beyond nitrite oxidation. Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira . Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO 2 . Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Nitrospira -like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches. The recently discovered comammox bacteria have the potential to completely oxidize ammonia to nitrate. These microorganisms are part of the Nitrospira genus and are present in a variety of environments, including biological nutrient removal (BNR) systems. However, the physiological traits within and between comammox and nitrite-oxidizing bacterium (NOB)-like Nitrospira species have not been analyzed in these ecosystems. In this study, we identified Nitrospira strains dominating the nitrifying community of a sequencing batch reactor (SBR) performing BNR under microaerobic conditions. We recovered metagenome-derived draft genomes from two Nitrospira strains: (i) Nitrospira sp. strain UW-LDO-01, a comammox-like organism classified as “ Candidatus Nitrospira nitrosa,” and (ii) Nitrospira sp. strain UW-LDO-02, a nitrite-oxidizing strain belonging to the Nitrospira defluvii species. A comparative genomic analysis of these strains with other Nitrospira -like genomes identified genomic differences in “ Ca . Nitrospira nitrosa” mainly attributed to each strain’s niche adaptation. Traits associated with energy metabolism also differentiate comammox from NOB-like genomes. We also identified several transcriptionally regulated adaptive traits, including stress tolerance, biofilm formation, and microaerobic metabolism, which might explain survival of Nitrospira under multiple environmental conditions. Overall, our analysis expanded our understanding of the genetic functional features of “ Ca . Nitrospira nitrosa” and identified genomic traits that further illuminate the phylogenetic diversity and metabolic plasticity of the Nitrospira genus. IMPORTANCE Nitrospira -like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches. Author Video : An author video summary of this article is available.

The discovery of comammox in the genus Nitrospira changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and—among nitrifiers—unsurpassed ecophysiological versatility could make comammox Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations. The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and in situ transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other Nitrospira genome and might confer a selective advantage to this strain over other Nitrospira strains in anoxic conditions. IMPORTANCE The discovery of comammox in the genus Nitrospira changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and—among nitrifiers—unsurpassed ecophysiological versatility could make comammox Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations.

Purpose Nitrification, the microbial oxidation of ammonia to nitrate via nitrite, is a pivotal component of the biogeochemical nitrogen cycle. Nitrification was conventionally assumed as a two-step process in which ammonia oxidation was thought to be catalyzed by ammonia-oxidizing archaea (AOA) and bacteria (AOB), as well as nitrite oxidation by nitrite-oxidizing bacteria (NOB). This long-held assumption of labour division between the two functional groups, however, was challenged by the recent unexpected discovery of complete ammonia oxidizers within the Nitrospira genus that are capable of converting ammonia to nitrate in a single organism (comammox). This breakthrough raised fundamental questions on the niche specialization and differentiation of comammox organisms with other canonical nitrifying prokaryotes in terrestrial ecosystems. Materials and methods This article provides an overview of the recent insights into the genomic analysis, physiological characterization and environmental investigation of the comammox organisms, which have dramatically changed our perspective on the aerobic nitrification process. By using quantitative PCR analysis, we also compared the abundances of comammox Nitrospira clade A and clade B, AOA, AOB and NOB in 300 forest soil samples from China spanning a wide range of soil pH. Results and discussion Comammox Nitrospira are environmentally widespread and numerically abundant in natural and engineered habitats. Physiological data, including ammonia oxidation kinetics and metabolic versatility, and comparative genomic analysis revealed that comammox organisms might functionally outcompete other canonical nitrifiers under highly oligotrophic conditions. These findings highlight the necessity in future studies to re-evaluate the niche differentiation between ammonia oxidizers and their relative contribution to nitrification in various terrestrial ecosystems by including comammox Nitrospira in such comparisons. Conclusions The discovery of comammox and their broad environmental distribution added a new dimension to our knowledge of the biochemistry and physiology of nitrification and has far-reaching implications for refined strategies to manipulate nitrification in terrestrial ecosystems and to maximize agricultural productivity and sustainability.

The recently discovered complete ammonia oxidizers (comammox Nitrospira ) have fundamentally changed the perceptions of two-step nitrification. Previous studies suggest that comammox Nitrospira may outcompete canonical ammonia oxidizers under ammonia-limited conditions (e.g., acidic soils). However, there is no convincing evidence that links comammox Nitrospira activity to nitrification in acidic soils. Here, we report the relative contributions of ammonia-oxidizing bacteria (AOB), archaea (AOA), and comammox Nitrospira to nitrification in four orchard soils with different ages (5Y, 10Y, 20Y, and 30Y) and one adjacent natural forest soil (NF) in China. DNA-based stable-isotope probing demonstrated that both AOA and AOB assimilated 13 CO 2 in the nonacidic NF, 5Y, and 10Y soils, although a small shift from the light fractions to heavy fractions was observed for comammox Nitrospira clade A in the 10Y soil. Comammox Nitrospira clade A rather than AOA and AOB was labeled by 13 CO 2 in strongly acidic 30Y soil. In strongly acidic 20Y soil, comammox Nitrospira clade A was much more heavily labeled than AOB. These results suggest the different contributions of ammonia oxidizers to nitrification in orchard soils with different ages and a greater functional importance of comammox Nitrospira than canonical ammonia oxidizers in ammonia oxidation in the tested strongly acidic orchard soils.

The increasing production of nitrogen-containing fertilizers is crucial to meet the global food demand, yet high losses of reactive nitrogen associated with the food production/consumption chain progressively deteriorate the natural environment. Currently, mesophilic nitrogen-removing microbes eliminate nitrogen from wastewaters. Although thermophilic nitrifiers have been separately enriched from natural environments, no bioreactors are described that couple these processes for the treatment of nitrogen in hot wastewaters. Samples from composting facilities were used as inoculum for the batch-wise enrichment of thermophilic nitrifiers (350 days). Subsequently, the enrichments were transferred to a bioreactor to obtain a stable, high-rate nitrifying process (560 days). The community contained up to 17% ammonia-oxidizing archaea (AOAs) closely related to 'Candidatus Nitrososphaera gargensis', and 25% nitrite-oxidizing bacteria (NOBs) related to Nitrospira calida. Incorporation of super(13)C-derived bicarbonate into the respective characteristic membrane lipids during nitrification supported their activity as autotrophs. Specific activities up to 198 plus or minus 10 and 894 plus or minus 81 mg N g super(-1) VSS per day for AOAs and NOBs were measured, where NOBs were 33% more sensitive to free ammonia. The NOBs were extremely sensitive to free nitrous acid, whereas the AOAs could only be inhibited by high nitrite concentrations, independent of the free nitrous acid concentration. The observed difference in product/substrate inhibition could facilitate the development of NOB inhibition strategies to achieve more cost-effective processes such as deammonification. This study describes the enrichment of autotrophic thermophilic nitrifiers from a nutrient-rich environment and the successful operation of a thermophilic nitrifying bioreactor for the first time, facilitating opportunities for thermophilic nitrogen removal biotechnology.

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

A performance comparison between the conventional anaerobic/anoxic/oxic (A super(2)/O) process and the anoxic/anaerobic/oxic (reverse A super(2)/O) process was conducted in lab-scale at the hydraulic retention time of 0.75 d and solids retention time of 10 d for more than 200 days. Both processes demonstrated excellent removal efficiency for organic matter (> 96%) and total nitrogen (> 85%), with no significant difference in microbial activities, sludge phosphorus (P) content, sludge settling property, and nitrifying community (dominated by Nitrosomonas and Nitrospira). However, there was significant difference in P removal, with the total P removal efficiencies in the reverse A super(2)/O and A super(2)/O systems of 76 plus or minus 6% and 70 plus or minus 6%, respectively. Placing an anoxic stage before the anaerobic stage in the reverse A super(2)/O process resulted in a lower oxidation-reduction potential (ORP = -268 plus or minus 45 mV) of the anaerobic zone, which contributed to higher P uptake by bacteria under subsequent aerobic respiration.

Nitrous oxide (N 2 O) 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 N 2 O and NO globally. However, nothing is known about N 2 O 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 N 2 O, and emits N 2 O at levels that are comparable to AOA but much lower than AOB. Furthermore, we demonstrate that N 2 O formed by N. inopinata formed under varying oxygen regimes originates from abiotic conversion of hydroxylamine. Our findings indicate that comammox microbes may produce less N 2 O 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.

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 (K m(app)_NH3  ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (K m(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 .