Ecophysiological Characterization of Ammonia-Oxidizing Archaea and Bacteria from Freshwater

ABSTRACTAerobic biological ammonia oxidation is carried out by two groups of microorganisms, ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). Here we present a study using cultivation-based methods to investigate the differences in growth of three AOA cultures and one AOB culture enriched from freshwater environments. The strain in the enriched AOA culture belong to thaumarchaeal group I.1a, with the strain in one enrichment culture having the highest identity with “CandidatusNitrosoarchaeum koreensis” and the strains in the other two representing a new genus of AOA. The AOB strain in the enrichment culture was also obtained from freshwater and had the highest identity to AOB from theNitrosomonas oligotrophagroup (Nitrosomonascluster 6a). We investigated the influence of ammonium, oxygen, pH, and light on the growth of AOA and AOB. The growth rates of the AOB increased with increasing ammonium concentrations, while the growth rates of the AOA decreased slightly. Increasing oxygen concentrations led to an increase in the growth rate of the AOB, while the growth rates of AOA were almost oxygen insensitive. Light exposure (white and blue wavelengths) inhibited the growth of AOA completely, and the AOA did not recover when transferred to the dark. AOB were also inhibited by blue light; however, growth recovered immediately after transfer to the dark. Our results show that the tested AOB have a competitive advantage over the tested AOA under most conditions investigated. Further experiments will elucidate the niches of AOA and AOB in more detail.

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Enrichment and Characterization of an Autotrophic Ammonia-Oxidizing Archaeon of Mesophilic Crenarchaeal Group I.1a from an Agricultural Soil

Applied and Environmental Microbiology ◽

10.1128/aem.05787-11 ◽

2011 ◽

Vol 77 (24) ◽

pp. 8635-8647 ◽

Cited By ~ 154

Author(s):

Man-Young Jung ◽

Soo-Je Park ◽

Deullae Min ◽

Jin-Seog Kim ◽

W. Irene C. Rijpstra ◽

...

Keyword(s):

16S Rrna ◽

Agricultural Soil ◽

Ammonia Oxidizing Bacteria ◽

Sequence Comparisons ◽

Soil Nitrification ◽

Content Type ◽

Group I ◽

Single Sequence

ABSTRACTSoil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizingArchaeafrom soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain MY1) in a highly enriched culture derived from agricultural soil. Fluorescencein situhybridization microscopy showed that, after 2 years of enrichment, the culture was composed of >90% archaeal cells. Clone libraries of both 16S rRNA and archaealamoAgenes featured a single sequence each. No bacterialamoAgenes could be detected by PCR. A [13C]bicarbonate assimilation assay showed stoichiometric incorporation of13C intoArchaea-specific glycerol dialkyl glycerol tetraethers. Strain MY1 falls phylogenetically within crenarchaeal group I.1a; sequence comparisons to “CandidatusNitrosopumilus maritimus” revealed 96.9% 16S rRNA and 89.2%amoAgene similarities. Completed growth assays showed strain MY1 to be chemoautotrophic, mesophilic (optimum at 25°C), neutrophilic (optimum at pH 6.5 to 7.0), and nonhalophilic (optimum at 0.2 to 0.4% salinity). Kinetic respirometry assays showed that strain MY1's affinities for ammonia and oxygen were much higher than those of ammonia-oxidizing bacteria (AOB). The yield of the greenhouse gas N2O in the strain MY1 culture was lower but comparable to that of soil AOB. We propose that this new soil ammonia-oxidizing archaeon be designated “CandidatusNitrosoarchaeum koreensis.”

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Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01478-10 ◽

2010 ◽

Vol 76 (22) ◽

pp. 7575-7587 ◽

Cited By ~ 148

Author(s):

Byoung-Joon Park ◽

Soo-Je Park ◽

Dae-No Yoon ◽

Stefan Schouten ◽

Jaap S. Sinninghe Damsté ◽

...

Keyword(s):

16S Rrna ◽

Marine Sediments ◽

Ammonia Oxidation ◽

Enrichment Culture ◽

Gene Copy ◽

Initial Growth ◽

Ammonia Oxidizing Archaea ◽

Group I ◽

Sulfur Oxidizing ◽

Sulfur Oxidizing Bacteria

ABSTRACT The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “Candidatus Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [13C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.

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“CandidatusNitrosotenuis aquarius,” an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter

Applied and Environmental Microbiology ◽

10.1128/aem.01430-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 17

Author(s):

Laura A. Sauder ◽

Katja Engel ◽

Chien-Chi Lo ◽

Patrick Chain ◽

Josh D. Neufeld

Keyword(s):

Genome Sequence ◽

Ammonia Oxidation ◽

Waste Product ◽

Optimal Growth ◽

Protein Glycosylation ◽

Ammonia Toxicity ◽

Ammonia Oxidizing Bacteria ◽

Common Belief ◽

Content Type ◽

Group I

ABSTRACTAmmonia is a metabolic waste product excreted by aquatic organisms that causes toxicity when it accumulates. Aquaria and aquaculture systems therefore use biological filters that promote the growth of nitrifiers to convert ammonia to nitrate. Ammonia-oxidizing bacteria (AOB) have been isolated from aquarium biofilters and are available as commercial supplements, but recent evidence suggests that ammonia-oxidizing archaea (AOA) are abundant in aquarium biofilters. In this study, we report the cultivation and closed genome sequence of the novel AOA representative “CandidatusNitrosotenuis aquarius,” which was enriched from a freshwater aquarium biofilter. “Ca. Nitrosotenuis aquarius” oxidizes ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells and a generation time of 34.9 h. “Ca. Nitrosotenuis aquarius” has an optimal growth temperature of 33°C, tolerates up to 3 mM NH4Cl, and grows optimally at 0.05% salinity. Transmission electron microscopy revealed that “Ca. Nitrosotenuis aquarius” cells are rod shaped, with a diameter of ∼0.4 μm and length ranging from 0.6 to 3.6 μm. In addition, these cells possess surface layers (S-layers) and multiple proteinaceous appendages. Phylogenetically, “Ca. Nitrosotenuis aquarius” belongs to the group I.1aThaumarchaeota, clustering with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants. The complete 1.70-Mbp genome contains genes involved in ammonia oxidation, bicarbonate assimilation, flagellum synthesis, chemotaxis, S-layer production, defense, and protein glycosylation. Incubations with differential inhibitors indicate that “Ca. Nitrosotenuis aquarius”-like AOA contribute to ammonia oxidation within the aquarium biofilter from which it originated.IMPORTANCENitrification is a critical process for preventing ammonia toxicity in engineered biofilter environments. This work describes the cultivation and complete genome sequence of a novel AOA representative enriched from a freshwater aquarium biofilter. In addition, despite the common belief in the aquarium industry that AOB mediate ammonia oxidation, the present study suggests anin siturole for “Ca. Nitrosotenuis aquarius”-like AOA in freshwater aquarium biofilters.

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Core and Intact Polar Glycerol Dibiphytanyl Glycerol Tetraether Lipids of Ammonia-Oxidizing Archaea Enriched from Marine and Estuarine Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.02758-10 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3468-3477 ◽

Cited By ~ 138

Author(s):

Angela Pitcher ◽

Ellen C. Hopmans ◽

Annika C. Mosier ◽

Soo-Je Park ◽

Sung-Keun Rhee ◽

...

Keyword(s):

San Francisco ◽

Marine Sediments ◽

Membrane Lipids ◽

Hot Spring ◽

Enrichment Culture ◽

Estuarine Sediments ◽

Content Type ◽

Ammonia Oxidizing Archaea ◽

Coastal Marine ◽

Group I

ABSTRACTGlycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary (“CandidatusNitrosoarchaeum limnia”), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and “CandidatusNitrosopumilus maritimus”; however, they were absent in the group I.1b AOA “CandidatusNitrososphaera gargensis.”

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Measurements of nitrite production in and around the primary nitrite maximum in the central California Current

Biogeosciences ◽

10.5194/bg-10-7395-2013 ◽

2013 ◽

Vol 10 (11) ◽

pp. 7395-7410 ◽

Cited By ~ 47

Author(s):

A. E. Santoro ◽

C. M. Sakamoto ◽

J. M. Smith ◽

J. N. Plant ◽

A. L. Gehman ◽

...

Keyword(s):

Ammonia Oxidation ◽

Heterotrophic Bacteria ◽

California Current ◽

Euphotic Zone ◽

Ammonia Oxidizing Bacteria ◽

Central California ◽

Oxidation Rates ◽

Ammonia Oxidizing Archaea ◽

Nitrite Production ◽

Nh3 Oxidation

Abstract. Nitrite (NO2−) is a substrate for both oxidative and reductive microbial metabolism. NO2− accumulates at the base of the euphotic zone in oxygenated, stratified open-ocean water columns, forming a feature known as the primary nitrite maximum (PNM). Potential pathways of NO2− production include the oxidation of ammonia (NH3) by ammonia-oxidizing bacteria and archaea as well as assimilatory nitrate (NO3−) reduction by phytoplankton and heterotrophic bacteria. Measurements of NH3 oxidation and NO3− reduction to NO2− were conducted at two stations in the central California Current in the eastern North Pacific to determine the relative contributions of these processes to NO2− production in the PNM. Sensitive (< 10 nmol L−1), precise measurements of [NH4+] and [NO2−] indicated a persistent NH4+ maximum overlying the PNM at every station, with concentrations as high as 1.5 μmol L−1. Within and just below the PNM, NH3 oxidation was the dominant NO2− producing process, with rates of NH3 oxidation to NO2− of up to 31 nmol L−1 d−1, coinciding with high abundances of ammonia-oxidizing archaea. Though little NO2− production from NO3− was detected, potentially nitrate-reducing phytoplankton (photosynthetic picoeukaryotes, Synechococcus, and Prochlorococcus) were present at the depth of the PNM. Rates of NO2− production from NO3− were highest within the upper mixed layer (4.6 nmol L−1 d−1) but were either below detection limits or 10 times lower than NH3 oxidation rates around the PNM. One-dimensional modeling of water column NO2− production agreed with production determined from 15N bottle incubations within the PNM, but a modeled net biological sink for NO2− just below the PNM was not captured in the incubations. Residence time estimates of NO2− within the PNM ranged from 18 to 470 days at the mesotrophic station and was 40 days at the oligotrophic station. Our results suggest the PNM is a dynamic, rather than relict, feature with a source term dominated by ammonia oxidation.

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Transcriptional Response of the Archaeal Ammonia Oxidizer Nitrosopumilus maritimus to Low and Environmentally Relevant Ammonia Concentrations

Applied and Environmental Microbiology ◽

10.1128/aem.02028-13 ◽

2013 ◽

Vol 79 (22) ◽

pp. 6911-6916 ◽

Cited By ~ 28

Author(s):

Tatsunori Nakagawa ◽

David A. Stahl

Keyword(s):

Ammonia Oxidation ◽

Physiological State ◽

Transcriptional Response ◽

Ammonia Oxidizer ◽

Mrna Levels ◽

Marine Microorganisms ◽

Transcript Levels ◽

Content Type ◽

Ammonia Oxidizing Archaea ◽

Ammonia Transport

ABSTRACTThe ability of chemoautotrophic ammonia-oxidizing archaea to compete for ammonia among marine microorganisms at low ambient concentrations has been in part attributed to their extremely high affinity for ammonia, but as yet there is no mechanistic understanding of supporting metabolism. We examined transcription of selected genes for anabolic functions (CO2fixation, ammonia transport, and cell wall synthesis) and a central catabolic function (ammonia oxidation) in the thaumarchaeonNitrosopumilus maritimusSCM1 growing at two ammonia concentrations, as measured by combined ammonia and ammonium, one well above theKmfor ammonia oxidation (∼500 μM) and the other well below theKm(<10 nM). Transcript levels were generally immediately and differentially repressed when cells transitioned from ammonia-replete to ammonia-limiting conditions. Transcript levels for ammonia oxidation, CO2fixation, and one of the ammonia transport genes were approximately the same at high and low ammonia availability. Transcripts for all analyzed genes decreased with time in the complete absence of ammonia, but with various rates of decay. The new steady-state mRNA levels established are presumably more reflective of the natural physiological state of ammonia-oxidizing archaea and offer a reference for interpreting message abundance patterns in the natural environment.

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Isotopic Signature of N2O Produced by Marine Ammonia-Oxidizing Archaea

Science ◽

10.1126/science.1208239 ◽

2011 ◽

Vol 333 (6047) ◽

pp. 1282-1285 ◽

Cited By ~ 276

Author(s):

Alyson E. Santoro ◽

Carolyn Buchwald ◽

Matthew R. McIlvin ◽

Karen L. Casciotti

Keyword(s):

Ammonia Oxidation ◽

Stratospheric Ozone ◽

Isotopic Signature ◽

Primary Sources ◽

Ammonia Oxidizing Bacteria ◽

Isotopic Signatures ◽

Ozone Destruction ◽

Ammonia Oxidizing Archaea ◽

Isotope Measurements ◽

Archaeal Ammonia Oxidation

The ocean is an important global source of nitrous oxide (N2O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N2O, but the isotopic signatures of N2O produced by these processes are not consistent with the marine contribution to the global N2O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N2O. Natural-abundance stable isotope measurements indicate that the produced N2O had bulk δ15N and δ18O values higher than observed for ammonia-oxidizing bacteria but similar to the δ15N and δ18O values attributed to the oceanic N2O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N2O source.

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Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream

Applied and Environmental Microbiology ◽

10.1128/aem.00250-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4492-4504 ◽

Cited By ~ 14

Author(s):

Manabu Nishizawa ◽

Sanae Sakai ◽

Uta Konno ◽

Nozomi Nakahara ◽

Yoshihiro Takaki ◽

...

Keyword(s):

Oxygen Isotope ◽

Ammonia Oxidation ◽

Isotope Effects ◽

Isotope Ratios ◽

Oxidation Activity ◽

Content Type ◽

Ammonia Oxidizing Archaea ◽

Nitrogen Pools ◽

Archaeal Ammonia Oxidation

ABSTRACTAmmonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in thein situquantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ15NNO2−and δ18ONO2−, respectively) are geochemical tracers for evaluating the sources and thein siturate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilicThaumarchaeotapopulations composed almost entirely of “CandidatusNitrosocaldus.” The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ18O value of nitrite produced from ammonia oxidation varied with the δ18O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilicThaumarchaeotacould be estimated using δ18ONO2−in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments.IMPORTANCEBecause ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilicThaumarchaeotaand elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

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Malonic Semialdehyde Reductase from the Archaeon Nitrosopumilus maritimus Is Involved in the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

Applied and Environmental Microbiology ◽

10.1128/aem.03390-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1700-1707 ◽

Cited By ~ 16

Author(s):

Julia Otte ◽

Achim Mall ◽

Daniel M. Schubert ◽

Martin Könneke ◽

Ivan A. Berg

Keyword(s):

Succinic Semialdehyde ◽

Alcohol Dehydrogenases ◽

Content Type ◽

Metallosphaera Sedula ◽

Ammonia Oxidizing Archaea ◽

The Family ◽

Terrestrial Environments ◽

Identification And Characterization ◽

Low Activity

ABSTRACTThe recently described ammonia-oxidizing archaea of the phylumThaumarchaeotaare highly abundant in marine, geothermal, and terrestrial environments. All characterized representatives of this phylum are aerobic chemolithoautotrophic ammonia oxidizers assimilating inorganic carbon via a recently described thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Although some genes coding for the enzymes of this cycle have been identified in the genomes ofThaumarchaeota, many other genes of the cycle are not homologous to the characterized enzymes from other species and can therefore not be identified bioinformatically. Here we report the identification and characterization of malonic semialdehyde reductase Nmar_1110 in the cultured marine thaumarchaeonNitrosopumilus maritimus. This enzyme, which catalyzes the reduction of malonic semialdehyde with NAD(P)H to 3-hydroxypropionate, belongs to the family of iron-containing alcohol dehydrogenases and is not homologous to malonic semialdehyde reductases fromChloroflexus aurantiacusandMetallosphaera sedula. It is highly specific to malonic semialdehyde (Km, 0.11 mM;Vmax, 86.9 μmol min−1mg−1of protein) and exhibits only low activity with succinic semialdehyde (Km, 4.26 mM;Vmax, 18.5 μmol min−1mg−1of protein). Homologues ofN. maritimusmalonic semialdehyde reductase can be found in the genomes of allThaumarchaeotasequenced so far and form a well-defined cluster in the phylogenetic tree of iron-containing alcohol dehydrogenases. We conclude that malonic semialdehyde reductase can be regarded as a characteristic enzyme for the thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle.

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Multidimensional Reveal of Nitrogen Regulation on Comammox

10.21203/rs.2.21888/v1 ◽

2020 ◽

Author(s):

Yuxiang Zhao ◽

Jiajie Hu ◽

Weiling Yang ◽

Jiaqi Wang ◽

Zhongjun Jia ◽

...

Keyword(s):

Nitrogen Source ◽

Nitrate Reduction ◽

Ammonia Oxidation ◽

Enrichment Culture ◽

Ammonia Oxidizer ◽

Nitrification Rate ◽

Ammonia Oxidizing Bacteria ◽

Metagenomic Sequencing ◽

Sole Nitrogen Source ◽

Total Bacteria

Abstract Background The discovery of complete ammonia oxidizer (comammox) was groundbreaking. Comammox can use ammonia as the sole nitrogen source and turn it to nitrate. Moreover, genomic data indicated that comammox contained genes which can metabolize urea and nitrite. However, the feasibility of enriching comammox with urea and nitrite in long term has not been proved. This study enriched comammox’s culture by using nitrite in reactor SA and urea in reactor SB. Results The nitrification rate of reactor SB (1.29 mg N·g -1 biofilm · d -1 ) was higher than that in reactor SA (0.6 mg N · g -1 biofilm · d -1 ) at the 390 th day. Comammox outnumbered ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in both reactor SA (9.04 × 10 9 copies / g biofilm) and reactor SB (5.34×10 10 copies/ g biofilm). In reactor SA, comammox’s amoA accounted for 92% of the total amoA, which was higher than that in reactor SB (85%). However, the percentage of comammox (4%) in total bacteria was much lower than reactor SB (14%). The results of metagenomic sequencing showed that all the pathways of nitrogen cycle including nitrification, nitrogen fixation, denitrification, assimilation nitrate reduction, and dissimilation nitrate reduction can be detected in both reactor SA and reactor SB except the anammox pathway. The genes related to nitrite oxidation and nitrate reduction in reactor SA (TPM = 5099; TPM = 3329) was higher than that of in reactor SB (TPM = 4071; TPM = 2984), presumably due to the demand of turning nitrite to nitrate and turning nitrate to ammonia. While genes related to ammonia oxidation and urea metabolism in reactor SB (TPM = 3915; TPM = 3638) was higher than that in reactor SA (TPM = 2708; TPM = 3002). Conclusion Nitrite and urea can regulate the enrichment culture of comammox by converting its metabolic pathway. Using nitrite as sole nitrogen source can improve the proportion comammox’s amoA in total amoA while using urea as the sole nitrogen source may increase comammox’s proportion in total bacteria. These results can accelerate the enrichment of comammox and facilitate the promotion of comammox’s engineering operation.

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