Complete nitrification by a single microorganism

31 Aug.,2022

 

nitrosomonas converts

Nitrification is a two-step process where ammonia is considered to first be oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and/or archaea (AOA), and subsequently to nitrate by nitrite-oxidizing bacteria (NOB). Described by Winogradsky already in 1890 1 , this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle 2 . 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 3 . 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 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 on 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.

Nitrification, the aerobic oxidation of ammonium to nitrate is divided into two subsequent reactions: ammonium oxidation to nitrite (equation (1)) and nitrite oxidation to nitrate (equation (2)). These two reactions are catalysed by physiologically distinct clades of microorganisms.

NH4++1.5O2→NO2-+H2O+2H+(ΔG0,=−274.7kJ⋅mol−1)

(1)

NO2-+0.5O2→NO3-(ΔG0,=−74.1kJ⋅mol−1)

(2)

NH4++2O2→NO3-+H2O+2H+(ΔG0,=−348.9kJ⋅mol−1)

(3)

Even though the existence of a single microorganism capable of oxidizing ammonium to nitrate (equation (3)) was not previously reported, it was proposed that such a microorganism could have a competitive advantage in biofilms and other microbial aggregates with low substrate concentrations3.

In this study, to characterize the microorganisms responsible for nitrogen transformations in an ammonium-oxidizing biofilm, we sampled the anaerobic compartment of a trickling filter connected to a recirculation aquaculture system (RAS)4 with an ammonium effluent of less than 100 μM. To enrich for the N-cycling community, a bioreactor was inoculated and supplied with low concentrations of ammonium, nitrite and nitrate under hypoxic conditions (≤ 3.1 µM O2). Within 12 months, we obtained a stable enrichment culture, which efficiently removed ammonium and nitrite from the medium ( ). The culture showed anaerobic ammonium-oxidizing (anammox) activity ( ), and fluorescence in situ hybridization (FISH) revealed that anammox organisms of the genus Brocadia constituted ~45% of all FISH-detectable bacteria. Surprisingly, Nitrospira-like NOB accounted for ~15% of the community and co-occurred with the Brocadia species in flocs ( ). This tight clustering with anammox bacteria was unexpected as both microorganisms require nitrite for growth. Together with the presence of Nitrospira at very low oxygen concentrations, this indicated that there could be a functional link between these organisms.

To determine the function of Nitrospira in the community, we extracted and sequenced total DNA from the enrichment culture biomass. In total 4.95 Giga base pairs of trimmed metagenomic sequence were obtained and used for de novo assembly. By differential-coverage and sequence composition-based binning5 it was possible to extract high-quality draft genomes of two Nitrospira species. The two strains had genomic pairwise average nucleotide identities (ANI)6 of 75% and thus clearly represented different species (Nitrospira sp.1 and sp.2, and ). Surprisingly, both genomes contained the full set of AMO and hydroxylamine dehydrogenase (HAO) genes for ammonia oxidation, in addition to the nitrite oxidoreductase (NXR) subunits necessary for nitrite oxidation in Nitrospira7. In both species all these genes were localized on a single contiguous genomic fragment, along with general housekeeping genes that allowed reliable phylogenetic assignment. Consequently, these Nitrospira species had the genetic potential for the complete oxidation of ammonia to nitrate. No AMO of canonical ammonia-oxidizing bacteria or archaea could be detected in the trimmed metagenomic reads or by amoA-specific PCR8,9 on DNA extracted from reactor biomass, and no other indications for the presence of ammonia-oxidizing microorganisms were found in the metagenome or by FISH analyses. The AMO structural genes (amoCAB) of both Nitrospira species, along with the putative additional AMO subunits amoEDD210,11, formed one gene cluster with haoAB-cycAB (encoding HAO, the putative membrane anchor protein HaoB, electron transfer protein cytochrome c554 and quinone reducing cytochrome cm552, respectively)12 and showed highest similarities to their counterparts in betaproteobacterial AOB (60% average amino acid identity to the Nitrosomonas europaea genes; and Supplementary Table 1). The same genomic region also contained genes for copper and heme transport, cytochrome c biosynthesis, and iron storage. These accessory genes were highly conserved in ammonia-oxidizing bacteria but not in other Nitrospira7,13, indicating their involvement in AMO and HAO biosynthesis or activation. Nitrospira sp.1 encoded three discrete amoC genes, one of which was clustered with a second, almost identical copy of amoA (97.7% amino acid identity). Nitrospira sp.2 lacked the second amoA, but contained four additional amoC and a second haoA gene (Supplementary Table 1). Unlike other Nitrospira7,13, both species lacked enzymes for assimilatory nitrite reduction, indicating adaptation to ammonium-containing habitats. For ammonium uptake, they encoded low affinity Rh-type transporters most closely related to Rh50 found in Nitrosomonas europea14, in contrast to most AOB and NOB that have the high affinity AmtB-type proteins. Both species encoded ureases and the corresponding ABC transport systems, indicating that urea could be used as an alternative ammonium source. Interestingly, Ca. N. inopinata, the moderately thermophilic ammonia-oxidizing Nitrospira described by Daims et al.15, encoded a similar set of AMO, HAO and urease proteins, and also lacked genes for assimilatory nitrite reduction. Unlike the two species described here, however, it contained a periplasmic cytochrome c nitrite reductase (NrfA) that could allow it to conserve energy by dissimilatory nitrite reduction to ammonium (DNRA), but might also provide ammonium for assimilation. The evolutionary divergence of these organisms was also reflected in the low ANI values of 70.3 - 71.6% between Ca. N. inopinata and the two species described here. Concerning their genetic repertoire for nitrite oxidation, sp.2 had four almost identical (>99% amino acid identity) NXR alpha and beta (NxrAB) subunits. Sp.1 had two nxrAB copies encoding identical NxrB subunits, but NxrA subunits with amino acid identities of 89.6%, which were separated into distinct clusters in phylogenetic analyses. One homolog branched with sequences from N. moscoviensis, while the other formed a novel sequence cluster together with the sequences from sp.2 ( ).

To ascertain that ammonia oxidation occurred under hypoxic conditions in the enrichment culture, we supplied the bioreactor with 15N-labelled ammonium. While the anammox bacteria consumed 15NH4+ and converted it into 29N2, a steady increase of 30N2 was also observed ( ). This formation of 30N2 could only be explained by the production of 15N-labelled nitrite derived through aerobic ammonium oxidation. As metagenomic analyses confirmed that the Nitrospira species were the only organism in the enrichment harbouring AMO and HAO, this clearly showed that they were able to perform this reaction even at O2 concentrations lower than 3.1 µM. To unambiguously link this activity to Nitrospira, we visualized the AMO protein in situ using batch incubations with reactor biomass and fluorescein thiocarbamoylpropargylamine (FTCP), a fluorescently labelled acetylene analogue that acts as suicide substrate for AMO16 and covalently binds to the enzyme17. When counterstained with Nitrospira-specific FISH probes, including a newly designed probe specifically targeting the 16S rRNA-defined phylogenetic group comprising spp.1 and 2 ( and ), strong FTCP labelling of Nitrospira cells was observed, providing strong support for the presence of the ammonia-oxidizing enzyme at single-cell level ( and ).

Batch incubations were performed at ambient oxygen concentrations to determine conversion rates of ammonium and nitrite, the level of inhibition by allylthiourea (ATU; a potent inhibitor of bacterial ammonia oxidation18,19), and the use of urea as ammonium source for nitrification. Flocs were mechanically disrupted to ensure complete exposure of the biomass to oxygen, which inhibits the anammox and denitrification processes20,21. This inhibition was confirmed by the lack of labelled N2 formation in incubations with 15NH4+. In these incubations ( and ), the culture oxidized ammonium (6.0 ± 1.0 µM NH4+/h) and nitrite (23 ± 4.7 µM NO2-/h) to nitrate. ATU selectively inhibited ammonia oxidation, but did not affect nitrite oxidation rates. Urea was converted to ammonium, which was subsequently oxidized to nitrate (7.8 ± 1.1 µM nitrate/h) suggesting that these Nitrospira species were capable of using urea as source of ammonia to drive nitrification, as was also reported for some AOA22 and AOB23. This trait could enable them to thrive in environments like fertilized soils, wastewater treatment plants (wwtps), and many aquatic systems where urea is often present at micromolar levels24. However, it should be noted that the two Nitrospira spp. were not the only organisms in the enrichment culture that encoded ureases.

To investigate substrate-dependent inorganic carbon fixation as a proxy for energy conservation from ammonia and nitrite oxidation, we used FISH in combination with microautoradiography (FISH-MAR)25. Aerobic incubations with mechanically disrupted flocs were performed in the presence of 500 µM ammonium, 500 µM ammonium with 100 µM ATU, or 500 µM nitrite. Nitrospira incorporated carbon from 14C-labelled bicarbonate in the presence of either ammonium or nitrite, and ammonia-dependent carbon fixation was strongly inhibited by the addition of ATU ( and ). Only flocs containing Nitrospira were labelled during all incubations, suggesting that these were the only chemolithoautotrophic nitrifying organisms present in the culture and indeed could conserve energy from the oxidation of ammonia and nitrite.

In 16S rRNA-based phylogenetic analyses, the two ammonia-oxidizing Nitrospira species from our enrichment culture formed two separate lineages within one strongly supported sequence cluster affiliated with Nitrospira sublineage II26 ( ). They both grouped with highly similar sequences (>99% nucleotide identity) from a diverse range of habitats, including soil, groundwater, RAS, wastewater treatment plants (wwtps) and drinking water distribution systems. The formation of distinct clusters containing sp.1 and sp.2 indicated that the last common ancestor encoded genes for complete nitrification and that this lifestyle might be conserved in most organisms affiliated with this sequence group.

To explore the environmental relevance of these Nitrospira, we searched the NCBI nr database27 for closely related amoA genes. Surprisingly, we found the AmoA proteins of the two Nitrospira species to be phylogenetically divergent from the described bacterial AmoA sequences. Nitrospira sp.2 AmoA was 97-98% identical to the so-called “unusual” methane monooxygenase (PMO) proteins of Crenothrix polyspora28. The two AmoA copies from Nitrospira sp.1 had lower similarities to Crenothrix PmoA (90-91% identity), but also affiliated with this group ( ). Sequences within this group cannot be amplified by standard amoA primers, but only by pmoA primers when used at reduced stringency29. Therefore the public databases only contain few closely related sequences, which mainly were derived from habitats studied for their bacterial methane-oxidizing (MOB) communities. Highly similar sequences derived from wwtps and drinking water systems, however, indicated occurrence of ammonia-oxidizing Nitrospira in a range of engineered and natural environments. We furthermore screened all publicly available shotgun datasets on MG-RAST30. Indeed, 168 metagenomes (out of 6255) and 28 metatranscriptomes (out of 1051) contained at least two reads affiliated with this amoA group, yielding a total of 3727 reads that were obtained mainly from soil, sediments and wwtps ( ). Thus, our results showed that the Crenothrix sequence group consists of so far unrecognized AMO sequences overlooked in nitrification studies based on amoA gene detection. Based on these findings, it is highly likely that the PCR-based determination of the Crenothrix pmoA gene from an environmental sample28 was erroneous, and this cluster only contains genes encoding AMOs. Nevertheless, with the currently available information it cannot be excluded that certain Crenothrix species attained an amoA gene through lateral gene transfer and use the encoded protein as a surrogate PMO.

In conclusion, here we demonstrated the existence of complete nitrification in a single organism (comammox) and identified two Nitrospira species capable of catalysing this process (equation (3)). In 16S rRNA or amoA/pmoA-based studies these organisms would have been classified as NOB or MOB, respectively. Hence, our results show that a whole group of ammonia-oxidizing organisms was previously overlooked. Our findings furthermore disprove the long-held assumption that nitrification (ammonia oxidation via nitrite to nitrate) is catalysed by two distinct functional groups, thus redefining a key process of the biogeochemical nitrogen cycle.

Based on their physiology, differences in genome content, and separation in different phylogenetic groups in 16S rRNA-based analyses, we propose tentative names for both Nitrospira species present in our enrichment: “Candidatus Nitrospira nitrosa” (Etymology: L. fem. adj. nitrosa, full of natron; the nitrite and nitrate forming Nitrospira) for sp.1 and “Candidatus Nitrospira nitrificans” (N.L. part. adj. nitrificans, nitrifying; the nitrifying Nitrospira) for sp.2. Both species are chemolithoautotrophic and fully oxidize ammonia via nitrite to nitrate.