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.

A pure culture of the complete nitrifier Nitrospira inopinata shows a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers and genomic potential for alternative metabolisms, probably reflecting an important role in nitrification in oligotrophic environments. Nitrospira inopinata was the first bacterium identified that is capable of catalysing complete ammonia oxidization (referred to as comammox). Holger Daims and colleagues now report a pure culture of this organism, which enabled a characterization of its physiology. The authors find that N. inopinata has a high affinity for ammonia, a low maximum rate of ammonia oxidation, a high growth yield compared to canonical nitrifiers, and the genomic potential for alternative metabolisms. The team compare the nitrification kinetics of N. inopinata to that of four ammonia-oxidizing archaea. The results suggest that N. inopinata is likely to have an important role in nitrification, especially in oligotrophic environments. 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 Nitrospira1,2, 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, 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 enable more encompassing future studies of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems.

Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with 14C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70–86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with 14C-bicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up >50% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that—like for AOB—ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

To date, very little is known about the bacterial core community of marine sediments. Here we study the environmental distribution, abundance and ecogenomics of the gammaproteobacterial Woeseiaceae/JTB255 marine benthic group. A meta-analysis of published work shows that the Woeseiaceae/JTB255 are ubiquitous and consistently rank among the most abundant 16S rRNA gene sequences in diverse marine sediments. They account for up to 22% of bacterial amplicons and 6% of total cell counts in European and Australian coastal sediments. The analysis of a single-cell genome, metagenomic bins and the genome of the next cultured relative Woeseia oceani indicated a broad physiological range, including heterotrophy and facultative autotrophy. All tested (meta)genomes encode a truncated denitrification pathway to nitrous oxide. The broad range of energy-yielding metabolisms possibly explains the ubiquity and high abundance of Woeseiaceae/JTB255 in marine sediments, where they carry out diverse, but yet unknown ecological functions.