Do it yourself nitrification

Credit: Getty / Photodisc

Microbial oxidation of ammonia (or ammonium) via nitrite to nitrate is a central step in the nitrogen cycle. This process, termed nitrification, was thought to involve two separate steps mediated by distinct microorganisms: the initial oxidation of ammonia to nitrite, which is catalysed by ammonia-oxidizing microorganisms (AOMs; including bacteria (AOB) or archaea (AOA)); followed by the oxidation of nitrite to nitrate, which is catalysed by nitrite-oxidizing bacteria (NOB). Two recent studies challenged this notion by describing Nitrospira spp. that can carry out both reactions and therefore are capable of complete nitrification.

The functional separation of ammonia and nitrite oxidation has puzzled microbiologists for decades, particularly as it has been predicted that microorganisms that are capable of complete nitrification (termed complete ammonia oxidizers, or comammox) may have an advantage over AOMs and NOB under substrate-limiting conditions, such as in microbial aggregates and biofilms. Therefore, van Kessel et al. and Daims et al. enriched cultures for comammox by growing biofilm samples in the presence of low concentrations of ammonium.

Nitrospira spp. (...) are capable of complete nitrification

van Kessel et al. sampled the anaerobic compartment of a trickling filter connected to a recirculation aquaculture system and enriched this community during 1 year, after which the culture was mostly composed of microorganisms belonging to the genus Brocadia (which are known anaerobic ammonium oxidizers) and Nitrospira (which are known NOB). By sequencing the total DNA from the enrichment culture, the authors were able to assemble draft genomes of two Nitrospira species. Surprisingly, both genomes contained not only the genes involved in nitrite oxidation, but also all the genes necessary for ammonia oxidation (including genes encoding the different subunits of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase). Furthermore, both species lacked some of the genes necessary for assimilatory nitrite reduction, suggesting that these species have the potential for complete nitrification and may have adapted to ammonium-containing environments; these species were named Candidatus Nitrospira nitrosa and Candidatus Nitrospira nitrificans.

Daims et al. sampled a biofilm from the walls of a pipe in a deep oil exploration well and enriched this community for comammox during 4 years, after which the culture was capable of complete nitrification and composed of a single Nitrospira species and a non-nitrifying betaproteobacterium affiliated with the family Hydrogenophilaceae. DNA sequencing and reconstruction of the complete genome of the Nitrospira species identified the presence of genes involved in nitrite oxidation and ammonia oxidation, and the absence of some of the genes necessary for assimilatory nitrite reduction; this species was named Candidatus Nitrospira inopinata.

To further investigate the ability of Ca. N. nitrosa and Ca. N. nitrificans to carry out complete nitrification, van Kessel et al. combined the use of a fluorescently labelled ammonia analogue that binds to AMO with Nitrospira-specific fluorescence in situ hybridization (FISH) probes, which revealed that these bacteria express a functional AMO. Furthermore, combining FISH and ¹⁴C-labelling, the authors demonstrated that the two Nitrospira species are capable of inorganic carbon fixation during ammonia oxidation. Similarly, Daims et al. demonstrated full nitrification by Ca. N. inopinata by growing their enriched culture in media containing ammonium as the sole energy source and CO₂ as the sole carbon source; Ca. N. inopinata growth was coupled with the oxidation of ammonium to nitrate. Furthermore, metaproteomic analysis revealed that all of the Ca. N. inopinata enzymes involved in both ammonia and nitrite oxidation were expressed during incubation with ammonium.

Finally, both studies investigated the distribution of comammox Nitrospira spp. in public databases and by carrying out metagenomic screening of environmental samples, based on the presence of amoA (which encodes the A subunit of AMO), which is a functional and phylogenetic marker for AOMs. Notably, amoA from these newly discovered Nitrospira spp. that are capable of complete nitrification are distinct from amoA present in AOB. Furthermore, several highly similar amoA sequences were found in Nitrospira genomes present in different environments, including agricultural soils, freshwater environments, wastewater treatment plants and drinking water systems, suggesting that comammox Nitrospira bacteria are widely distributed.