The do-it-all nitrifier

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Science  22 Jan 2016:
Vol. 351, Issue 6271, pp. 342-343
DOI: 10.1126/science.aad9839

With numerous amendments to the microbial nitrogen cycle over the past two decades, it seems at times that nothing is certain. Yet one aspect of the nitrogen cycle seemed clear: that the labor of nitrification—the oxidation of ammonia (NH3) to nitrite (NO2) and ultimately nitrate (NO3)—is divided between two separate groups of microorganisms. Sergei Winogradsky first showed this in the late 1800s when he isolated the organisms responsible for the two steps of nitrification, ammonia oxidizers and nitrite oxidizers. But a series of recent papers upends this 100-year-old dogma with the description of three different cultivated bacteria (1, 2) and an uncultivated bacterium (3) that can each carry out the complete oxidation of ammonia to nitrate.

We should know by now that if a reaction is thermodynamically possible, microbes will find a way. A complete nitrifier should gain more energy per mole of substrate (1), but may grow at a slower rate, than organisms carrying out the individual steps of the pathway. Ten years ago, Costa et al. (4) modeled the trade-off between growth rate (favored by short metabolic pathways) and growth yield (favored by longer pathways). They found that this trade-off should favor the existence of a complete nitrifier when microbes grow slowly in clonal colonies. Such conditions are found in the biofilms that cover many natural and engineered surfaces.

It is perhaps no surprise, then, that the complete ammonia oxidizers, or comammox bacteria, come from biofilms in an aquaculture treatment system (1), a deep subsurface pipe (2), and a bioactive filter at a drinking water treatment plant (3). In all cases, the comammox bacteria belong to the genus Nitrospira, members of which were until recently believed to rely on nitrite for growth.

In one of the studies, van Kessel et al. (1) enriched two Nitrospira bacteria using a bioreactor fed with a steady stream of low concentrations of ammonium. The resulting consortium contained the anammox (anaerobic ammonium-oxidizing) bacteria Brocadia as well as two newly discovered Nitrospira bacteria, provisionally named “Candidatus N. nitrosa” and “Ca. N. nitrificans.”

The genomes of the two Nitrospira species contain the genes necessary for ammonia oxidation, including ammonia monooxygenase (amo) and hydroxylamine oxidoreductase (hao), and for nitrite oxidation, including the molybdoprotein nitrite oxidoreductase (nxr). Careful stable-isotope and radioisotope incubations confirmed ammonia oxidation and carbon fixation. Using the newfound amo sequences to probe public databases, the researchers discovered an entire clade of putative comammox amo sequences previously identified as particulate methane monooxygenase (pmo). These findings suggest that the newly discovered bacteria were previously detected in the environment but had been mistakenly identified as methane-oxidizing bacteria.

Similarly, Daims et al. (2) enriched a thermophilic comammox bacterium, “Ca. Nitrospira inopinata,” in coculture with a proteobacterium. The authors sequenced the complete genome of Ca. N. inopinata and combined this analysis with proteomics to show that both ammonia- and nitrite-oxidizing enzymes are abundant during growth. They further explored whether ammonia-oxidizing capability was lost by extant Nitrospira species that are capable of nitrite oxidation only, or whether the newly discovered comammox Nitrospira acquired ammonia-oxidizing capability through lateral gene transfer. Nucleotide usage in the amo gene sequences is different from the rest of the genome, suggesting acquisition of the amo genes through a recent lateral gene transfer event.

The newly cultivated comammox bacteria provide support for comammox potential in uncultivated bacteria, such as the Nitrospira genome assembled by Pinto et al. (3). As with its cultivated counterparts, this genome contains genes for the transport and degradation of urea, potentially providing an additional source of ammonia for energy and biosynthesis.

These discoveries contribute to a growing appreciation that nitrifiers are more than we believed them to be (see the figure). Historically characterized as reliant on ammonia or nitrite for energy and inorganic carbon fixation, both ammonia oxidizers and nitrite oxidizers have been proven by recent research to be much more metabolically versatile. Select nitrite oxidizers, such as Nitrobacter, can use acetate or pyruvate for growth in the absence of nitrite. Ammonia-oxidizing thaumarchaea not only transport organic carbon sources (5) but may even require these sources (6). Thaumarchaea may also use cyanate (7) as a source of ammonia to fuel growth. Nitrite-oxidizing Nitrospira may degrade urea or cyanate to feed ammonia oxidizers, which in turn feed the Nitrospira with nitrite (7, 8). Perhaps the most surprising recent finding is that some Nitrospira grow not as nitrite oxidizers but as aerobic hydrogen oxidizers (9). The implication is that the carbon and nitrogen cycles are entwined in complex ways.

Metabolic diversity of nitrifiers.

Nitrification has long been held to be a two-step process divided between two groups of organisms, some representatives of which are shown. Recent studies (13) have uncovered nitrifiers that are capable of complete nitrification, as well as novel substrates for the generation of ammonium.


The approach used to uncover these complete nitrifiers highlights an elegant combination of an old strategy—enrichment of microbial consortia—with the latest bioinformatic techniques for reconstructing complete genomes from uncultivated organisms. This strategy enables researchers to mimic conditions in the environment, where both substrates and products are kept at low concentrations by other members of the consortium. It thus allows for the cultivation of novel organisms that may be missed when high concentrations of substrate are provided (10). Comammox Nitrospira, like other recently described nitrite oxidizers (11), require ammonium for biosynthesis, meaning that they would have been missed by typical strategies enriching for nitrite oxidizers using nitrite as the sole nitrogen source. Comammox bacteria and nitrite oxidizers may actually compete with ammonia oxidizers (their previously assumed partners) for ammonium, highlighting a potentially underappreciated role for competition in the nitrogen cycle (12).

How does the discovery of comammox change our perception of the global nitrogen cycle? Conventional methods of measuring nitrification rates in the environment likely capture the activity of these organisms, and nitrification is already treated as a one-step process in many global biogeochemical models. It is uncertain, however, whether existing measurement methods capture direct nitrification from the organic nitrogen pool. Nitrification is a major source of nitric oxide (NO) and the greenhouse gas nitrous oxide (N2O) to the atmosphere; it remains to be shown whether comammox organisms make either of these compounds, and if so, under what conditions. Irrespective of their quantitative importance, the discovery of comammox emphasizes that our perception of nitrification (and of many other biogeochemical processes) is based on physiological characterization of a small fraction of extant microbial diversity.

References and Notes

Acknowledgments: W. Haskell provided feedback on the figure. C. Deutsch provided early access to modeling results. A.E.S. is an associate fellow in the Integrated Microbial Biodiversity program of the Canadian Institute for Advanced Research and is partially supported by United States National Science Foundation award OCE-126000.6
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