Physiology and Environmental Importance of Comammox

Nitrification, the microbially driven two-step oxidation of ammonia via nitrite to nitrate, is a key process of the biogeochemical nitrogen cycle. Its ecological importance is immense. Nitrification forms most of the nitrate in the worlds oceans, impairs the efficiency of nitrogen fertilization in agriculture, is a crucial step of biological wastewater treatment to prevent nitrogen pollution of aquatic ecosystems, and produces the greenhouse gas nitrous oxide as a by-product. According to textbooks nitrification is catalyzed by two guilds of microorganisms, which separately oxidize ammonia or nitrite. Huge gaps in our knowledge of nitrification were revealed by the recent discovery of complete ammonia oxidizers (comammox) in the bacterial genus Nitrospira, which alone catalyze both steps of nitrification. This finding has radically changed our picture of nitrification, but comammox Nitrospira are difficult to cultivate in the lab and remain largely unexplored. This project aims to gain a fundamental understanding of the ecophysiology, diversity, and environmental distribution of comammox Nitrospira. Yet uncultured comammox bacteria will be double-labeled with isotopes based on their ability to carry out complete nitrification. The labeled cells will be sorted by an innovative method based on Raman microspectroscopy and propagated by robot-assisted mass cultivation. The new cultures will be used to measure for the first time the nitrification kinetics of comammox Nitrospira from different habitats by using an elegant microrespirometry method. These experiments will inform on the ecological niches of comammox and test the hypothesis that comammox Nitrospira thrive best in oligotrophic habitats and gain flexibility by adapting their kinetic properties to environmental shifts. Genome analyses of the new comammox cultures will show if these bacteria possess genes for energy metabolisms other than nitrification. Genome-based hypotheses on such metabolisms will be tested with incubation experiments and single-cell isotope labeling and chemical imaging tools. The results will illuminate the metabolic repertoire of comammox Nitrospira and reveal whether these bacteria play multiple ecological roles. Little is known about the habitats and importance of comammox for nitrification. A large- scale census, which uses a unique gene of comammox as molecular marker, will inform on the distribution of uncultured comammox Nitrospira in terrestrial, aquatic, and engineered ecosystems. The relevance of comammox for nitrification will be assessed in isotope labeling experiments with environmental samples from different habitats. This project promises ground-breaking insights into the biology of the novel comammox bacteria. The results will be highly relevant to scientists and practitioners in microbiology, biogeochemistry, agriculture, and wastewater and drinking water treatment.

Nitrification is a key process of the biogeochemical nitrogen cycle in natural and engineered ecosystems. Traditionally, it was assumed to be catalyzed by two distinct functional groups of microorganisms, the ammonia oxidizers and the nitrite oxidizers. Recently, however, we and others discovered novel bacteria that carry out nitrification on their own and were thus named 'complete ammonia oxidizers' (comammox). The goal of this project was to gain more insights into the ecophysiology of the novel comammox organisms and their distribution and biological roles and adaptations in different habitats. Nitrous oxide (N2O) is the third most abundant greenhouse gas in the atmosphere and the primary substance responsible for ozone depletion nowadays. Its concentration in the atmosphere has consistently increased due to human activities, especially the agricultural sector. As the demand for nitrogen fertilizers rises to sustain the growing human population, N2O emissions will likely further escalate. Canonical ammonia-oxidizing microorganisms living in fertilized soils are known to be a major source of N2O, but nothing was known about comammox in this context. In this project, we discovered that comammox bacteria release significantly less N2O than canonical ammonia-oxidizing bacteria, and that these small amounts of N2O are not formed by enzymatic processes but result from the abiotic conversion of hydroxylamine (a nitrification intermediate) that leaks out of comammox cells. Thus, future measures to increase the relative abundance of comammox over canonical nitrifiers in agricultural soils (e.g., by adapted fertilization procedures) could become effective strategies to reduce climate change and preserve the ozone layer. Another surprising result was that comammox bacteria can use the nitrogen compound guanidine as their sole substrate for growth. Guanidine is used in various industrial processes and occurs naturally (e.g., in human urine), but no organism had been known to use guanidine as an energy source. We showed that comammox bacteria possess a guanidinase enzyme, which is essential for guanidine degradation, and that they use an efficient biochemical pathway not found in other nitrifying microbes to gain energy from guanidine. Growth of comammox on guanidine is a unique ecological niche of these bacteria. It may open new possibilities to support comammox bacteria in various settings to reduce N2O emissions and enable the biodegradation of important micropollutants. We also discovered an unexpected metabolic versatility of comammox organisms occurring in wastewater treatment plants. Aside from nitrification, these comammox can gain energy from oxidizing hydrogen and sulfite, and some species seem to survive anoxic conditions by fermentation (a kind of metabolism not found in other nitrifiers). Intriguingly, we also found that comammox are key nitrifiers in extreme habitats like hot springs (up to 80C) and saline alkaline lakes (Austrian national park Neusiedler See-Seewinkel).