Nitrate- and metal dependent methane oxidation by Archaea: a new greenhouse gas sink in freshwater ecosystems

Methane (CH4) is a powerful greenhouse gas and an abundant energy source for men and microorganisms. It is well established that aerobic bacteria and sulfate-reducing archaeal-bacteria consortia oxidize methane to the 25x less climate-forcing CO2, whereas anaerobic oxidation of methane (AOM) in freshwater ecosystems constitutes an underexplored sink. Recently, I have characterized a bacterium (Candidatus "Methylomirabilis oxyfera"), which oxidizes methane at the expense of nitrite (NO2-). Nitrate (NO3-), however, and not nitrite, is the most abundant nitrogen compound and pollutant in the environment. It is not clear how and to which extent nitrate can serve as an electron acceptor for methane oxidation. In this proposal I describe how I will use nitrate-reducing methanotrophic enrichment cultures to explore the biochemical mechanism and environmental relevance of this biogeochemically new and important process. In contrast to the purely bacterial nitrite-fed cultures, the nitrate-dependent cultures enriched by me consist of approximately equal amounts of M. oxyfera and archaea closely related to methanogens (AOM-associated archaea, AAA). A preliminary metagenomic analysis of the culture suggests that the AAA are metabolically very versatile, potentially capable of (reverse) methanogenesis and electron transfer to nitrate and possibly metals. The hypotheses I deduced from the metagenome will be tested by a combination of physiological experiments using stable-isotope labelled substrates, possible intermediates and inhibitors, mRNA-sequencing and expression studies. Then I will use the knowledge gained in this first phase of the project for the two following research lines: I will develop molecular markers for and survey representative field sites to gain insight into the environmental relevance of archaeal, nitrate-dependent methane oxidation. Additionally, I will explore the predicted ability of AAA to couple methane oxidation to iron and manganese reduction, a scarcely described metabolism for which the responsible organisms have not been identified.