Methane is a major greenhouse gas, accounting for up to 20-30% of global warming effect . Aerobic methanotrophic bacteria constitute the main microbial methane sink through their ability to derive energy from its oxidation to carbon dioxide using the key enzyme methane monooxygenase . Cultured representatives of this functional guild are phylogenetically positioned within the Gammaproteobacteria (Type I methanotrophs, forming two clades, i.e. Type Ia and Ib), the Alphaproteobacteria (Type II methanotrophs) [3, 4], and the Verrucomicrobia. These methanotrophs appear physiologically diverse, especially in regard to their nitrogen metabolism , with strains demonstrating co-metabolic oxidization of ammonia , nitrate reduction to nitrite , detoxification of hydroxylamine and nitrite to the greenhouse gas nitrous oxide via a multitude of pathways [8–10], and fixation of atmospheric nitrogen [11–14].
The effect of nitrogen, under the form of either ammonium- or nitrate-based fertilizers, on methane fluxes in soils is a widely studied topic of global concern. Unfortunately, available reports are not in agreement, with methanotrophic activity after nitrogen addition either showing no effect, inhibition (by non-specific ionic effects , competitive inhibition between methane and ammonia or the formation of toxic intermediates [10, 16]) or stimulation (by relief of nitrogen limitation), resulting in identical or altered associated community composition [17–20]. The currently accepted thesis assumes niche partitioning among methanotrophic species, with methane oxidation activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure [19, 21].
Unfortunately, widely applied tools for microbial community assessment, based on short read 16S rRNA gene sequencing techniques or proxies thereof (such as denaturating gradient gel electrophoresis (DGGE) or terminal-restriction fragment length polymorphism (T-RFLP)), only have a limited phylogenetic resolution mostly restricted to genus level diversity , and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. Nevertheless, we know from other organisms that ecophysiology can be strain-specific, and closely related bacteria can occupy distinct niches [23, 24]. This was for example elaborately demonstrated in Prochlorococcus, with cultured strains having distinct pigmentation, maximum growth rates, metal tolerances, nutrient utilizations and photophysiological characteristics . Also, among methanotrophic genera of the same type (I or II), ammonia co-metabolisation and product inhibition was found to be organism-specific, although in this study it is difficult to ascertain the taxonomic level at which differences occur as only two strains from different genera were tested for each type .
We hypothesized that strains within the same methanotrophic genus, thus below the level at which most techniques assign operational taxonomic units (OTU), can demonstrate a large physiological versatility in their nitrogen metabolism. If so, this would make current microbial community structure analyses less suitable for evaluating changes in in situ methanotrophic communities after for example fertilization. To this end, we explored the differential response of various aspects of the nitrogen metabolism within fourteen genotypically distinct members of the genus Methylomonas (Type Ia), including the species M. methanica and M. koyamae. Their tolerance to high ammonium, nitrate, nitrite and hydroxylamine concentrations, their ability to fix nitrogen, and their capacity to produce nitrite and nitrous oxide from nitrate or ammonium were qualitatively evaluated. Methylomonas is a relevant genus for such a comparison, since Methylomonas strains are ubiquitous in nature [4, 7, 25–27] and nitrogenous fertilization was reported to stimulate some of its members in rice field soils . Two non-Methylomonas strains belonging to Type Ib and Type II were included as references.