Anaerobic oxidation of methane in lake environments: rates, pathways, environmental controls and microorganisms

Freshwater lakes represent an important source of methane to the atmosphere, and large amounts of methane are produced in anoxic sediments by anaerobic methanogens. The produced methane dissolves and accumulates in the anoxic sediment pore water, from where it may escape, by ebullition or diffusion, into the near-bottom waters of the lake. Microbial methane oxidation, including aerobic methane oxidation, catalyzed by methane-oxidizing bacteria in oxic environments, and anaerobic oxidation of methane (AOM) by anaerobic methanotrophs under anoxic condition, is the only biological process mitigating methane emissions from natural systems. So far, most studies in freshwater lakes focussed on aerobic methane oxidation at the sediment surface or in oxic water layers. Methane oxidation within anoxic sediments, or within waters of, e.g. eutrophic or permanently stratified freshwater bodies, remains largely unexplored. Recently, however, AOM has been shown to also occur in freshwater environments, raising questions regarding its potential to reduce methane emission from sediments. In addition to sulfate (typically inveolved in AOM in marine settings), oxidants such as nitrite, nitrate, iron and manganese oxides may serve as terminal electron acceptors in AOM. However, the knowledge about the exact pathways and the microorganisms mediating AOM in anoxic freshwater habitats, as well as the environmental controls are still rudimentary. Understanding these controls is crucial for predicting future changes in lacustrine methane emissions under changed climatic/environmental conditions.
In this PhD project, I evaluated different methods to quantify AOM rates in lake sediments and scrutinized different freshwater systems for the presence of AOM. I then selected two representative lake environments, where I could verify the presence of AOM, for greater-detail investigations into the exact modes of, and controls on, AOM: I studied AOM in the anoxic waters in Lake Lugano and within sediments of Lake Cadagno, both lakes in southern Switzerland.
In Lake Lugano, I investigated methane oxidation in the anoxic waters of the two hydrologically connected basins of this lake, which display very different mixing regimes. In both basins, I measured maximum methane oxidation rates below the redoxcline. In the seasonally stratified South Basin, putatively aerobic methane oxidizing (MOx) bacteria belonging to the Methylococcaceae family (Type I MOB) dominated the methanotrophic community, and were mostly responsible for the methane oxidation in the water column. In the permanently stratified North Basin, methane consumption at the oxic-anoxic interfaces can be attributed to both Methylococcaceae and Candidatus Methylomirabilis, i.e., bacteria that were previously reported to perform nitrite-depedent AOM (i.e. AOM coupled to nitrite reduction/denitrification). A secondary methane turnover maximum was observed well within the anoxic water column, where both Methylococcaceae and Candidatus Methylomirabilis were present and ammonium oxidation was apparently indicated. Hence, both (micro-) aerobic methane oxidation and nitrite-dependent, true, AOM appear to be important methane-consuming processes in the water column of the North Basin. Most intriguingly, I could demonstrate that water column stability is the prime environmental factor that controls the growth and abundance of the denitrifying methanotrophs. The stably anoxic conditions in the North Basin are particularly conducive to the proliferation of denitrifying AOM bacteria, whereas the seasonal mixing and shorter-term fluctuations in the redox regime in the South Basin seem to prevent the thriving of nitrite dependent AOM bacteria.
In anoxic lake sediments, maximum AOM rates of ~15 nmol/cm3/d were observed in the deep sediments of sulfate-rich Lake Cadagno, as well as in the surface sediments of Lake Sempach and Lake Lugano. For Lake Cadagno, I present a conclusive data set (radiolabel-based AOM rate measurements, stable isotope probing of lipid biomarkers, 16S rRNA gene-sequencing) which highlights that AOM is coupled to sulfate reduction, and carried out by uncultured archaea of the candidate genus Methanoperedens. Depth distributions of Candidatus Methanoperedens and potential sulfate reducing ANME partners in the AOM zones suggests that methane oxidation is most likely performed in archaea-bacteria association. Furthermore, I could demonstrated that this process is indirectly supported by the continuous sulfate production via oxidation of reduced sulfur compounds with other oxidants (e.g., manganese oxide). In this way, sulfate-dependent AOM (involving a cryptic sulfur cycle) may be “disguised” as AOM coupled to manganese reduction. Our study suggest that methanotrophic archaea in syntrophy with sulfate-reducing bacterial partners play an important role in mitigating methane emissions from terrestrial freshwater environments to the atmosphere, analogous to ANME-1, -2, and -3 in marine settings.
In both Lake Sempach and Lake Lugano, in situ AOM rate measurements combined with the molecular data and slurry incubation experiments suggest that putative anaerobic methane oxidation is likely performed by the Genus Methylobacter. I hypothesized that these tentative “aerobic” methanotrophs may switch to an anaerobic respiration mode, and are able to utilize electron acceptors other than O2, such as humic substances in the surface lake sediments.
The presented research significantly expands the range of freshwater habitats where AOM activity could be verified, highlighting its ecological importance of anaerobic methane oxidizing microorganisms (bacteria and archaea, benthic and pelagic) as sentinels of methane emission in freshwater environments. I speculate that both sulfate and nitrite-dependent AOM may act as an important biological methane filter both in the water column of permanently stratified lakes worldwide, as well as in anoxic lacustrine sediments.