Copper availability, methanobactin production and methane oxidation in Swiss lakes

Methane (CH4) is a potent greenhouse gas with a much higher global warming potential than CO2. A vast amount of methane is produced and stored in natural wetlands and lakes. The multiple factors that can control aerobic methane oxidation in these environments are still not fully understood. We propose an international, interdisciplinary research program between the Universities of Basel and Vienna, in which hyphenated HPLC-MS, trace-metal geochemical, isotope, biomarker and molecular microbiological techniques, applied to experimental and field samples, are combined to allow for an in-depth investigation of the role of copper (Cu) as a functional constituent of a key enzyme in bacterial methane oxidation. Of particular interest are the mechanisms of, and controls on, bacterial Cu acquisition through the release of methanobactins (MB), Cu specific compounds produced by methanotrophic bacteria to increase Cu availability and uptake. The existence of such a high affinity Cu uptake system implies that low Cu availability influences methanotrohic diversity and activity in natural environments. We propose to assess for the first time the distribution and temporal dynamics of methanobactin in two Swiss lakes, expecting new insights into the environmental controls on chalcophore production by methanotrophic bacteria and, in turn, methane oxidation under micro-aerobic conditions in lacustrine redox-transition zones. Anticipating the important role of reduced sulfur compounds in modulating Cu speciation in aquatic environments, our main goals will be to (1) address the role of sulfide as an important constraint on Cu-availability in freshwater, (2) to investigate the potential of MB exudation to increase the solubility and bioavailability of Cu-sulfides, and in turn (3) to assess whether MB production can enhance methane oxidation rates under Cu-limiting conditions in lakes. Within the frame of two PhD projects we propose the following research questions: Does Cu-availability impact and possibly limit aerobic methane oxidation in lakes? Can methanotrophic bacteria actively overcome Cu limitation through the production of methanobactin? Can we observe active methanobactin production in lakes and are there links between Cu, methanobactin concentrations, and methane oxidation rates? How does Cu/sulfide interaction influence Cu speciation in a fresh water environment, and does the kinetic stability of soluble Cu-sulfide complexes at low oxygen levels decrease the bioavailability of Cu for methanotrophs? Can Cu limitation trigger shifts of the lacustrine methanotrophic community composition? We will address these questions in a series of laboratory experiments and field measurements in the redox-transition zones of two lakes in Switzerland (Lugano and Cadagno lakes). We will search for links between Cu availability and speciation, sulfide concentrations, methanobactin production, suboxic methane oxidation rates and microbial population structure, and we will elucidate the geochemical mechanisms of bacterial copper acquisition from sulfides. Results from the proposed study will be directly applicable to other freshwater and marine environments, and can provide a basis for extrapolation. The efficiency of Cu acquisition by methanotrophic bacteria may have profound effects on the cycling of carbon and, possibly, the global climate. Furthermore, this study may be one of the starting points for research that addresses whether biochemical strategies developed by aerobic methane oxidizers to overcome Cu limitation may have been the evolutionary response to the competition for methane between anaerobic and aerobic methane oxidation.

Project Summary Methane is an important greenhouse gas and its production and removal by microbial processes is therefore of key importance in the estimation of carbon cycling and climate change. One of the important processes in this context is methane oxidation by methanotrophic bacteria. The rate of methanotrophic methane oxidation does not only depend on the presence of substrates but also on the availability of nutrients. A key nutrient for methanotrophic activity is copper, since it is a cofactor of the metallo-enzyme 'particulate monomethaneoxygenase' (pmmo) which catalyses the oxidation of methane by oxygen as terminal electron acceptor. The presence of a high affinity copper acquisition system involving the exudation of the copper specific metallophore methanobactin (mb) by the microaerophilic methanotroph Methylosinus trichosporium OB3b and other methanotrophs bears witness to the importance of copper as an essential nutrient for methanotrophs and to the fact that copper availability in key natural environments may be limiting methanotrophic activity. Aerobic methanotrophs reside close to environmental oxic-anoxic interfaces where the formation of Cu-sulphide phases, like the Cu(I)-sulphide covellite, can limit the bioavailability of Cu due to their high stability and low solubility. The reactivity of chalkophores towards Cu-sulphide mineral phases has not yet been investigated. The project has been addressing the question if methanotrophic bacteria can actively overcome Cu limitation through the production of methanobactin. We particularly focussed on a specific geochemical aspect in methanotrophic copper acquisition, namely, how Cu/sulfide interactions influence Cu speciation in a fresh water environment, and if the kinetic stability of soluble Cu-sulfide complexes at low oxygen levels decrease the bioavailability of Cu for methanotrophs. We could clearly demonstrate that the bacterial Cu acquisition mechanism involving methanobactin is highly efficient. However, the efficacy of mb was pH dependent and differed between sulfide mineral phases. Cu-mobilisation by mb was most significant in the presence of thermodynamically stable Cu-sulfide nanoparticles that may be suspended in lake water columns or the sediment. While Cu-sulfide oxidation can also increase the bioavailability of Cu, we demonstrated that mb enhanced Cu-mobilisation from Cu-sulfides in the presence of O2, indicating that mb is a necessary constituent to increase dissolved Cu concentrations in methanotrophic habitats where the oxidation of Cu-sulfides is expected to be slow. Consistently with these findings, we also showed that mb can mobilize Cu from sulfidic sediments. The results of this project identified the role of Cu-acquisition as a controlling factor in the destruction of methane, an important greenhouse gas. We could show that the mechanism is efficient and that we therefore expect a small impact of Cu-sulfide formation in sediments or the acquatic water column on methanotrophic activities.