Combining field-sampling and incubation-experimental efforts, and employing stable isotopic, radio-labelling, and molecular analyses applied to samples from Lake Lugano (Southern Switzerland), we specifically proposed to investigate CH₄ production related to the exploitation and decomposition of methylated organic compounds by establishing functional links between epilimnetic CH₄ accumulation, concentrations of methanogenetic substrates and nutrients, the molecular composition of dissolved organic matter (DOM), and the planktonic community structure in this eutrophic lake. During the tenure of the original project, we have broadened the scope of the original research plan by establishing a regular sampling of plankton and CH₄ biogeochemistry in another Swiss Lake, Lake Cadagno. During the first 2.5 years of the project, we generated a comprehensive data set that allowed us to answer important questions that we proposed in the parent proposal. Based on results from both lakes, we could demonstrate clear seasonal variability in the magnitude of the lacustrine methane paradox, and establish links between the accumulation of CH_4, feeding zooplankton, and the degradation of planktonic detritus. We could experimentally show that CH₄ can be produced in oxygenated lake waters from methylated compounds both under nutrient-limited and nutrient-replete conditions. At the same time, we were able to exclude the possibility that the lacustrine “methane paradox” in the two studied lakes results from an upwelling or lateral transport of sedimentary gas. Moreover, we were able to prove that aerobic CH₄ accumulation varied with different plankton communities, confirming close links to the lacustrine productivity cycle and general limnological conditions. For example, we observed putative links between the development of picocyanobacterial blooms (Synechoccocus), the concentration of dimethylsulfide (DMS, a potential substrate in CH₄ formation) in subsurface waters, and the accumulation of CH₄, yet, the overall amount of DMS seemed insufficient to fully account for the observed methane paradox. While canonical methanogens are virtually absent from the epilimnetic microbial communities of both lakes (less than 0.05% of OTUs), methylotrophic bacteria as well as photoautotrophs are abundant, and are potentially capable of exploiting methylated nutrients (methylphosphonate - MPn, methylamines-MA). In fact, the most recent set of incubation experiments within SNSF project 3623910 supported that both methylphosponate (MPn) and methylamine (MA) are suitable substrates for CH₄ generation in oxic waters of the studied lakes. Most importantly, and in contrast to literature reports, the production of CH₄ was not inhibited by inorganic phosphorous (as PO₄³-P), indicating that P-limitation is not a pre-requisite for the exploitation of methylated P to occur in nature.

Although the data so far are highly promising and provide a solid basis for at least three publications, not all of the questions originally posed could be addressed unambiguously (due to the highly complex nature of analytical techniques required to identify and quantify natural concentrations of methylated P and N compounds, as well as molecular characterization of DOM). Specifically, uptake of MPn and/or MA in the context of lacustrine aerobic CH₄ production, the identity of the microbes/algae involved, and the specific role of picocyanobacteria still needs further investigation. Given the comprehensiveness and great variety of data already generated, the project extension will provide the time required for a more in-depth statistical analysis and exploitation of the already existing data set. In addition, we propose complementary bio-geochemical analyses of samples that have already been collected during past samplings to confirm the robustness of existing results (characterization of DOM by FTIR-ICMS, characterization and quantification of polysaccharide esters of phosphonic acids by NMR and methylated amines by solid-phase microextraction techniques), as well as incubation experiments with ¹³C labelled MPn and ¹⁵N labelled MA to assess uptake and metabolism of these compounds by isolated Synechoccocus strains using isotope-ratio mass-sepctrometry and NanoSims imagining. Prime objective of the project extension will be to establish whether, and to what extend, the exploitation of dissolved organic phosphorus (DOP) and methylated-N compounds occurs naturally under different trophic conditions, and what the potential rates of CH₄ generation are. We will also investigate further the C isotopic fingerprint (including clumped CH₄ isotope signatures) of MPn or MA-based aerobic methane production. This project extension thus promises to provide additional milestones in our efforts to understand the “methane paradox” in lakes, helping us to gain insight into the biogeochemical controls on global CH₄ emissions from aquatic environments.