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Quantifying the Contribution of Methylotrophy to Methane Production from Freshwater Wetlands

EMSL Project ID


Developing a holistic understanding of carbon cycling in wetlands - which is needed to improve the predictive abilities of process based biogeochemical models for estimating terrestrial net methane fluxes - requires mechanistic understanding of soil microbial physiology. Microorganisms have been historically oversimplified in such models, and historical paradigms about the relevance of select methane pathways have resulted in overlooking methane generation pathways not believed to be important to methane fluxes. Particularly, there is growing recognition of the importance of methanogenesis from methylated compounds (direct methylotrophic methanogenesis) in wetlands, however historical assumptions – and thus models - represent methanogenesis from acetate (acetoclastic) and hydrogen/carbon dioxide (hydrogenotrophic) over the former. In addition to direct methylotrophic methanogenesis, non-methanogenic bacteria are now well known to catabolize methylated compounds and produce substrates for methanogens (e.g., acetate), thus suggesting an indirect pathway of methanogenesis via an under-appreciated methylotrophic food web. Yet, mechanistic studies quantifying the fluxes and community metabolism from both these direct and indirect routes of methane formation from methylated compounds are lacking. Thus, the overarching objective of this proposed EMSL-enabled research is to resolve both the relevance of methylotrophic metabolisms and the complexity of the microbial methylotrophic food web in wetland soils. Here, we propose to operate laboratory reactors using soils from a high methane emitting freshwater wetland (Old Woman Creek Reserve, OH) and fed with 13C-istopically labeled substrates (13C-Acetate, 13C-Dimethyl Sulfide, 13C-Trimethyllysine). Enabled by EMSL knowledge and facilities, we propose to temporally track the flow of labeled carbon into: (1) methane and carbon dioxide produced by microcosms via GC-MS (2) metabolite pools via NMR and (3) cellular biomass via metaproteomics. Our laboratory will provide paired geochemistry, metagenomics, and 16S rRNA analyses. Specifically, isotopically-resolved GC-MS data will allow for quantification of the methane formed from labeled methylotrophic compounds – via the sum of the direct and indirect routes – providing the first absolute measurement of methylotrophy to wetland methane production. Further, the NMR and metaproteomic data will shed critical insight onto the specific physiology of the methylotrophic food web, informing both relevant metabolisms (e.g., what pathways are active in the indirect route) and key members of the methylotrophic community utilizing the labeled carbon. We postulate that methylotrophy will produce as much methane as acetate but yield a more complex methane cycling networked community. Our findings will illuminate biochemical knowledge into soil methylotrophy, but also lay a foundation for translation to models by making a clear case for the importance of this metabolism to methane production. We will use these data with biogeochemical modelers, to build representation of this pathway in wetland process-based model that currently includes acetoclastic and hydrogenotrophic pathways. Ultimately, these quantitative and physiological data represent a critical need to better inform and further their predictive abilities with regards to understand terrestrial freshwater methane fluxes.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

Jared Ellenbogen
Colorado State University


Kelly Wrighton
Colorado State University