Rivers and their adjacent riparian forests are intimately linked by the exchange of water, nutrients, and organic matter. Both riparian cottonwood trees and adjacent river sediments host microbial communities including archaeal methanogens, supporting methane production and emission to the atmosphere. Here we combine microbial community and stable isotope analyses to characterize the drivers of methane cycling in distinct anoxic habitats (river sediments versus riparian cottonwood stems) in the Oldman River, southern Alberta (Canada). We demonstrate that, differences in the chemical characteristics of organic matter support divergent microbial communities that generate methane from distinct metabolic pathways. Organic matter in river sediments had C/N ratios approximately 50-fold lower than in tree stems and had more diverse dissolved organic components. Contrasting substrate availability between river sediment and tree stems was likely the primary mechanism for the observed differences in bacterial and methanogen community compositions, and greater microbial diversity in river sediments than in tree stems. The methane carbon isotope composition (δ¹³C values) differed for the tree stem (-103.6 to -70.6‰) and river sediment (-55.1 to -48.4‰) environments, suggesting that methane was primarily produced via CO₂-reduction in tree stems by Methanobacteriales, while river sediments produced more methane through acetate fermentation primarily by Methanosarcinales. This study demonstrates the importance of organic matter quality and microbial community composition in driving metabolic processes contributing to methane production and emission in rivers and adjacent riparian forests.

Tree stem, river sediment and alluvial groundwater samples were collected and used for DNA extraction and Illumina sequencing of bacterial and methanogen communities. Sequence data were processed using the DADA2 package in R to create amplicon sequence variants. Tree stem and river sediment samples were sealed in mason jars and flushed with nitrogen gas to provide an anaerobic environment where methane production could occur, and these were left to incubate in the dark at room temperature. At intervals, gas samples were removed from the headspace and measured for methane and carbon dioxide gas concentrations and stable carbon isotope composition using cavity ring-down spectroscopy. Measured values were adjusted based on repeated measures (n=2) of reference gases measured prior to, and following, sample measurements. Tree stem and river sediment samples were also dried, and used to measure the organic carbon and nitrogen and their associated isotopic compositions by Continuous Flow-Elemental Analysis-Isotope Ratio Mass Spectrometry. Tree stem and river sediment samples were also used for leachates and analysis of dissolved organic matter. Chromophoric dissolved organic matter was characterized by UV-Vis absorption, while additional dissolved organic matter properties were determined using a spectrofluorometer. All additional methodology for these analyses is present within the ReadMe file "Smits_Nov2021_riparian_methane_production_ReadMe.txt"

Values presented in the datasets are final and have undergone normalization methods to standards or controls. Any missing values are presented as "%". Samples across datasets were collected from the same trees, groundwater well and river sediment locations, and have matching sample names in the files.

Additional data collection/processing, equipment and variables involved in these analyses are discussed in more depth in the ReadMe file "Smits_Nov2021_riparian_methane_production_ReadMe.txt"