Methylphosphonate metabolism by Pseudomonas sp populations contributes to the methane oversaturation paradox in an oxic freshwater lake

The 'CH4 oversaturation paradox' has been observed in oxygen-rich marine and lake waters, and viewed to significantly contribute to biosphere cycling of methane, a potent greenhouse gas. Our study focused on the intriguing well-defined pelagic methane enriched zone (PMEZ) in freshwater lakes. Spiking Yellowstone Lake PMEZ samples with 13 C-labeled potential methanogenesis substrates found only 13 C-methylphosphonate (MPn) resulted in (CH4)-C-13 generation. In 16S rRNA gene Illumina libraries, four Pseudomonas sp. operational taxonomic units surprisingly accounted for similar to 11% abundance in the PMEZ community. Pseudomonas sp. isolates were also obtained from MPn enrichments with PMEZ water; they were most aggressive in MPn metabolism and their 16S rRNA gene sequences matched 35% of the Illumina PMEZ Pseudomonas reads. Further, two key genes encoding C-P lyase (phnJL, an important enzyme for dealkylation of MPn), were only amplifiable from PMEZ DNA and all PCR generated phnJL clones matched those of the Pseudomonas sp. isolates. Notably, methanogen 16S rRNA signatures were absent in all Illumina libraries and mcrA was not detected via PCR. Collectively, these observations are consistent with the conclusion that MPn metabolism contributes significantly to CH4 oversaturation in Yellowstone Lake and likely other oxic freshwater lake environments, and that Pseudomonas sp. populations are critical participants.