Incomplete Denitrifying Bacteria Drive N2O Fluxes in Ancient Siberian Permafrost Microcosms

Nitrous oxide (N2O) contributes to stratospheric ozone depletion and global warming. Knowledge about microbial formation and consumption of N2O in old permafrost remains limited. Permafrost samples collected on the East Siberian Sea coast of Russia from a single borehole at depths of 5.4 and 16.9 m, which showed presence of nitrogen substances and nitrogen cycling genes, were used to establish microcosms supplemented with NO3-- and N2O to assess denitrification and N2O consumption at 4 and 20°C. Rapid N2O formation was observed in NO3--supplemented microcosms, but N2O consumption was slow and incomplete over a 1-year incubation in all microcosms. Twenty-three qualified metagenome-assembled genomes (MAGs) harboring genes involved in NO3- and/or N2O reduction were recovered from both NO3-- and N2O-supplemented microcosms. Twenty MAGs represent novel taxa. Four MAGs, two of each from NO3-- and N2O-supplemented microcosms, contained nosZ genes indicating N2O consumption potential, however the complete denitrification (i.e., NO3-→N2) gene sets were not detected in these MAGs. Though, N2O production exceeded N2O consumption in NO3--supplemented microcosms at 4°C. Our microcosm experiments suggest N2O formation surpasses its consumption in newly-thawed ~120 kyr old permafrost, emphasizing the importance of using integrated approaches to assess and predict N turnover in response to permafrost degradation.