Anoxic seagrass leaf environments as potential hotspots for toxin production and N2O emission

Epiphytes on seagrass leaves can render parts of the leaf phyllosphere anoxic in darkness owing to leaf/epiphyte respiration and O₂ diffusion constraints. In such anoxic microenvironments, anaerobic microbes can potentially produce phytotoxins and greenhouse gases, but the actual occurrence of such processes in seagrass epiphytic biofilms remain uncertain. We used microsensors to measure O₂, NO, N₂O and H₂S concentration gradients, as well as NO and O₂ dynamics within epiphytic biofilms on seagrass (Zostera marina) leaves under changing environmental conditions. The bacterial community composition of epiphytic biofilms was analyzed with 16S rRNA gene amplicon sequencing. Flavobacteriaceae and Rhodobacteraceae were dominant bacterial community members accounting for ˃50 % of the relative abundance, and sulfate-reducing bacteria (Desulfobacterota) were omnipresent in the epiphytic biofilms. We found pronounced production of NO, N₂O and H₂S in anoxic parts of the seagrass phyllosphere, with NO and H₂S reaching maximal concentrations of 1.0 and 4.4 μmol L^-1, respectively, under slow flow and hypoxic seawater conditions, while the highest N₂O concentration in the epiphytic biofilms reached 5.9 μmol L^-1 in hypoxic, nitrate-rich seawater. Part of the phytotoxic NO and H₂S diffused into the seagrass leaves, while no NO escaped the biofilm. In contrast, N₂O emission from the biofilm in hypoxic and eutrophic seawater reached 9.6 μmol N₂O m^-2 day^-1. Such release of the potent greenhouse gas N₂O from seagrass leaves with epiphytic biofilms under eutrophic conditions could potentially offset the carbon burial capacity of seagrass meadows. Ocean eutrophication can thus stimulate denitrification and sulfate reduction within anoxic leaf microenvironments, negatively impacting seagrass fitness and ecological function.