Shell biofilm-associated nitrous oxide production in marine molluscs: Processes, precursors and relative importance

Emission of the greenhouse gas nitrous oxide (N₂O) from freshwater and terrestrial invertebrates has exclusively been ascribed to N₂O production by ingested denitrifying bacteria in the anoxic gut of the animals. Our study of marine molluscs now shows that also microbial biofilms on shell surfaces are important sites of N₂O production. The shell biofilms of Mytilus edulis, Littorina littorea and Hinia reticulata contributed 18-94% to the total animal-associated N₂O emission. Nitrification and denitrification were equally important sources of N₂O in shell biofilms as revealed by ¹⁵N-stable isotope experiments with dissected shells. Microsensor measurements confirmed that both nitrification and denitrification can occur in shell biofilms due to a heterogeneous oxygen distribution. Accordingly, ammonium, nitrite and nitrate were important drivers of N₂O production in the shell biofilm of the three mollusc species. Ammonium excretion by the animals was found to be sufficient to sustain N₂O production in the shell biofilm. Apparently, the animals provide a nutrient-enriched microenvironment that stimulates growth and N₂O production of the shell biofilm. This animal-induced stimulation was demonstrated in a long-term microcosm experiment with the snail H.reticulata, where shell biofilms exhibited the highest N₂O emission rates when the animal was still living inside the shell.