In Vivo Emission of Dinitrogen by Earthworms via Denitrifying Bacteria in the Gut

Earthworms emit the greenhouse gas nitrous oxide (N₂O), and ingested denitrifiers in the gut appear to be the main source of this N₂O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N₂), the end product of complete denitrification. When [¹⁵N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N₂ (and also labeled N₂O) under in vivo conditions; emission of N₂ by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N₂ per h per g (fresh weight), respectively. Isolated gut contents also produced [¹⁵N]nitrate-derived N₂ and N₂O under anoxic conditions. N₂ is formed by N₂O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [¹⁵N]nitrate-derived N₂ by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N₂O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N₂ (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N₂ per h per g (fresh weight), respectively; these emission rates approximated that of N₂O. These collective observations indicate that (i) earthworms emit N₂ concomitant with the emission of N₂O via the in situ activity of denitrifying bacteria in the gut and (ii) N₂O is quantitatively an important denitrification-derived end product under in situ conditions.