Evidence for Involvement of Gut-Associated Denitrifying Bacteria in Emission of Nitrous Oxide (N₂O) by Earthworms Obtained from Garden and Forest Soils

<jats:title>ABSTRACT</jats:title><jats:p>Earthworms (<jats:italic>Aporrectodea caliginosa</jats:italic>,<jats:italic>Lumbricus rubellus</jats:italic>, and<jats:italic>Octolasion lacteum</jats:italic>) obtained from nitrous oxide (N<jats:sub>2</jats:sub>O)-emitting garden soils emitted 0.14 to 0.87 nmol of N<jats:sub>2</jats:sub>O h<jats:sup>−1</jats:sup>g (fresh weight)<jats:sup>−1</jats:sup>under in vivo conditions.<jats:italic>L. rubellus</jats:italic>obtained from N<jats:sub>2</jats:sub>O-emitting forest soil also emitted N<jats:sub>2</jats:sub>O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878–1882, 1997). In contrast, commercially obtained<jats:italic>Lumbricus terrestris</jats:italic>did not emit N<jats:sub>2</jats:sub>O; however, such worms emitted N<jats:sub>2</jats:sub>O when they were fed (i.e., preincubated in) garden soils.<jats:italic>A. caliginosa</jats:italic>,<jats:italic>L. rubellus</jats:italic>, and<jats:italic>O. lacteum</jats:italic>substantially increased the rates of N<jats:sub>2</jats:sub>O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N<jats:sub>2</jats:sub>O emission by earthworms accounted for approximately 33% of the N<jats:sub>2</jats:sub>O emitted by garden soils. In vivo emission of N<jats:sub>2</jats:sub>O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N<jats:sub>2</jats:sub>O. In the presence of nitrate, acetylene increased the N<jats:sub>2</jats:sub>O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N<jats:sub>2</jats:sub>O. In vivo emission of N<jats:sub>2</jats:sub>O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N<jats:sub>2</jats:sub>O emission of dissected earthworm gut sections were substantially higher than the rates of N<jats:sub>2</jats:sub>O emission of dissected worms lacking gut sections, indicating that N<jats:sub>2</jats:sub>O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N<jats:sub>2</jats:sub>O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N<jats:sub>2</jats:sub>O-emitting earthworms produced N<jats:sub>2</jats:sub>O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N<jats:sub>2</jats:sub>O by earthworms and contribute to the N<jats:sub>2</jats:sub>O that is emitted from certain terrestrial ecosystems.</jats:p>