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

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