Emission of Denitrification-derived Nitrous Oxide and Dinitrogen by Earthworms from Brazil

The earthworm alimentary canal is an anoxic microzone in aerated soils. Small to medium sized earthworms of the family Lumbricidae emit the denitrification-derived greenhouse gas nitrous oxide (N2O) whereas the large Octochaetus multiporus (Megascolecidae) does not. To examine this paradoxical phenomenon, differently sized species of the families Glossoscolecidae (Rhinodrilus alatus, Glossoscolex paulistus, Glossoscolex sp., Pontoscolex corethrurus), Megascolecidae (Amynthas gracilis, Perionyx excavatus), Acanthodrilidae (Dichogaster annae, Dichogaster sp.), and Eudrilidae (Eudrilus eugeniae) were obtained in Brazil and analyzed. All small species and the large R. alatus emitted N2O whereas large Glossoscolex species did not. Acetylene, which blocks the reduction of N2O to N2, mostly increased the emission of N2O, indicating that denitrification was the main source of N2O. On a per worm basis, R. alatus emitted the highest amount of nitrogenous gases with N2 being predominant, indicating complete denitrification to N2 in its gut. These collective results demonstrated that the emission of denitrification derived N2O is more widespread in earthworm families than previously thought, and that large earthworms can emit nitrogenous N2O gases in vivo. Dissimilatory nitrate reducers and denitrifiers of gut content and soil of G. paulistus and A. gracilis were analyzed by barcoded amplicon pyrosequencing of the structural genes narG, nirK, and nosZ. Gene sequences in gut and soil of G. paulistus were highly similar whereas sequences in gut and soil of A. gracilis were significantly different from each other, and were likewise different when compared to those of the gut and soil of G. paulistus. However, gene analyses strongly indicated that the denitrifying gut microbiota for both earthworms was soil-derived and predominantly consisted of members of the Rhizobiales.