Earthworm gut possesses unique nosZ sequences for complete denitrification

The drilosphere, which includes the earthworm body and earthworm-worked soil, is an anoxic hotspot for denitrification that produces nitrous oxide (N 2 O) as a reaction intermediate and dinitrogen (N 2 ) as the final product. The objective was to document the diversity and activity of denitrifying bacteria in the drilosphere of the earthworm Lumbricus terrestris . Drilosphere components included live earthworms, gut homogenate, fresh and aged casts, middens and burrow lining, with bulk soil from the same field as a control. Denitrifier community in drilosphere components and bulk soil was evaluated by extracting genomic DNA and targeting the nosZ amplicon sequences with 454 pyrosequencing, and basal denitrification rates were determined with the acetylene block assay. Pyrosequencing detected 39 representative OTUs associated with Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and uncultured bacteria. Of these, 20 OTUs were unique to the drilosphere, with 70 % of the nosZ sequences found only in the gut homogenate, several of which belong to an undescribed phylogenetic clade, suggesting a novel group of endemic denitrifiers in the gut of L. terrestris . Basal denitrification rates ranged from 2.67 to 0.07 μg (N 2 O + N 2 )-N g soil -1 h −1 and ranked as: live earthworms ≥ fresh casts ≥ burrow lining ≥ middens = aged casts ≥ bulk soil ≥ gut homogenate (p < 0.05, Fisher’s LSD test). Complete denitrification in L. terrestris and its drilosphere produced more N 2 than N 2 O. Distinctive nosZ sequences in the gut of L. terrestris point to a clade with different N 2 O reduction potential than the denitrifiers in the L. terrestris drilosphere and bulk soil.