N 2 O-Producing Microorganisms in the Gut of the Earthworm Aporrectodea caliginosa Are Indicative of Ingested Soil Bacteria

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Abstract
The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N 2 O) and (ii) to characterize the N 2 O-producing bacteria of the earthworm gut. The production of N 2 O in the gut of garden soil earthworms ( Aporrectodea caliginosa ) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N 2 O were transient products when supplemental nitrate was reduced to N 2 by gut content homogenates. In contrast, nitrite and N 2 O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N 2 O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N 2 O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N 2 O or N 2 . The initial N 2 O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N 2 O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N 2 O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.