The in vivo production of nitrous oxide (N2O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N2O-producing soil bacteria. In situ measurement of N2O and O2 with microsensors demonstrated that the earthworm gut is anoxic and the site of N2O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N2O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N2O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N2O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N2O, and addition of buffer or ammonium had essentially no effect on the initial N2O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N2O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N2O-producing soil bacteria during gut passage.
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