N₂O Microsensor

A method to create an O2 insensitive and fast responding microsensor for N2O is presented. The elimination of O2 interference was obtained by attaching a capillary with an alkaline solution of ascorbate in front of the tip of an electrochemical microsensor that was sensitive to both O2 and N2O. A 0.1 mm layer of 2 M ascorbate at pH 12 was able to remove all O2 when measuring in air-saturated solutions (O2 partial pressure 21 kPa), and no reduction of N2O by the ascorbate was observed. The response of the sensor to N2O was linear from 0 to 1.2 mM, and the detection limit was less than 1 µM. The tip diameters of the sensors described in detail were 50-80 µm, but much smaller sensors can be constructed. The 90% response times were about 40-50 s. Interference of carbon dioxide was shown to be negligible with respect to environmental monitoring, and the sensitivity to acetylene, which is commonly used to inhibit nitrous oxide reductase in denitrification studies, could be kept sufficiently low for experimental work.

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.