Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

Abstract

Abstract In the C2H2-C2H4 assay for measurement of heterotrophic N2 fixation in water-logged soils, the diffusion of C2H2 into the soil and the recovery of C2H4 from it are critical factors regulating the assay result. To establish an C2H2-C2H4 assay technique suitable for waterlogged soils, the C2H2-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC2H2 of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared. A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC2H2 of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min. The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N2 fixation in waterlogged soils. A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C2H2-C2H4 assay.