Acetylene - ethylene assay for N/sub 2/ fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C/sub 2/H/sub 2/-C/sub 2/H/sub 4/ assay for N/sub 2/ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N/sub 2/ase-catalyzed reduction of C/sub 2/H/sub 2/ to C/sub 2/H/sub 4/, gas chromatographic isolation of C/sub 2/H/sub 2/ and C/sub 2/H/sub 4/, and quantitative measurement with a H/sub 2/-flame analyzer. As little as 1 ..mu mu..mole C/sub 2/H/sub 4/ can be detected, providing a sensitivity 10/sup 3/-fold greater than is possible with /sup 15/N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C/sub 2/H/sub 2/-reducing activity in the field. Applications to field samples included an evaluation of N/sub 2/ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicted a calculated seasonal N/sub 2/ fixation rate of 30 to 33 kg N/sub 2/ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements ofmore » N/sub 2/ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N/sub 2/ fixing activity of soybeans. The validity of measuring N/sub 2/ fixation in terms of C/sub 2/H/sub 2/ reduction was established through extensive comparisons of these activities using defined systems, including purified N/sup 2/ase preparations and pure cultures of N/sub 2/-fixing bacteria.« less