In situ studies on N2 fixation using the acetylene reduction technique.

The measurement of in situ N2-fixation rates on the basis of total nitrogen changes or 15N2 uptake is not entirely satisfactory-the former method is insufficiently sensitive and accurate, and the '5N method is time consuming, expensive, and requires a mass spectrometer. The discovery in 1966 by Sch6llhorn and Burris' and by Dilworth2 that the nitrogen-fixing complex (nitrogenase) reduces acetylene to ethylene2 suggested that the rate of acetylene reduction may be used as an index of the rate of N2 fixation. Subsequently, the measurement of ethylene production from acetylene'-' and the measurement of cyanide,8 isocyanide,9 and azide reduction8' 10 have been used to aid in laboratory studies of N2 fixation. However, the potential of the method for field investigations of N2 fixation generally has not been appreciated by limnologists, marine biologists, and soil scientists. In this paper, data obtained in experiments designed to test the feasibility of employing a simple method for measuring acetylene reduction as an index of N2 fixation in the field illustrate that the method is practical and extremely sensitive. Sites Studied.-Lake studies were performed in Lake Mendota, Madison, Wisconsin, either offshore or in mid-lake; algal samples also were obtained from Lakes Monona and Wingra. Soil studies were made in the Madison area, studies on nonlegumes on land surrounding Plummer Lake, Vilas County, Wisconsin, and studies on soybeans at the University of Wisconsin experimental farm, Arlington, Wisconsin. Gases.-Acetylene (purified grade) and a gas mixture of 02 (22%), CO2 (0.04%), and argon (78%, high purity) were obtained commercially (Matheson Co.). Field Method.-Experiments were carried out in 5.0-ml capacity glass serum bottles fitted with rubber serum stoppers. Lake samples (1.0 ml with or without prior concentration, depending on the experiment and the density of the algal population) were added to each bottle. Soil samples 0.78-cm in area and 1-cm deep were taken with a cork borer. Root nodules were detached immediately after the plants were dug, and 100-500 mg fresh weight of nodules was added per bottle. With lake samples, air was removed by flushing the liquid with the premixed gas phase for 1.5 min; then each bottle was stoppered and flushed for a further 1.5 min by introducing gas through a no. 22 hypodermic needle and venting it through a second needle. With soil and with root nodule samples, air was removed from the stoppered bottles through a hypodermic needle with a hand vacuum pump prior to flushing with the premixed gas phase. Evacuation and flushing were performed twice. Acetylene (0.5 ml) or 15N2 (1.0 ml) was then injected from a hypodermic syringe, and the samples were incubated in situ for the desired period. Generally, 18 samples could be prepared, gassed, and replaced in situ for incubation within 30 min of sampling the material. Reactions were terminated by the injection of 50% trichloroacetic acid (0.2 ml to lake water samples and 0.5 or 1.0 ml to root nodule and soil samples). A covering of RTV sealant (General Electric Co.) was applied to each stopper after gassing and after injection of trichloroacetic acid. Samples were returned to the laboratory and analyzed for total nitrogen, for ethylene production, or for 15N enrichment as required. Laboratory Studies.-Samples of lake algae were exposed in the laboratory using the methods described above or as detailed later. During incubation, the samples were continuously shaken at 300 in a water bath tinder a constant light intensity of 320 ft-c. Analysis.-Ethylene formation was detected by gas chromatography with a Varian-Aerograph model 600D gas chromatographic apparatus (H-flame ionization detector) fitted with a 9-ft long,