The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(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(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(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 indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(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(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Communities of microscopic plant life, or phytoplankton, dominate the Earth's aquatic ecosystems. This important new book by Colin Reynolds covers the adaptations, physiology and population dynamics of phytoplankton communities in lakes and rivers and oceans. It provides basic information on composition, morphology and physiology of the main phyletic groups represented in marine and freshwater systems and in addition reviews recent advances in community ecology, developing an appreciation of assembly processes, co-existence and competition, disturbance and diversity. Although focussed on one group of organisms, the book develops many concepts relevant to ecology in the broadest sense, and as such will appeal to graduate students and researchers in ecology, limnology and oceanography.

/pdf/the-ecology-of-phytoplankton-4ev70n3jsq.pdf

The Ecology of Phytoplankton

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Seven years of time-series observations of biogeochemical processes in the subtropical North Pacific Ocean gyre have revealed dramatic changes in the microbial community structure and in the mechanisms of nutrient cycling in response to large-scale ocean–atmosphere interactions. Several independent lines of evidence show that the fixation of atmospheric nitrogen by cyanobacteria can fuel up to half of the new production. These and other observations demand a reassessment of present views of nutrient and carbon cycling in one of the Earth′s largest biomes.

/pdf/the-role-of-nitrogen-fixation-in-biogeochemical-cycling-in-4a4oehaobz.pdf

The role of nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean

The surface water of the marine environment has traditionally been viewed as a nitrogen (N) limited habitat, and this has guided the development of conceptual biogeochemical models focusing largely on the reservoir of nitrate as the critical source of N to sustain primary productivity. However, selected groups of Bacteria, including cyanobacteria, and Archaea can utilize dinitrogen (N2) as an alternative N source. In the marine environment, these microorganisms can have profound effects on net community production processes and can impact the coupling of C-N-P cycles as well as the net oceanic sequestration of atmospheric carbon dioxide. As one component of an integrated ‘Nitrogen Transport and Transformations’ project, we have begun to re-assess our understanding of (1) the biotic sources and rates of N2 fixation in the world’s oceans, (2) the major controls on rates of oceanic N2 fixation, (3) the significance of this N2 fixation for the global carbon cycle and (4) the role of human activities in the alteration of oceanic N2 fixation. Preliminary results indicate that rates of N2 fixation, especially in subtropical and tropical open ocean habitats, have a major role in the global marine N budget. Iron (Fe) bioavailability appears to be an important control and is, therefore, critical in extrapolation to global rates of N2 fixation. Anthropogenic perturbations may alter N2 fixation in coastal environments through habitat destruction and eutrophication, and open ocean N2 fixation may be enhanced by warming and increased stratification of the upper water column. Global anthropogenic and climatic changes may also affect N2 fixation rates, for example by altering dust inputs (i.e. Fe) or by expansion of subtropical boundaries. Some recent estimates of global ocean N2 fixation are in the range of 100−200 Tg N (1−2 × 1014 g N) yr −1, but have large uncertainties. These estimates are nearly an order of magnitude greater than historical, pre-1980 estimates, but approach modern estimates of oceanic denitrification.

Dinitrogen fixation in the world's oceans

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,

In situ studies on n2 fixation using the acetylene

https://www.pnas.org/content/pnas/58/5/2071.full.pdf

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

Known nitrogen-fixing species of blue-green algae are capable of reducing acetylene to ethylene, but acetylene is not reduced by Anacystis nidulans, which does not fix nitrogen. Cycad root nodules which contain blue-green algae as endophytes reduce acetylene. Acetylene reduction is inhibited by carbon monoxide. Nitrate or ammonium-nitrogen has no immediate effect on algae reducing acetylene, but algae grown on nitrate-nitrogen gradually lose their capacity to reduce acetylene. Nitrate-nitrogen also inhibits heterocyst formation in these algae and there is a fairly direct correlation between the abundance of heterocysts in a particular sample and its capacity to reduce acetylene. Aphanizomenon flosaquae reduces acetylene and fixes nitrogen in unialgal culture and there is strong presumptive evidence that these reductions are carried out by the alga rather than by associated bacteria. The molar ratios of ethylene: ammonia produced vary within the range 1.4–1.8.

Acetylene reduction by nitrogen-fixing blue-green algae.

Summary
Nitrogen fixation in various Wisconsin lakes was compared using the C2H2 reduction technique. Rates of C2H2 reduction were unaffected by N2 when sufficient C2H2 was added to the gas phase. Thus in field studies the natural gas phase need not be removed before the addition of C2H2. C2H2 was reduced rapidly in eutrophic waters (Little Arbor Vitae Lake, Lake Mendota and southern Green Bay) but more slowly in oligotrophic waters (Crystal Lake, Trout Lake, Lake Mary and northern Green Bay). C2H2 reduction was greatest in the surface waters, was largely light-dependent and showed a marked diurnal variation. There was also significant variation with area and with time, and in general acetylene reduction was detected only when heterocystous algae were present. The rate of nitrogen fixation in Lake Mendota, calculated on the basis of weekly measurements taken during most of the ice-free season, was approximately 2.4 kg/ha/year.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.1971.tb02551.x

Nitrogenase activity in wisconsin lakes of differing degrees of eutrophication

Phosphorus-starved cells of Anabaena flos-aquae rapidly increase their capacity to reduce acetylene to ethylene when they receive phosphorus. This response may be used as a bioassay for detecting available phosphorus in aquatic ecosystems. The sensitivity of the method compares favorably with conventional methods for measuring dissolved orthophosphate, and has the additional advantage that it measures available phosphorus. Studies on Wisconsin lakes show that available phosphorus generally is present, that the concentrations are higher at the lower depths than at the surface, and that there may be diurnal variations in the available phosphorus content of surface waters. Important sources of available phosphorus in Lake Mendota are the waters below the thermocline and the input from storm sewers.

https://www.pnas.org/content/pnas/66/4/1104.full.pdf

G. P. Fitzgerald

Papers

In situ studies on n2 fixation using the acetylene

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

Acetylene reduction by nitrogen-fixing blue-green algae.

Nitrogenase activity in wisconsin lakes of differing degrees of eutrophication

Acetylene reduction assay for determination of phosphorus availability in wisconsin lakes.