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Journal ArticleDOI

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

01 Aug 1968-Plant Physiology (American Society of Plant Biologists)-Vol. 43, Iss: 8, pp 1185-1207
TL;DR: This 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.
Abstract: 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.
Citations
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Journal ArticleDOI
TL;DR: How isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen the understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies is reviewed.
Abstract: ▪ Abstract The use of stable isotope techniques in plant ecological research has grown steadily during the past two decades. This trend will continue as investigators realize that stable isotopes can serve as valuable nonradioactive tracers and nondestructive integrators of how plants today and in the past have interacted with and responded to their abiotic and biotic environments. At the center of nearly all plant ecological research which has made use of stable isotope methods are the notions of interactions and the resources that mediate or influence them. Our review, therefore, highlights recent advances in plant ecology that have embraced these notions, particularly at different spatial and temporal scales. Specifically, we review how isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen our understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies. Where possible we also...

1,710 citations

Journal ArticleDOI
TL;DR: This paper reviews and update long-standing and more recent estimates of biological N2 fixation for the different agricultural systems, including the extensive, uncultivated tropical savannas used for grazing.
Abstract: Biological dinitrogen (N2) fixation is a natural process of significant importance in world agriculture. The demand for accurate determinations of global inputs of biologically-fixed nitrogen (N) is strong and will continue to be fuelled by the need to understand and effectively manage the global N cycle. In this paper we review and update long-standing and more recent estimates of biological N2 fixation for the different agricultural systems, including the extensive, uncultivated tropical savannas used for grazing. Our methodology was to combine data on the areas and yields of legumes and cereals from the Food and Agriculture Organization (FAO) database on world agricultural production (FAOSTAT) with published and unpublished data on N2 fixation. As the FAO lists grain legumes only, and not forage, fodder and green manure legumes, other literature was accessed to obtain approximate estimates in these cases. Below-ground plant N was factored into the estimations. The most important N2-fixing agents in agricultural systems are the symbiotic associations between crop and forage/fodder legumes and rhizobia. Annual inputs of fixed N are calculated to be 2.95 Tg for the pulses and 18.5 Tg for the oilseed legumes. Soybean (Glycine max) is the dominant crop legume, representing 50% of the global crop legume area and 68% of global production. We calculate soybean to fix 16.4 Tg N annually, representing 77% of the N fixed by the crop legumes. Annual N2 fixation by soybean in the U.S., Brazil and Argentina is calculated at 5.7, 4.6 and 3.4 Tg, respectively. Accurately estimating global N2 fixation for the symbioses of the forage and fodder legumes is challenging because statistics on the areas and productivity of these legumes are almost impossible to obtain. The uncertainty increases as we move to the other agricultural-production systems—rice (Oryza sativa), sugar cane (Saccharum spp.), cereal and oilseed (non-legume) crop lands and extensive, grazed savannas. Nonetheless, the estimates of annual N2 fixation inputs are 12–25 Tg (pasture and fodder legumes), 5 Tg (rice), 0.5 Tg (sugar cane), <4 Tg (non-legume crop lands) and <14 Tg (extensive savannas). Aggregating these individual estimates provides an overall estimate of 50–70 Tg N fixed biologically in agricultural systems. The uncertainty of this range would be reduced with the publication of more accurate statistics on areas and productivity of forage and fodder legumes and the publication of many more estimates of N2 fixation, particularly in the cereal, oilseed and non-legume crop lands and extensive tropical savannas used for grazing.

1,355 citations


Additional excerpts

  • ...Both gases can be readily detected and quantified using gas chromatography (Schollhorn and Burris 1967; Hardy et al. 1968)....

    [...]

Journal ArticleDOI
TL;DR: It is concluded that biological invasion by Myrica faya alters ecosystem-level properties in this young volcanic area; at least in this case, the demography and physiology of one species controls characteristics of a whole ecosystem.
Abstract: Myrica faya, an introduced actinorhizal nitrogen fixer, in invading young volcanic sites in Hawaii Volcanoes National Park. We examined the population biology of the invader and ecosystem-level consequences of its invasion in open-canopied forests resulting from volcanic cinder-fall. Although Myrica faya is nominally dioecious, both males and females produce large amounts of fruit that are utilized by a number of exotic and native birds, particularly the exotic Zosterops japonica. In areas of active colonization, Myrica seed rain under perch trees of the dominant native Metrosideros polymorpha ranged from 6 to 60 seeds m{sup {minus}2} yr{sup {minus}1}; no seeds were captured in the open. Planted seeds of Myrica also germinated an established better under isolated individuals of Metrosideros than in the open. Diameter growth of Myrica is > 15-fold greater than that of Metrosideros, and the Myrica population is increasing rapidly. Rates of nitrogen fixation were measured using the acetylene reduction assay calibrated with {sup 15}N. Myrica nodules reduced acetylene at between 5 and 20 {mu}mol g{sup {minus}1} h{sup {minus}1}, a rate that extrapolated to nitrogen fixation of 18 kg ha{sup {minus}1} in a densely colonized site. By comparison, all native sources of nitrogen fixation summed to 0.2 kg ha{sup {minus}1}more » yr{sup {minus}1}, and precipitation added < 4 kg ha{sup {minus}1} yr{sup {minus}1}. Measurements of litter decomposition and nitrogen release, soil nitrogen mineralization, and plant growth in bioassays all demonstrated that nitrogen fixed by Myrica becomes available to other organisms as well. We conclude that biological invasion by Myrica faya alters ecosystem-level properties in this young volcanic area; at least in this case, the demography and physiology of one species controls characteristics of a whole ecosystem.« less

1,139 citations

Journal ArticleDOI
TL;DR: Estimates based on this method compare favourably with other methods for field evaluation of N2-fixation, provided that the site and the sampling strategy are appropriate for application of the method.
Abstract: This paper reviews a growing body of literature on the use of variations in the natural abundance of 15N to estimate the fractional contribution of N2-fixation to N2-fixing systems. This method is based on the small difference in 15NN abundance which frequently occurs between N derived from N2-fixation and N derived from other sources. The requirement of the method is that this difference be significant. Whether this requirement is met is site specific and must be empirically established at each site of interest. Advantages and disadvantages of this method are compared with those of more conventional methods. Sources of error, including heterogeneity of 15NN abundance of non-atmospheric N sources are considered. Tests of the method, under both greenhouse and field conditions, are described. Estimates based on this method compare favourably with other methods for field evaluation of N2-fixation, provided that the site and the sampling strategy are appropriate for application of the method. Applications of the method in several ecosystems are described.

1,037 citations

Journal ArticleDOI
TL;DR: The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H 2]-fixing activities obtained with various systems, and methods of measurement of N2 fixation are compared.
Abstract: 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.

1,021 citations

References
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01 Jan 2016
TL;DR: 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.
Abstract: 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,

858 citations

Journal ArticleDOI
TL;DR: It is suggested that the first step in N 2 reduction is a two-electron reduction leading to a non-dissociable intermediate at the oxidation level of diimide.

548 citations

Journal ArticleDOI
TL;DR: Proper procedures have now been developed for the isolation of two enzyme fractions, both of which are required for N2 reduction, ATP-dependent H2 evolution, and the related release of inorganic phosphate.
Abstract: The requirement for both an electron donor and ATP for the enzymatic reduction of N2 to ammonia was demonstrated with extracts of Clostridium pasteurianum,1-4 Azotobacter vinelandii,5 7 and Rhodospirillum rubrum.' 8 With hydrosulfite as the electron donor and an ATP-generating system (creatine phosphokinase), N2-reducing preparations from A. vinelandii and R. rubrum were shown to catalyze a concomitant ATP-dependent H2-evolution reaction.6-9 This reaction was subsequently demonstrated with extracts of C. pasteurianum10' 11 by inhibiting the classical hydrogenase with carbon monoxide. The use of hydrosulfite as the electron donor, in combination with an ATP-generating system, provides a useful assay reaction for enzyme purification since both required components can be added exogenously. We reported previously7 that the N2-reducing and ATP-dependent H2-evolving activities could be obtained from azotobacter extracts by precipitation with protamine followed by pH fractionation, and that these fractions contained a high level of nonheme iron as well as molybdenum. Procedures have now been developed for the isolation of two enzyme fractions, both of which are required for N2 reduction, ATP-dependent H2 evolution, and the related release of inorganic phosphate. The first enzyme contains both nonheme iron and molybdenum; the second contains nonheme iron but no molybdenum. The purification and some properties of these two enzymes are described in this report. A preliminary report was presented previously.12 Mlortenson'3 14 recently reported evidence suggesting the requirement for at least two enzymes from C. pasteurianum extracts for the catalysis of N2 reduction and related reactions. Materials and Methods.-Azotobacter vinelandii 0 was cultured and harvested as previously described,6 and the cell paste either used when harvested or stored frozen in an argon atmosphere. Cells (100-120-gm cell paste) were ruptured in a French pressure cell, and the S1441/2 supernatant fraction (obtained by centrifugation at 144,000 X g for 30 min), containing 90-95% of the activity of crude extracts, was prepared as previously described.7 Since a sensitivity to oxygen developed during purification, all buffers and solutions were saturated with argon and contained 0.1 mg of dithiothreitol per ml. Protamine sulfate fractionation: Protamine was used both to remove nucleic acids and to precipitate the enzymatically active components. A 2% solution of the reagent (Sigma Chemical Co., Grade II) was prepared in water at room temperature, the pH adjusted to 6.0 with 1 N NaOH, and the precipitate removed by centrifugation. The supernatant solution was decanted and stirred mechanically under a stream of argon to remove dissolved oxygen. The S144-1/2 fraction, allowed to cool during ultracentrifugation, was held at 00 during protamine fractionation. Protamine sulfate solution was added at the rate of 5 ml per gm of protein, and the precipitated nucleic acids were removed by centrifugation. The decanted supernatant solution was adjusted to pH 6.5 with 0.5 N acetic acid, and the active components were precipitated upon the further addition of 1.2 ml of protamine sulfate solution per gm of S,44-i/, protein. After centrifugation, the precipitate was suspended in 0.01 M potassium phosphate, pH 7.0, at room temperature and stirred with purified7 cellulose phosphate (Sigma Chemical Co.) added at the rate of 100 mg per gm of S44-i/, protein. Cellulose phosphate with its bound protamine was removed on a fritted

308 citations

Journal ArticleDOI
TL;DR: Ethylene formation was detected by mass spectrometry or gas chromatography and the amounts of ethylene produced were determined by integration of the peaks obtained and comparison with a standard curve.
Abstract: Methods and Materials.-The gases H2, N2 (high purity), and acetylene (purified grade) were commercial cylinder gases. Acetylene was freed from traces of acetone by condensing the acetone in a trap cooled with dry ice. Enzyme preparations: Cultures of Clostridium pasteurianum (strain W-5) were grown in a nitrogen-deficient medium with N2, harvested, dried in a rotary evaporator, and stored in evacuated tubes at -20°. Extracts were obtained by autolyzing the dried cells for 1 hr with shaking in 0.05 M cacodylate buffer pH 6.8 at 320 in an atmosphere of H2. The resulting suspension was centrifuged at 20,000 g for 25 min, and the supernatant was used. Azotobacter vinelandii (strain 0) was grown in aerated liquid cultures in 180-liter glass-lined fermentors, and the cells were stored as a frozen paste. N2-fixing extracts were prepared as described by Bulen, Burns, and LeComte4 and the supernatant of successive centrifugations at 35,000 g for 30 min and 144,000 g for 60 min was used. Experimental conditions: Experiments for inhibition of N2 fixation in C. pasteurianum were run in 20-ml rubber-stoppered serum bottles containing 1 ml of reaction mixture and the desired atmosphere; H2 was used as the electron donor. The mixture contained 5 /moles ATP, 50 Jmoles acetylphosphate, 2 emoles MgCl2, 50 /Amoles cacodylate buffer at pH 6.8, and enzyme preparation containing about 8 mg protein. The bottles were shaken at 320 in a water bath, and the reaction was started by introducing the enzyme extract through a hypodermic needle. The reaction was stopped by addition of I ml of saturated K2CO3 solution, usually after 30 min. After microdiffusion to an acid-dipped glass rod inserted into the serum bottle, ammonia was determined spectrophotometrically by the Nessler method. Control reactions omitted H2 or substituted an argon atmosphere. The average specific activity of the preparations for N2 fixation was about 5 nanomoles N2 fixed/mg protein X min at a pN2 of 500 mm and a pH2 of 200 mm. Experiments for acetylene reduction were performed in 40-ml vessels with serum stoppers and 10 ml of reaction mixture, or in 100-ml vessels (containing 50 ml of reaction mixture stirred with a magnetic bar) equipped with a serum stopper and a three-way capillary stopcock. Reactions were stopped by introducing saturated ammonium sulfate solution. Ethylene formation was detected by mass spectrometry or gas chromatography. All quantitative ethylene determinations were performed with an Aerograph 700 gas chromatograph equipped with a 2-mm inside diameter aluminum column 160 cm long. The column was filled with activated alumina and was run at 1450. Helium served as carrier gas at flow rates of 0.5-2 cc/sec. The amounts of ethylene produced were determined by integration of the peaks obtained and comparison with a standard curve. The reactions to examine the inhibition of preparations from A. vinelandii were run in 20-ml rubber-stoppered serum bottles containing 2 ml of reaction mixture and the desired atmosphere. Each bottle contained 0.2 mg creatine phosphokinase, 50 jemoles creatine phosphate, 5 Mmoles ATP, 5 jsmoles Mg++, 80 jmoles cacodylate buffer pH 7.0, 40 jsmoles Na2S204, and about 9 mg of crude enzyme protein. Introduction of Na2S204 and the enzyme started the reaction; the bottles were shaken for 40 min at 320. The reactions were stopped with saturated K2CO3 solution, and the ammonia was determined as described for C. pasteurianum. Control reactions were run under argon.

220 citations