Problems of the Acetylene Reduction Technique Applied to Water-Saturated Paddy Soils

The acetylene reduction assay for the measurement of N₂ fixation in a water-saturated paddy soil is limited by the slow diffusion of acetylene and ethylene. In laboratory incubation tests, vigorous shaking after the assay period is needed to release ethylene into the gas within the assay vials. Shaking prior to the incubation is also effective for dissolving acetylene in the water-saturated soil. However, a water-saturated soil depth of less than 10 mm during incubation is recommended. In field assays, some amounts of ethylene remain in the water-saturated soil phase of the acetylene reduction assay chamber, but stirring the water-saturated soil before sampling reduces the amount of ethylene remaining in soil. Evidence of a downward movement of acetylene and an upward movement of ethylene through rice plants was obtained. Because of the rapid transfer of acetylene to rice plant roots, an in situ acetylene reduction assay covering a rice hill is likely to detect nitrogen fixation in the proximity of roots where acetylene is easily accessible. Acetylene introduction to the water-saturated soil phase prior to assay did not greatly increase the acetylene reduction rate. Carbon dioxide enrichment in the assay chamber did not enhance nitrogen fixation in a paddy including rice and algae during a 1-day cycle.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

Root Nodule Enzymes of Ammonia Assimilation in Alfalfa (Medicago sativa L.) : DEVELOPMENTAL PATTERNS AND RESPONSE TO APPLIED NITROGEN

Nitrogenase-dependent acetylene reduction activity of glasshouse-grown alfalfa (Medicago sativa L.) decreased rapidly in response both to harvesting (80% shoot removal) and applied NO₃⁻ at 40 and 80 kilograms N per hectare. Acetylene reduction activity of harvested plants grown on 0 kilogram N per hectare began to recover by day 15 as shoot regrowth became significant. In contrast, acetylene reduction activity of all plants treated with 80 kilograms NO₃⁻-N per hectare and harvested plants treated with 40 kilograms NO₃⁻-N per hectare remained low for the duration of the experiment. Acetylene reduction of unharvested alfalfa treated with 40 kilograms N per hectare declined to an intermediate level and appeared to recover slightly by day 15. Changes in N₂-fixing capacity were accompanied by similar changes in levels of nodule soluble protein.     

Continuous, automated acetylene reduction assays using intact plants

An automated method was developed for continuous, in situ determination of acetylene reduction (N₂ fixation) by intact soybean plants (Glycine max [L.]). The culture vessel containing the roots of intact plants grown in sand culture is sealed at the surface and an air-acetylene mixture continuously injected into the root chamber. The effluent gas is automatically sampled and injected into a gas chromatograph. Continuous acetylene assay at intervals as short as 3.5 min may be made over a period of several days, without attention, except for plant watering. Adverse effects of prolonged exposure of the root system to acetylene were mitigated by pulse injection of acetylene for 20 min followed by 40 min of acetylene-free air. Bare root systems can be suspended in a reaction chamber and sprayed with water or nutrient solution; this permits periodic removal of the root system for sampling nodules.     

In situ determination of nitrogen fixation in Antarctica using a high sensitivity portable gas chromatograph

A portable gas chromatograph was employed in the Vestfold Hills, Antarctica, during the austral summer of 1979-80 for determining nitrogenase activity of the blue-green alga Nostoc commune Vaucher by the acetylene reduction assay. Acetylene reduction was measured in samples taken along a transect where the vegetation changed with respect to differing topography and water availability. Submerged colonies of Nostoc recorded the highest fixation rates (6.89 nmol C₂H₄. cm^-2 h^-1). Damp mosscyanophyte associations growing on shallow slopes showed moderate rates of acetylene reduction (1.99 nmol C₂H₄. cm^-2 h^-1) whilst the drier vegetation of the steeper terrain was the least active (0.19 nmol C₂H₄. cm^-2 h^-1. The employment of a high sensitivity portable gas chromatograph provided an accurate and reliable method of measuring acetylene reduction.     

Reinvestigation of Coprosma for ability to fix atmospheric nitrogen

Summary Coprosma robusta Raoul plants grown in plus- and minus-nitrogen sand culture showed no significant response to removal of stipules. Acetylene reduction assays for nitrogenase on leaves and stipules gave negative results for six Coprosma species. Two unwashed root samples showed slight acetylene reduction and this was ascribed to casual aerobic rhizosphere organisms.     

Defence structure development inRhizopus nigricans during mycoparasitism byFusarium oxysporum F. sp.lycopersici

Summary Unfertilized and NPK-fertilized plots in the long term fertility trial at Los Baños, Philippines were used to observe the effect of nitrogen fertilizer on heterotrophic N₂-fixing activities in plow layer soil and in association with wetland rice. The activities were measured in the field byin situ acetylene reduction assays. Acetylene reduction activity of the plow layer soil in the paddy field was measured by soil cores. No appreciable difference was found-between fertilized and unfertilized plots.     

In situ acetylene-ethylene assay of biological nitrogen fixation in lowland rice soils

Summary An in situ device for assaying biological nitrogen fixation in flooded rice soils, using the acetylene reduction method, was developed. Diurnal variations in acetylene reduction by an inoculated field plot and by laboratory-grown cultures of nitrogen-fixing algae showed a prominent single-peak pattern of nitrogenase activity. The peak occurred at mid-day for laboratory-grown algae and at late afternoon for the algae grown in the field plot. Some nitrogenase activity was noted during the night. Acetylene reduction studies in rice fields of Albay province, Philippines, showed an estimated fixation of 18.5 to 33.3 kg N/ha each cropping season for the fields of Puro soil and 2.3 to 5.7 kg N/ha each cropping season for the fields of Santo Domingo soil. re]19751202     

Acetylene reduction and respiration of bacteroids isolated from French-beans receiving nitrate

The competition between combined nitrogen and nitrogen fixation in legumes was studied after a 24 h exposure of nodulated French-beans to nitrate. Acetylene reduction by bacteroids was significantly inhibited and even nitrogenase extracted from nitrate-treated plant nodules showed reduced activity. Sensitivity to nitrate was directly related to nodule age and also increased with increasing oxygen tensions in the bacteroid incubations with or without a gas phase; it was particularly marked when glucose was used in place of succinate as energy-yielding substrate. Bacteroid respiration was also depressed by nitrate-treatment of the plants, leading to diminished acetylene reduction and this effect increased with increasing oxygen concentrations. Added oxyleghemoglobin partly restored oxygen consumption and acetylene reduction by bacteroid suspensions.     

Contribution of basal portion of shoot to N2 fixation associated with wetland rice

Summary Oryza sativa grown in flooded soil were transferred to water culture solution and acetylene reduction activities (ARA) of intact plants and rootless plants were measured for 5 h. Relative rate of ARA associated with the rootless wetland rice plant as compared with an intact plant varied from 8 to 100 percent, depending on the growth stage and varieties of rice and highest at the early stage (3 weeks after transplanting) for all varieties tested (IR26, Latisail, Khao Lo, and JBS236). ARA of shoots was associated with basal parts of the shoots about 3 cm from the base of wetland cultivated rice andOryza australiensis. Phyllospheric ARA was negligible except for senescent outer leaf sheaths. Microaerophilic N₂-fixing bacteria also inhabited basal parts of shoots (outer leaf sheaths and stems) of wetland rice. These findings suggest that N₂-fixation is partly associated with the shoots of wetland rice plants.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

Effect of acetylene on root respiration and acetylene reducing activity in nodulated soya bean

Acetylene decreased root and nodule respiration, as measured by CO₂ evolution of nodulated or non-nodulated Glycine max. An inhibition of 25 to 35% in 15 to 30 minutes occurred when 13% C₂H₂ was introduced in the gas flux which aerated the root nutrient solution. When the light intensity was doubled to 800 microeinsteins per square meter per second, the inhibition increased to 50% and nodule acetylene reduction activity was inhibited 50%.     

Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

Impact of soil environmental factors on rates of N2-fixation associated with roots of intact maize and sorghum plants

We have evaluated the effects of oxygen partial pressure (pO₂), combined nitrogen, and the availability of organic substrates on nitrogen fixation (acetylene reduction) by bacteria associated with the roots of intact maize and sorghum plants. We also investigated the possibility of enhancing associative nitrogen-fixation by inoculating the soil in which the plants were grown withAzospirillum. Acetylene reduction (AR) activity was greatest when roots of intact plants were exposed to pO₂ between 1.3 and 2.1 kPa. Field-grown and greenhouse-grown plants supported similar levels of activity. Respiration inhibitors (2,4-dinitrophenol and sodium azide) eliminated AR activity at 2 kPa O₂, whereas a fermentation inhibitor (sodium fluoride) only partially reduced the activity. Acetylene reduction activity was rapidly (1–3 h) inhibited by NH ₄ ⁺ , NO ₃ ^– , and NO ₂ ^– at concentrations of 4–20 mg Nl^–1. Rates of AR varied substantially among individual plants in each experiment and between experiments. Amendment with any of several organic substrates greatly increased AR activity when rates were low, suggesting that the lack of activity was caused by a shortage of available carbon in the rhizosphere. Inoculation withAzospirillum failed to increase rates of AR associated with maize plants. In several experiments the indigenous bacteria associated with uninoculated plants exhibited greater activity than the bacteria associated with inoculated plants.     

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar `M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO₃ once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N₂. During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.     

Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.     

Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest

Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro

The acetylene reduction assay was used to measure presumed N₂-reducing activity in Rhizobium-soybean cell associations in vitro. No acetylene reduction was observed in liquid suspensions of these organisms, but cells plated onto an agar medium from a liquid suspension of Rhizobium and soybean cells exhibited acetylene-dependent production of ethylene after 7 to 14 days. Aggregates of soybean cells 0.5 to 2.0 mm in diameter were required for this activity. Decreasing oxygen from 0.20 atm to 0.10, 0.04, or 0.00 atm completely inhibited acetylene reduction. The presence of 2,4-dichlorophenoxyacetic acid or kinetin increased endogenous ethylene production and inhibited acetylene-dependent ethylene production. Acetylene reduction was observed with three out of four strains of R. japonicum tested, and three rhizobial strains, which produce root nodules on cowpeas but not soybeans, formed an association capable of acetylene-dependent ethylene production.