Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.     

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.     

Acetylene reduction with physiological electron donors by extracts and particulate fractions from nitrogen-fixing Azotobacter chroococcum

1. 1. Preparations were obtained from Azotobacter chroococcum which reduced acetylene to ethylene using physiological electron donors instead of sodium dithionite. These preparations fell into two categories: those which required catalytic amounts of benzyl viologen for acetylene reduction and those that did not.

2. 2. Acetylene reduction without benzyl viologen or sodium dithionite was observed only with particles that sedimented at 40 000 × g after disrupting bacteria in the French press or with preparations obtained by disrupting bacteria protected by a mixture of defatted bovine serum albumin-Ficoll-MgCl₂ with liquid N₂; supernatant fractions required benzyl viologen for acetylene reduction.

3. 3. Added ATP inhibited acetylene reduction by large particles; ATP and MgCl₂ were necessary for maximum acetylene reduction with bovine serum albumin-protected preparations.

4. 4. NADH and carbon substrates acted as electron donors but H₂ did not; NAD⁺ was necessary for maximum acetylene reduction with carbon substrates.

5. 5. Anaerobic conditions were necessary for maximum acetylene reduction in all cases.

N2-Fixation by chemoautotrophic hydrogen bacteria

The Gram-positive coryneform bacteria strains 14g and 7C were found to be able to grow with N₂ as sole nitrogen source when incubated under microaerobic conditions. Nitrogenase activity in whole cells was assayed by acetylene reduction. High rates of ethylene production (50–120 nmole/hxmg cell protein) were observed in N₂ or glutamate grown cell suspensions shaken in an atmosphere of 2.5% O₂, 10% acetylene and 87.5% argon.     

Inoculationin vitro of wheat seedlings and wheat straw cultures withBacillus azotofixans

Bacillus azotofixans is a recently described species capable of fixing molecular nitrogen efficiently.Ecological studies performed in monoxenic wheat cultures, both in 0.7% agar and in vermiculite-sand mixture, showed that no acetylene reduction occurred and that this bacteria did not grow when supplied only with the wheat plant root exudates. However, after glucose addition to the 0.7% agar cultures, acetylene reduction ability (ARA) was detected. Comparing ARA for media with glucose both with and without plants, it was observed that the plants supply some component leading to the increase of the nitrogenase activity, since the ARA doubled in the samples containing plants.In wheat straw cultures a fast growth of the bacteria was observed in the first 24 hours after inoculation, but no acetylene reduction was detected. After glucose addition to the media with and without straw, nitrogenase activity was detected.     

Acetylene Reduction Assay for Nitrogenase Activity: Gas Chromatographic Determination of Ethylene Per Sample in Less Than One Minute

Ammoniacal silver nitrate (10 mg/ml) was added to terminate acetylene reduction assays used to measure nitrogenase activity. Silver nitrate quantitatively precipitated acetylene as the carbide salt, but did not affect the ethylene formed. The vials containing ethylene can be analyzed gas chromatographically at the rate of about 13 samples in 10 min.     

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.     

Acetylene reduction by nitrogen-fixing blue-green algae

Summary 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.     

Respiration and nitrogen fixation in nodulated roots of soya bean and pea

Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules.     

Endogenous ethylene production is a potential problem in the measurement of nitrogenase activity associated with excised corn and sorghum roots

Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots.     

Estimation of nitrogenase using a colorimetric determination for ethylene

Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene.     

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.     

Enumeration of acetylene-reducing bacteria in strip-mined reclamation sites in a temperate grassland

Summary From 36 to 71% of bacteria, depending on the sampling site, that were isolated from the soil or rhizosphere of undisturbed prairie soil or reclamation sites of strip-mined grassland areas in western North Dakota were capable of reducing acetylene. These bacteria generally could be divided into two populations; one capable of acetylene reduction under aerobic conditions and another capable of acetylene reduction under anaerobic conditions. The reclamation site to which no topsoil had been applied, pH 8.5, had a bacterial population which generally was capable of higher levels of acetylene reduction than individual bacteria isolated from other sites.     

Nitrogen Fixation by Marine Photosynthetic Bacteria

S ummary . Potential nitrogen-fixing marine photosynthetic bacteria isolated from the coastal waters and sea-bed sediments of Cardigan Bay and adjoining mud flats, were investigated, using the acetylene reduction technique, to ascertain the magnitude of their contribution of fixed nitrogen to the coastal ecosystem. Aberystwyth harbour and Iceland fjord mud readily yielded Athiorhodaceae, the majority of which consistently reduced acetylene when grown as pure cultures on marine media. Only one such strain was isolated from local seawater, but they were more common in Iceland fjord water. No marine Thiorhodaceae were isolated. It was essential to monitor crude and pure culture systems for ethylene production in the absence of acetylene.     

Nitrogen Fixation in Seawater

S ummary . The acetylene reduction technique was used for a 3-year period to monitor potential nitrogen fixation by aerobic heterotrophic bacteria in the sea 2 miles offshore in Cardigan Bay. Samples from depths down to 15 m were membrane-filtered and the residues incubated aerobically or anaerobically in acetylene-containing gas mixtures in sealed Millipore Field Monitors. Pure cultures of aerobic heterotrophs isolated from spread-plates or monitor membranes supplied with 'nitrogen-free'media, were tested for ability to reduce acetylene. Glucose was the best of 14 substrates tested to support both growth and acetylene reduction by marine bacteria. The results suggested the presence of a few aerobic or facultatively anaerobic nitrogen fixers among much more numerous and efficient 'fixed-nitrogen-scavengers'. The acetylene-reducing capacity of pure cultures was inexplicably variable, even under closely-standardized conditions: this is discussed. The more consistent acetylene reducers included various-sized rods (Gram positive and negative), coccobacilli and yeasts, which latter may have had non-culturable bacteria associated with them. No recognizable Azotobacter sp. was isolated, despite the strongly-selective conditions imposed. Similar results were obtained for seawater samples from the Irish Sea and an Iceland fjord.     

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.     

NITROGEN FIXATION BY LEGUMES AND BLUE-GREEN ALGAL-LICHEN CRUSTS IN A COLORADO DESERT ENVIRONMENT

Potential sources of fixed nitrogen in a Colorado desert environment were examined by the acetylene reduction method at the Deep Canyon Desert Research Center, near Palm Desert, California. In field and greenhouse studies all members of the genera Astragalus, Dalea, Lotus, Lupinus, Melilotus, and Prosopis examined formed active nodules (acetylene reduction) with indigenous soil bacteria. No evidence of nodulation was found for Acacia greggii, Cercidium floridum, or Hoffmannseggia microphylla. Lotus tomentellus was estimated to fix 0.1 kg N ha⁻¹ by the time of flowering under field conditions. Several members of the genus Dalea showed substantial rates of acetylene reduction in the greenhouse: D. emoryi, 16.1 + 3.5, D. mollissima, 11.4 + 3.7, D. schottii 2.9 + 1.7, D. spinosa 2.5 + 0.4 μmoles ethylene plant⁻¹ hr⁻¹. In greenhouse assays where water was supplied continuously, blue-green algal-lichen crusts reduced acetylene at an average rate of 11.0 + 5.7 nmoles ethylene cm⁻² hr⁻¹ with a maximum of 57.1. But when in situ assays were done following irrigation of a field plot with 2.3 cm of water, much lower activities were observed with a maximum activity of only 6.4 nmoles cm⁻² hr⁻¹.     

Influence of Acetylene on Growth of Sulfate-Respiring Bacteria

At a concentration of 20% of the atmosphere of the culture flasks, acetylene inhibited growth and carbon dioxide production by Desulfovibrio desulfuricans and Desulfovibrio gigas. The bacteria did not reduce acetylene to ethylene, and neither acetylene dicarboxylic acid nor ethylene was inhibitory. At 10%, acetylene was partially inhibitory for the desulfovibrios. At 5%, acetylene impeded the rate but did not limit the extent of growth and catabolism of the desulfovibrios. Desulfotomaculum ruminis was affected only negligibly, if at all, by acetylene and ethylene at any of these concentrations.     

Invalidity of the acetylene reduction assay in alkane-utilizing, nitrogen-fixing bacteria.

The cause of the failure of the C2H2-C2H4 assay for nitrogen-fixing bacteria growing on lower alkanes was studied. Acetylene was a strong competitive inhibitor of methane oxidation for methane-utilizing bacteria, as well as for the oxidation of lower alkanes by other bacteria, so that energy and reducing power were no longer available for the reduction of acetylene by nitrogenase. Nitrogen-fixing bacteria grown on alkanes may reduce acetylene when intermediates of alkane-breakdown or other substrates oxidizable in the presence of acetylene are supplied. Ethylene co-oxidation is not responsible for the failure of the test, because acetylene also inhibits this co-oxidation along with methane oxidation.     

Effects of long-term treatment with acetylene on nitrogen-fixing microorganisms.

Long periods of experimental incubation with acetylene led to a multifold enhancement of acetylene-reducing activity in Anabaena cylindrica, Anabaenopsis circularis, Rhodospirillum rubrum, and Azotobacter vinelandii. Rates of acetylene reduction showed a gradual increase and reached a peak after 2 to 6 h of continuous incubation under acetylene. Thereafter, enzyme activity rapidly declined. A similar enhancement of ethylene production was observed when pretreatment with acetylene was interrupted periodically by a brief exposure to ambient (or oxygen-free) atmosphere without acetylene although the decline of acetylene-reducing activity was less rapid. Pretreatment with acetylene depressed photosynthetic 14CO2 fixation and 15N2 incorporation in Anabaena cylindrica. It is concluded that assessments based on long-term experimental incubation with acetylene may grossly overestimate the actual quantities of fixed nitrogen in the field.