The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ 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 N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ 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 N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.

     

Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation

The interaction between the ATP-dependent evolution of H₂ catalyzed by nitrogenase and the oxidation of H₂ via a hydrogenase has been postulated to influence the efficiency of the N₂-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H₂ in air and the H₂ produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N₂ fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H₂ in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C₂H₂ reduction and O₂ consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.

When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N₂ fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H₂-evolving strain 176A27 produced increases in plant dry weight and total N₂ fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N₂ fixation for nodulated legumes capable of reutilizing the H₂ evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H₂-recycling process in N₂-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

     

TheParasponia parviflora — Rhizobium symbiosis. Isotopic nitrogen fixation,hydrogen evolution and nitrogen-fixation efficiency,and oxygen relations

Summary Isotopic¹⁵N₂ experiments confirmed nitrogen fixation inParasponia parviflora. The conversion ratio C₂H₄/N₂ was 6.7 under the experimental conditions employed. Measurements of the δ¹⁵N in leaves of Parasponia and Trema showed on the basis of these determinations thatParasponia parviflora possesses N₂-fixing capacity and can be distinguished in this respect from the non-nitrogen-fixingTrema cannabina tested by the same method. Therefore, δ¹⁵N can be used to monitor N₂ fixation in natural ecosystems. Hydrogen evolution and the relative efficiency of N₂ fixation in this relation have been determined. DetachedParasponia parviflora root nodules grown in soil and tested in an argon/oxygen atmosphere produced appr. 4 μmol H₂.h⁻¹.g⁻¹ fresh weight root nodules. The relative efficiency of hydrogen utilization as measured in argon, air, and in the presence of C₂H₂ 10% (v/v) was for both equations used for to express this efficiency 0.96 and 0.97, respectively. This indicates that Parasponia like the root nodules of some actinorhizal symbioses (Alnus, Myrica, Elaeagnus) and some tropical legumes (Vigna sinensis) has evolved mechanisms of minimizing net hydrogen production in air, thus increasing the efficiency of electron transfer to nitrogen. The oxygen relation of nitrogen fixation (C₂H₂) inParasponia parviflora root nodules was determined. The nitrogenase activity of Parasponia root nodules increased at increasing oxygen concentrations up till c. 40% O₂. At oxygen levels above 40% O₂, the nitrogenase activity of the root nodules was nil or very erratic suggesting that at these oxygen levels the nitrogenase is not longer protected against the harmful effect of oxygen. In this respect Parasponia root nodules differ from actinorhizal root nodules in other nonlegumes, where optimal nitrogenase activity was observed in the range of 12–25% oxygen. Respiration experiments with Parasponia root nodules showed that in the range 10, 20, and 40% oxygen, the respiration rate (CO₂ evolution) increased concomitantly with an increase of the acetylene reduction rate. The CO₂/C₂H₄ values obtained varied between 8.1 and 19.2, being therefore 2–3 times higher than similar estimations in some actinorhizal and legume root nodules. The respiratory quotient (RQ) of detachedParasponia parviflora root nodules was in air initially approximately 2.0, but this value dropped to about 1.0 in a 3-hours period.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

Incorporation of Different N Sources and Light Response Curves of Nitrogenase and Photosynthesis by Cyanobacterial Blooms from Rice Fields

In this work, we estimate the contributions of the different sources of N incorporated by two N₂-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N₂-fixing cyanobacterial blooms in rice fields comes from N₂ fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N₂ fixation (only about 33–41% of the N incorporated came from N₂ fixation). Our results on the photodependence of C and N₂ fixation indicate that in both cyanobacterial blooms, N₂ fixation showed a steeper initial slope (α) and was saturated with less irradiance than C fixation, suggesting that N₂ fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N₂ fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Nitrogen fixation in grassland and associated cultivated ecosystems

Summary Nitrogen fixation in the natural, Agropyron-Koeleria grassland ecosystem was studied using the C₂H₂-C₂H₄ and N¹⁵ assays. Small soil samples and also undisturbed soil cores were used for analyses. Both techniques indicated that grassland and associated cultivated soils had low fixation rates (0.6–1.8 kg/ha per 28 days in the laboratory and, 1 kg/ha per season under actual field conditions). Algal colonies (Nostoc spp.) on the soil surface were active fixers when the surface of the grassland was moist. However, their small biomass limits the extent of fixation in most areas. In native grassland, 16 legumes bore nodules. The three most common speciesVicia americana, Thermopsis rhombifolia andOxytropis sericea, all of which had active nodules, contributed 10 per cent of the total nitrogenase activity. The non-legumesElaeagnus commutata andShepherdia argentea were profusely nodulated with active nodules, but were confined to specific habitats. No nodules were found onArtemisia orOpuntia spp. The major, heterotrophic, asymbiotic bacteria in the soil were clostridia. These utilize substrates produced by aerobic cellulose and hemicellulose degrading organisms to fix N in anaerobic microsites. The C₂H₂:N₂ reduction ratio was 3 to 1 in large, aerobic core samples, but was greater under water-logged conditions where high fixation rates occurred.     

Nutrient Limitation to Nitrogen Fixation in Young Volcanic Sites

I used measures of ¹⁵N natural abundance and of nitrogenase activity (acetylene reduction) to examine whether the supply of non-N nutrients limits rates of N₂ fixation on young volcanic substrates in Hawaii. Leaves of the dominant tree (Metrosideros polymorpha, a nonfixer) were strongly depleted in ¹⁵N in control plots (–10.8 to –11.1⁰/₀₀). More than 5 y of repeated fertilization with P increased δ¹⁵N to –8.9 to –9.9⁰/₀₀, and the addition of all other essential plant nutrients (except N) together with P further increased ¹⁵N to –8.1 to –9.3⁰/₀₀. This pattern is consistent with enhanced N₂ fixation, because newly fixed N would have a δ¹⁵N near 0⁰/₀₀. Assays of nitrogenase activity in the experimental plots demonstrated that potential N fixation associated with nonvascular plants and with tree and fern litter were increased significantly by additions of P and by the combined nutrient treatment; when these were added together, the increase in nitrogenase activity was 6- to 11-fold over control plots. The supply of P and other weathering-derived nutrients constrains rates of N₂ fixation in these young volcanic sites and thereby contributes to the maintenance of N limitation to primary production and other ecosystem processes. Received 7 January 1999; accepted 3 May 1999.     

The biotechnology of hydrogen production by Nostoc flagelliforme grown under chemostat conditions

The potential of using N₂-fixing cyanobacteria to produce hydrogen photobiologically has stimulated research on the physiology and biotechnology of species exhibiting high H₂ production rates over long periods of time. In this work Nostoc flagelliforme, a terrestrial N₂-fixing cyanobacterium, has been examined to establish its physiology and potential for H₂ production under controlled conditions. Cell filaments of N. flagelliforme were purified and grown in liquid culture to optimize its H₂ metabolism. In batch-grown cultures the activity of nitrogenase, the key enzyme for H₂ production in N₂-fixing organisms, was found to be high only during a short phase of exponential growth. A chemostat system was thus constructed for long-term experiments using continuous cultures, with the aim of exploiting the exponential growth phase. The dilution rate (D) and environmental factors, such as N₂ concentration in the gas phase and temperature, significantly influenced H₂ production. Cells grown continuously under the optimized conditions of D = 0.022 h⁻¹, 34 °C and 5.1 kPa N₂ in the gas phase exhibited H₂ production rates that were more than four times higher than the maximal rates under standard batch growth conditions. Received: 14 October 1996 / Received revision: 18 February 1997 / Accepted: 22 February 1997     

Nitrogenase activity in the papyrus swamps of Lake Naivasha,Kenya

Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively anaerobic N₂-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10⁶ CFU g d.wt. roots^-1). Fewer numbers of N₂-fixing bacteria were detected in the sediments (1.9 × 10³-3.2 × 10⁴ CFU g d.wt. sediment^-1).     

Calorimetry of nitrogenase-mediated reductions in detached soybean nodules

Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N₂ fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.

There was major heat output by nodules for all of the nitrogenase substrates tested (H⁺, N₂, N₂O, and C₂H₂) further establishing the in vivo energy inefficiency of biological N₂ fixation. Exposure to a short burst of 100% O₂ partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H⁺ reductions was 171 ± 6 kilocalories per mole H₂ evolved in an Ar-O₂ atmosphere, that for N₂ fixation was 784 ± 26 kilocalories per mole H₂ evolved and N₂ fixed, and that for C₂H₂ reduction was 250 ± 12 kilocalories/mole C₂H₄ formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e⁻ is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N₂ fixation cost of 9.5 grams glucose per gram N₂ fixed or 3.8 grams C per gram N₂, which is in close agreement with earlier calculations based on nodular CO₂ production.

     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO(2)

The N₂-fixing legume nodule requires O₂ for ATP production; however, the O₂ sensitivity of nitrogenase dictates a requirement for a low pO₂ inside the nodule. The effects of long term exposures to various pO₂s on N₂[C₂H₂] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO₂ of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O₂ initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO₂ of 0.32 atmospheres had little effect on N₂[C₂H₂] fixation; a pO₂ of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O₂ controls by 95 hours. Re-exposure to ambient pO₂ of plants adapted to nonambient pO₂s reduced N₂[C₂H₂] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO₂ conditions, and a time period was required to readapt to ambient O₂. It is concluded that the N₂[C₂H₂]-fixing system of intact soybean plants is able to adapt to a wide range of external pO₂s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO₂ optimal for nitrogenase activity.     

Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants

Corn (Zea mays L.) plants were assayed for nitrogenase activity (C₂H₂ reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O₂ resulted in lower rates of C₂H₂ reduction. The time course of C₂H₂ reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn × teosinte) and indicated that a long preincubation at reduced O₂ is not absolutely required for early detection of nitrogenase activity. Isolation of N₂-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N₂-fixing bacteria.     

Acetylene reduction (N2-fixation) by Enterobacteriaceae isolated from industrial wastewaters and biological treatment systems

Summary Acetylene reducing (N₂-fixing) Entero-bacteriaceae have been isolated from activated sludge plants treating waste from the paper and food industries (10³ to 10⁶ cells per ml) and from composting plants handling forest waste (10⁵ to 10⁶ cells per g wet weight). Detailed studies on se-lected strains of all taxa showed that: (1) pure cul-tures were able to utilize a range of carbohy-drates, polyols, amino acids and carboxylic acids as sole sources of carbon (2) high levels of nitro-genase were attained during growth with a range of carbon substrates: highest levels (12—66 n mole C₂H₄.min⁻¹.mg protein⁻¹) were found for glucose and sucrose, variable levels for polyols, and lower levels for citrate and fumarate (1—23 n mole C₂H₄.min⁻¹.mg protein⁻¹) (3) organic ni-trogen compounds which were utilized as sole sources of nitrogen did not generally repress the synthesis of nitrogenase, although low levels were found for some strains during growth with glu-cosamine. Samples from a laboratory model acti-vated sludge system showed a mean rate of acety-lene reduction corresponding to the fixation of 26 μg N.h⁻¹.1⁻¹, and direct analysis of the in-fluent and effluent waters and sludge showed a net increase in nitrogen. These observations corre-lated with the presence of a population of N₂-fix-ing Enterobacteriaceae of ca. 10⁵ cells per ml and pure strains isolated from the system had a mean nitrogenase specific activity of 88 n mole C₂H₄.min⁻¹.mg protein⁻¹. It is therefore con-cluded that endogenous N₂-fixing Enterobacteria-ceae contained in some kinds of industrial waste-waters could successfully be used to diminish the addition of combined nitrogen to activated sludge treatment plants.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Reduction of acetylene by nodules of Trifolium subterraneum as affected by root temperature,Rhizobium strain and host cultivar

Summary The nitrogenase activity (measured by reduction of C₂H₂ to C₂H₄) of nodules of Trifolium subterraneum grown at root temperatures from 7°C–19°C was broadly correlated with nitrogen fixation. Root temperature did not affect enzyme activity per se but did affect the amount of enzyme formed. Exposure of nodules to 7°C for 24 h did not decrease activity cf. 19°C. Activity was greatest when nodules were about 4 days old, before swollen bacteroid forms were produced, and then declined. The effectiveness of a bacterial strain at a given temperature was related to the amount of enzyme produced and to its persistence. Nitrogenase activity should be measured throughout the plant growth cycle for valid comparisons of strain effectiveness.     

Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans

An apparatus was designed for simultaneous measurement of rates of N₂ fixation estimated by C₂H₂-C₂H₄ assay (N₂[C₂H₂] fixation) and NO₃⁻ absorption by roots of intact, nodulated soybeans (Glycine max [L.] Merr.). The principal design features include: (a) a gas-tight mist chamber in which nodulated roots can be exposed simultaneously to C₂H₂ in the gas phase and to a liquid phase containing NO₃⁻ sprayed in a fine mist; and (b) provision for sampling the gas phase for C₂H₄ determination, and the liquid phase for NO₃⁻ determination.     

Ecology of heterotrophic dinitrogen fixation in the rhizosphere of mangrove plant community at the Ganges river estuary in India

Summary Heterotrophic dinitrogen fixation in root associations of successional stages of the tropical mangrove plant community at the Ganges river estuary in India was investigated by excised-root acetylene reduction assay, and enumeration and identification of diazotrophic bacteria from sediment, root and tidal water samples. High to very high rates of nitrogenase activity (64–130 nmol C₂H₄/g dry root/h) were associated with washed excised roots of seven common early-successional mangrove species at the inundated swamps. Declining, late-successional mangroves at the occasionally inundated ridges had considerably lower values and the “declined” mangroves and other non-littoral species at embankment protected highlands had very low to insignificant values of root nitrogenase activity. Total and inorganic nitrogen contents of the mangrove sediments were low and were positively related to the stages of physiographic succession. Plant-associated sediments of particularly the old formation swamps had very high C/N ratios. Nine isolates of nitrogen-fixing bacteria belonging to all known O₂ response groups were distinguished from a large population of diazotrophs associated with roots of mangroves and other associate plant species of the community. The isolates differed with respect to their N₂-fixation efficiency and halotolerance in pure culture. There was no specificity of any of the bacterial isolates to any of the plant species of the community but a higher number of efficient isolates were seen to be associated with mangroves at the swampy succession. Sediment-free tidal water also contained a large population of microaerophilic and anaerobic N₂-fixing bacteria.     

Growth characteristics of non-heterocystous cyanobacterium Plectonema boryanum with N2 as nitrogen source

Strains of filamentous, non-heterocystous cyanobacteria from the Pasteur Culture Collection (PCC), able to synthesize nitrogenase under anaerobic test conditions, were tested for growth with N₂ as sole nitrogen source at low O₂ partial pressure (less than 0.05%). Plectonema boryanum (PCC 73110) exhibited exponential growth under these conditions. This capacity was restricted to light intensities not exceeding 500 lux. Growth rates were 0.014/h at 200 and 0.023 at 500 lux and similar to those of anaerobic and aerobic control cultures with nitrate as N-source. For N₂-fixing cultures incubated at 200 and 500 lux, acetylene reduction rates were 4–8 and 5–14 nmol C₂H₄ per mg protein per min, respectively. The ratio of phycocyanine to chlorophyll was higher (200 lux) or slightly reduced (500 lux) in N₂-fixing cultures as compared to control cultures with nitrate as N-source. On the basis of epifluorescence microscopy and microfluorimetry, no differences in pigment contents were found between individual cells or filaments of N₂-fixing cultures. Also no noteworthy differences were observed between the pycobiliprotein composition of individual cells in N₂ fixing cultures as compared to nitrate-grown controls. Thus the observed exponential growth of P. boryanum at low light intensities implies simultaneous nitrogen fixation and oxygenic photosynthesis. Additional continuous culture experiments showed that N₂-fixing exponential growth was dependent on O₂ partial pressures lower than 0.2–0.4%.The other strains tested (PCC 6412, 6602, 7403, 7104) did not grow under such conditions.Abbreviations Chl chlorophyll - PBP phycobiliproteins - PC phycocyanin - PCC Pasteur Culture Collection - OD optical density