BIOLOGICAL NITROGEN INFLUX IN AN OHIO RELICT PRAIRIE

The principal contributors of biologically fixed N in natural grassland ecosystems appear to be asymbiotic bacteria and heterocystous cyanobacteria. The environmental factors of light, moisture, and temperature play important roles in the magnitude of the N₂-fixation activity. Biological N₂-fixation was measured in the Elizabeth's Prairie section of the Lynx Prairie Preserve, Adams County, Ohio, during 15 site visits beginning 29 March through 8 November 1980. In situ N₂-fixation activity was measured using the acetylene-reduction technique. The percentage cover of cyanobacterial colonies (Nostoc sp.) was determined using Point-Frame Analysis. Soil and air temperatures and soil water potentials also were measured. Intact soil cores with a surface cover of Nostoc were collected and returned to the laboratory to quantify the effect of decreasing water potential on the N₂(C₂H₂)ase activity of Nostoc. The N₂(C₂H₂)ase activity of Nostoc on the intact soil cores displayed a linear response of approximately 10% decrease in N₂(C₂H₂)ase activity per one bar decrease in soil water potential. The cyanobacteria contributed almost all of the biologically fixed N at the site until late June. From late June through to mid September, heterotrophic diazotrophs played the major role in the N₂-fixation activity. These changes are attributed to fluctuations in Nostoc sp. colony cover, temperature, and soil water potentials. Extrapolation of the measured rates, and assuming an average of 10 hr per day of activity, Nostoc sp. is shown to have contributed 4.60 ± 1.17 kg N ha⁻¹ yr⁻¹. Heterotrophic diazotrophs contributed an estimated 3.19 ± 1.18 kg N ha⁻¹ yr⁻¹. The total biological N₂-fixation for the site was calculated at 8.2 ± 2.55 kg N ha⁻¹ yr⁻¹, from additional measurements which estimated total diazotrophic activity of the site. These rates of N₂-fixation are among the highest reported for temperate grassland habitats.     

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.     

Biological nitrogen fixation: A key to world protein

Summary 1. Characteristics and methodology of the C₂H₂-C₂H₄ assay forin situ measurement of N₂ fixation are outlined. 2. Electron micrographic analysis of the developmental morphology of the natural soybean symbiosis and C₂H₂-C₂H₄ analysis indicate that increasing N₂-fixing activity from 12–35 days of age is accompanied by an increase in bacteroid number per cell, bacteroid number per vesicle and inclusions per bacteroid. The mole ratio of leghemoglobin to nitrogenase also increases from 50 to a relatively constant plateau of 500 to 1500 during this period. The quantitative validity of the C₂H₂-C₂H₄ assay as a measure of N₂ fixation during a complete growth cycle of soybeans on nitrogen-free medium is demonstrated by Σ (C₂H₂→C₂H₄)×28/3 values which are 75–95% of the values determined for N₂ fixed by Kjeldahl analyses. 3. A technique for the establishment of the first callus N₂-fixing symbiosis in mixed cultures ofRhizobium legume provides a defined experimental system for exploration of legume symbiosis. N₂-fixing activity is about 1% of the natural system and is influenced by exogenous auxins and cytokinins. Morphology, including infection threads and vesicle enclosed bacteroids, is similar to the nodule system. 4. N₂-fixing activity of field-grown soybeans, including varieties which differed in flowering characteristics and maturity dates, and of peanuts was determined biweekly with the C₂H₂-C₂H₄ assay. Activity extended from nodule initiation to senescence and correlated with the nitrogen demands of the plant and in most cases >90% of the N₂ fixed during the 60–70 day period of fruit formation and maturation. A logarithmic relationship between N₂-fixing activity and age, and N₂ fixed and age was demonstrated as a fundamental characteristic of these annual symbionts,i.e. log N₂ fixed =k(t−t ₀), wheret ₀ is age at activity initiation. The resultant parameters: 1) age at activity initiation, 2) calculated rate of daily increase (7–9% for soybeans and 7–10% for peanuts), 3) age at end of logarithmic phase (about 80 days for soybeans), and 4) total N₂ fixed (about 250 mg per soybean plant) are useful bases for evaluation of environmental, bacterial and host effects on N₂ fixation. Various N fertilizers applied at planting and flowering inhibited N₂ fixation of soybeans by decreasing the rate of daily increase. 5. Physical and chemical characteristics of nitrogenase, including those of crystalline Mo-Fe protein, reactions of nitrogenase, and model studies are consistent with a proposed mechanism. 6. Potential utilities of N₂ fixation research include increased food protein production via initially enhanced N₂ fixation of legumes such as soybeans and eventually extension of N₂-fixing symbioses to non-legumes and new chemistry of N₂, including the direct incorporation of aerial N₂ into important organic compounds. Contribution No. 1748.     

PHOTOAUTOTROPHIC GROWTH OF A RECENTLY ISOLATED N2-FIXING MARINE NON-HETEROCYSTOUS FILAMENTOUS CYANOBACTERIUM, SYMPLOCA SP. (CYANOBACTERIA)

Photoautotrophic growth of a marine non-heterocystous filamentous cyanobacterium, Symploca sp. strain S84, was examined under nitrate-assimilating and N₂-fixing conditions. Under continuous light, photon flux density of 55 μmol photons·m⁻² ·s⁻¹ was at a saturating level for growth, and light did not inhibit the growth rate under N₂-fixing conditions even when the photon flux density was doubled (110 μmol photons·m⁻² ·s⁻¹). Doubling times of the N₂-fixing cultures under 55 and 110 μmol photons·m⁻² ·s⁻¹ were about 30 and 31 h, respectively. Under 110 μmol photons·m⁻² ·s⁻¹ during the light phase of an alternating 12:12-h light:dark (L:D) cycle, the doubling time of the N₂-fixing culture was also about 30 h. When grown diazotrophically under a 12:12-h L:D regime, C₂H₂ reduction activity was observed mainly during darkness. In continuous light, relatively large cyclic fluctuations in C₂H₂ reduction were observed during growth. The short-term (<4 h) effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; 5 μM) indicated that C₂H₂ reduction activity was not influenced by photosynthetic O₂ evolution. Long-term (24 h) effects of DCMU indicated that photosynthesis and C₂H₂ reduction activity occur simultaneously. These results indicate that strain S84 grows well under diazotrophic conditions when saturating light is supplied either continuously or under a 12:12-h L:D diel light regime.     

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.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C₂H₂ at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C₂H₂ at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N₂ fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C₂H₂ than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N₂-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them. Only Bacillus strain C-11-25 has potential to benefit both temperate and tropical crops because it reduced significant acetylene over a wide temperature range.     

Growth, denitrification and nitrate ammonification of the rhizobial strain TNAU 14 in presence and absence of C2H4 and C2H2

Soil-N (NO₃ ^?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N₂-fixation. Organic N and ammonium oxidised to NO₃ ^? means oxygen depletion. Plants suffering under O₂ or infection stress start to excrete ethylene (C₂H₄). C₂H₄ widens the root intercellulars that O₂-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C₂H₄. Surplus nitrate and C₂H₄ inhibit nodulation of leguminous plants. Excess NO₃ ^? in the nodulesphere could be diminished by N₂-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C₂H₄ interferes with the potential of N₂-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO₃-mannitol-medium that was incubated under N₂-, 1% (v/v) N₂?C₂H₄-, and 1% (v/v) N₂?C₂H₂-atmosphere in the laboratory. C₂H₂ was included into the experiments because it is frequently used to quantify N₂-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 toRhizobium radiobacter andRhizobium tumefaciens. Strain TNAU 14 released N₂O into the bottle headspace in all treatments, surprisingly significantly less in presence of C₂H₂. Nitrate-ammonification was even completely blocked by C₂H₂. C₂H₄, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH₄ ⁺ during continuing incubation.     

The Absorption and Fluorescence Spectra of the Cyanobacterial Phycobionts of Cryptoendolithic Lichens in the High-Polar Regions of Antarctica

The algologically pure cultures of the green–brown cyanobacterium Chroococcidiopsissp. and three cyanobacteria of the genus Gloeocapsa, the blue–green Gloeocapsa sp.₁, the brown Gloeocapsa sp.₂, and the red–orange Gloeocapsa sp.₃, were isolated from sandstones and rock fissures in the high-polar regions of Antarctica. These cyanobacteria are the most widespread phycobionts of cryptoendolithic lichens in these regions. The comparative analysis of the absorption and the second-derivative absorption spectra of the cyanobacteria revealed considerable differences in the content of chlorophyll a and in the content and composition of carotenoids and phycobiliproteins. In addition to phycocyanin, allophycocyanin, and allophycocyanin B, which were present in all of the cyanobacteria studied, Gloeocapsa sp.₂ also contained phycoerythrocyanin and Gloeocapsa sp.₃ phycoerythrocyanin and C-phycoerythrin (the latter pigment is typical of nitrogen-fixing cyanobacteria). The fluorescence spectra of Gloeocapsa sp.₂ and Gloeocapsa sp.₃ considerably differed from the fluorescence spectra of the other cyanobacteria as well. The data obtained suggest that various zones of the lichens may be dominated either by photoheterotrophic or photoautotrophic cyanobacterial phycobionts, which differ in the content and composition of photosynthetic pigments.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

Effects of inorganic nitrogen on C2H2 reduction and CO2 exchange in the Peltigera praetextata-Nostoc and Peltigera aphthosa-Coccomyxa-Nostoc symbioses

Uptake of NH ₄ ⁺ and NO ₃ ^- by the N₂-fixing lichens Peltigera praetextata (two-component lichen) and P. aphthosa (three-component lichen) was studied. In addition, the effects of these ions, separately and in combination, on C₂H₂ reduction and CO₂ exchange were examined. Both NH ₄ ⁺ and NO ₃ ^- were utilized by the lichens. NH₄NO₃ caused an increased liberation of NO ₃ ^- from the lichens as compared to the release observed in untreated lichen thalli. NH ₄ ⁺ and NO ₃ ^- led to reduced C₂H₂ reduction by P. praetextata, which, however, was less pronounced than when the two ions were given in combination. In P. aphthosa the C₂H₂ reduction was inhibited by NH ₄ ⁺ and NH₄NO₃, but not by NO ₃ ^- alone. NH ₄ ⁺ and NO ₃ ^- had no effect on the net photosynthesis of P. praetextata, while, in combination, they led to inhibition, although only at a concentration higher than that inhibitory to the C₂H₂ reduction of P. aphthosa. The photsynthesis was inhibited by all salts, but only initially, probably a salt effect. Effects of NH ₄ ⁺ on the membrane potential of the cyanobiont are suggested as an important factor causing the depression of net photosynthesis.     

Effect of Bentazon, a Hill Reaction Inhibitor, on Symbiotic Nitrogen-fixing Capability and Apparent Photosynthesis

Symbiotic associations of bean plants (Phaseolus vulgaris L. cv. Blue Lake) and Rhizobium phaseoli strain 127K17 were treated with the Hill reaction inhibitor bentazon (3-isopropyl-1 H-2,1,3-benzothiadiazin-4-(3H)-one-2,2-dioxide). Plants receiving foliar and root treatments of 1.8 kilograms per hectare bentazon were assayed at 6 hour intervals for N₂-fixing capacity by measuring C₂H₂-dependent C₂H₄ production and H₂ evolution and for CO₂ exchange rates. In foliar treated plants greatest measured inhibition of CO₂ exchange rates and N₂-fixing capacity occurred 6 and 12 hours after treatment, respectively. In root-treated plants maximum inhibition of both processes was delayed by 6 hours, and was less severe than in foliar treated plants. Nitrogen-fixing capacity and CO₂ exchange rate recovered to control levels in all plants. Application of higher rates of bentazon resulted in greater inhibition of CO₂ exchange rate and N₂-fixing capacity. Inhibition of the two processes was positively correlated (r = 0.985). The results indicate that inhibition of N₂-fixing capacity was not caused by bentazon directly, but indirectly through limiting the availability of photosynthate to support root nodule activity.     

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     

Relationship between abundance of N2-fixing cyanobacteria and environmental features of Spanish rice fields

In order to estimate the potential utilization of N₂-fixing (heterocystous) cyanobacteria as natural biofertilizers in the Valencian rice fields (Spain), the distribution and seasonal variation of these microorganisms in water and sediment samples were evaluated, and the relationships among cyanobacterial abundance and physical and chemical characteristics of soil and water were investigated. N₂-fixing cyanobacteria were present in all the samples analyzed (25 sampling points sampled three times per year during two years). The relative cyanobacterial abundance in soil and water followed contrasting patterns, maximum presence in soil coincided with minimum abundance in water. Correlation analysis showed that cyanobacterial abundance in the two phases (water and sediment) was influenced more by water than by soil properties. Salinity, mineralization variables, and soluble reactive phosphate (SRP) correlated positively with heterocystous cyanobacteria presence. Furthermore, dissolved inorganic nitrogen (DIN) and the ratio DIN: SRP correlated negatively with cyanobacterial abundance. However DIN: SRP ratio better described the cyanobacterial distribution, with a threshold effect: below the Redfield ratio value (7.2 in mass units) cyanobacterial abundance was clearly higher. Correspondence to: A. Quesada.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

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).     

Enumeration of Free-Living Aerobic N2-Fixing H2-Oxidizing Bacteria by Using a Heterotrophic Semisolid Medium and Most-Probable-Number Technique

A heterotrophic semisolid medium was used with two sensitive assay methods, C₂H₂ reduction and O₂-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C₂H₂, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N₂-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N₂-fixing and H₂-oxidizing bacteria in some natural systems. The numbers of H₂-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H₂ transformation.     

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     

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.

     

Nitrogen fixation in lichens is important for improved rock weathering

It is known that cyanobacteria in cyanolichens fix nitrogen for their nutrition. However, specific uses of the fixed nitrogen have not been examined. The present study shows experimentally that a mutualistic interaction between a heterotrophic N₂ fixer and lichen fungi in the presence of a carbon source can contribute to enhanced release of organic acids, leading to improved solubilization of the mineral substrate. Three lichen fungi were isolated fromXanthoparmelia mexicana, a foliose lichen, and they were cultured separately or with a heterotrophic N₂ fixer in nutrient broth media in the presence of a mineral substrate. Cells of the N₂-fixing bacteria attached to the mycelial mats of all fungi, forming biofilms. All biofilms showed higher solubilizations of the substrate than cultures of their fungi alone. This finding has bearing on the significance of the origin and existence of N₂-fixing activity in the evolution of lichen symbiosis. Further, our results may explain why there are N₂-fixing photobionts even in the presence of non-fixing photobionts (green algae) in some remarkable lichens such asPlacopsis gelida. Our study sheds doubt on the idea that the establishment of terrestrial eukaryotes was possible only through the association between a fungus and a phototroph.