Showing papers on "Nitrogen fixation published in 1969"

Growth and physiology of Azotobacter chroococcum in continuous culture.

Abstract: SUMMARY: Azotobacter chroococcum (ncib 8003) organisms, grown in continuous culture without fixed nitrogen, had chemical compositions at various dilution rates characteristic of nitrogen-limited populations. Fast-growing variants were selected for at high dilution rates; the efficiency of nitrogen fixation decreased with decreasing growth rate. In suitable media, carbon- and phosphate-limited populations were obtained and showed different compositions; they were very sensitive to inhibition by oxygen. Carbon-limited populations utilizing NH4 under argon were not oxygen sensitive; they formed nitrogenase when they were N-limited. The chemical compositions of the various populations corresponded to theory for the nutritional state considered. Nitrogen fixation entrained a maintenance coefficient of 1 -06 g. substrate/g. organism/hr compared with about 0.40 for ammonia assimilation. Assuming most of this maintenance was directed to respiratory protection of nitrogenase, an extrapolated maximum requirement of 4 moles ATP/mole N2 fixed was observed. Attempts to repeat reports of (1) dependence of cytochrome pattern on nitrogen fixation and (2) increased efficiency of fixation with ultraviolet-irradiated N2 were not successful with the strain of A. chroococcum used.

Nitrogen fixation by gloeocapsa.

Abstract: The continuous growth in a medium free of combined nitrogen and the experimental production of ethylene via acetylene reduction indicate that nitrogen fixation by blue-green algae is not solely confined to filamentous genera with heterocysts. Axenic cultures of Gloeocapsa sp., adapted to nitrate-free medium, form ethylene at rates comparable to those of species known to fix nitrogen.

Symbiotic Effectiveness and N2 Fixation in Nodulated Soybean

Abstract: Highly efficient nitrogen fixation by nodulating bacteria is required for maximum production by leguminous plants. The symbiotic relationship between nodulating bacteria and legumes in nodule formation and nitrogen fixation is complex and variable (2). The Rhizobium and legume genotypes (strains and varieties) exert influences which determine the growth response of the host (3,4, 10, 11). The growth response may be poor or moderate to luxuriant, and these symbioses are referred to as ineffective and effective, respectively. Three ranges of effectiveness based on nitrogen content were used by Parker and Allen (12) to compare the growth response of clover inoculated with 35 strains of R. trifolii. Abel and Erdman (1) evaluated the response of field grown Lee soybeans to different strains of R. japonicum and concluded that some symbioses exhibited more effectiveness than others in increasing seed yield, protein percentage of seed, nodulation,, plant appearance and fresh plant weight. Effectiveness in nodulated soybean has not been compared with activity of the nitrogen fixing enzyme. Nitrogen fixing activity ini nodulated leguminous plants can be estimated rapidly and conveniently by the acetylene reduction assay using gas chromatographic techniques (7). Requirements for the reduction of acetylene and molecular nitrogen have been shown to be the same for intact soybean nodules (9) and for partially purified cell-free extracts of bacteroids (8). Using this assay would indirectly determine nitrogenase activity for particular soybeanRhizobium symblioses exhibiting ineffectiveness or effectiveness. The present report provides evidence that the interaction of soybean and R. japonicum in some way controls nitrogenase activity, which results in a particular growth response in the plant.

Failure of putative nitrogen-fixing bacteria to fix nitrogen.

Abstract: SUMMARY: Analyses, tests with isotopic nitrogen and tests for acetylene and isocyanide reduction, using both continuous and batch cultures, were made with seven strains of putative nitrogen-fixing bacteria and three local isolates. Only two (single strains of Mycobacterium flavum and Pseudomonas azotogensis) fixed nitrogen; the active pseudomonad differed in several respects from the organism originally reported. Other Pseudomonas, Nocardia and Azotomonas species and the three local isolates did not fix; some simulated nitrogen fixation in cultural tests most impressively, but proved simply to be very efficient scavengers of traces of fixed nitrogen.

Nitrogen fixation by cultures and cell-free extracts of Mycobacterium flavum 301.

Abstract: SUMMARY: Growth, nitrogen fixation and acetylene reduction by Mycobacterium flavum 301 (ncib 10,071) were increased with sodium lactate, pyruvate, gluconate or succinate as compared with ethanol, a recommended substrate. Yeast extract could be replaced with (NH4)2SO4; in continuous culture a source of fixed nitrogen could be omitted altogether. Growth, nitrogen fixation and acetylene reduction all increased at lowered pO2 values. Wholly anaerobic conditions did not support growth. Nitrogen fixation was confirmed isotopically. Cell-free extracts performed the following reductions: N2 to NH3, H+ to H2, C2H2 to C2H4, KCN to CH4, CH3NC to CH4 + C2H4 + C2H6. An ATP-generating system, Mg2+, Na2S2O4, and anaerobic conditions during preparation and assay of extracts were required. 3·5 mole ATP were hydrolysed to release 1 mole H2. Pyruvate, α-ketobutyrate, α-ketoglutarate, succinate, glucose and glucose-6-phosphate did not replace dithionite. ADP, AMP or high concentrations of ATP inhibited reduction. Activity was associated with a particle which sedimented at 145,000 g over 3·1/2 hr. The nitrogenase system of M. flavum thus resembles the particulate system of Azotobacter, rather than the soluble pyruvate-utilizing system of Clostridium pasteurianum.

Special aspects of nitrogen fixation by blue-green algae.

Abstract: When carbon dioxide fixation was over 90% inhibited by CMU, nitrogen fixation remained unaffected in nitrogen-starved cells of Anabaena cylindrica. In normal cells under the same conditions nitrogen fixation was about 50% inhibited by CMU. These data suggest, first, that nitrogen fixation in this organism is independent of reducing potential generated by non-cyclic photo-electron transport and, secondly, that nitrogen fixation is stimulated by photosynthetically produced carbon skeletons to assimilate the fixed nitrogen. Although nitrogen fixation occurred to a limited extent in the dark, increasing light intensity stimulated nitrogen fixation both in the presence and absence of CMU. This suggests that light-generated ATP is required for nitrogen fixation in this alga. A ratio of pyruvate decarboxylation to nitrogen fixation of 3:1 has been established for A. cylindrica. This accords with the hypothesis that pyruvate acts as a hydrogen donor for nitrogen reduction and that provision of the required reductant is independent of photosynthesis in blue-green algae.

Translocation of c14-labeled photosynthate in nodulated legumes as influenced by nitrate nitrogen'

Abstract: A B S T R A C T Translocation of C14-labeled photosynthate was studied in nodulated pea and subterraneanclover plants which were grown either continuously without combined nitrogen or exposed to NaNO3 in the nutrient solution for five days prior to C1402 assimilation. Supplying combined nitrogen decreased the proportion of photosynthate translocated to nodules with a corresponding increase in the proportion going to roots. Nodules on plants grown without combined nitrogen had a higher radioactivity than nodules on plants treated with sodium nlitrate. IT IS WELL KNOWN that high levels of combined nitrogeni will inhibit nodulation and nitrogen fixation in legumes and non-legumes (Allos and Bartholomew, 1955; Stewart and Bond, 1961; Small, in press). A satisfactory hypothesis to explain the inhibition of nitrogen fixation has not been advanced. A number of aspects, e.g., internal carbohydrate: nitrogen ratio (Wilson, 1940) and hormonal effects (see Stewart, 1966, p. 42), may be involved. The present investigation was carried out to try to ascertain whether combined nitrogen, applied after nitrogen fixation had proceeded, could affect translocation of photosynthate to nodules. This in turn may have an effect on nodule function by influencing substrate available for respiration, growth, and organic nitrogen synthesis.

Nitrogen metabolism in lichens. i. nitrogen fixation in the cephalodia of peltigera aphthosa

Abstract: Nitrogen fixation has been demonstrated to occur in the cephalodia of the lichen Peltigera aphthosa which contain a species of Nostoc. The rate of fixation is very high in comparison with the maximum rates for N. muscorum under optimum culture conditions. Virtually all of the nitrogen fixed is secreted to the lichen thallus at a rate equal to the rate of fixation. The possible control mechanisms of this secretion are discussed.

Regulation of Nitrogen Fixation in Azotobacter vinelandii OP: the Role of Nitrate Reductase

Abstract: A number of chlorate-resistant mutants were selected, and one of these, clr68-5, was studied in detail. This mutant cannot utilize nitrate in vivo to overcome the effect of nonmetabolizable repressors of nitrogenase. The reason for this inability was that strain clr68-5 lacked nitrate reductase. Nitrate inhibited the activity of nitrogenase but did not act as a corepressor of nitrogenase in strain clr68-5 as it does in the wild type. Ammonia seemed to act as corepressor of nitrogenase in both strains.

Progress in the biochemistry of nitrogen fixation.

Abstract: A discussion of progress in the biochemistry of nitrogen fixation is simplified by the thorough reviews which have been given by Professor P. W. Wilson and Professor J. Chatt. Professor Wilson has outlined the history of the subject and has indicated that a great deal of our current knowledge actually was established by the use of intact organisms. Detailed studies of the enzymology of the process were limited by the necessity for using whole cells, but with intact cells the specific inhibitors of nitrogen fixation, the role of ammonia as the key intermediate in nitrogen fixation, the involvement of molecular hydrogen, the physical constants characteristic of nitrogen fixation, the enhancement of the hydrogen exchange reaction by molecular nitrogen, and the demonstration that nitrous oxide can serve as an alternative substrate to N2 all had been established. Little was known about the electron donors in nitrogen fixation, the role of ATP in the process had not been shown, and nothing was known about the composition of the nitrogen fixing enzymes although it was assumed that they contained iron and molybdenum. It was of obvious importance to obtain an active preparation which could fix nitrogen in the absence of intact cells. Much work was directed to this end and instances of positive nitrogen fixation were reported in the literature (Burris 1966). However, the fixation obtained was not very helpful for mechanism studies, because it was inconsistent and poorly controlled. In late 1959 we obtained consistent fixation with cell-free extracts from the blue–green algae (Schneider et al 1960), and at approximately the same time Carnahan, Mortenson, Mower & Castle (1960 a ) obtained good cell-free fixation with extracts from Clostridium pasteurianum . Their preparation was considerably more active and convenient than ours, and so most work with cell-free preparations from blue-green algae was abandoned in favour of work with the anaerobic bacteria. Their process of drying the cells on a rotary vacuum evaporator and supplying very high concentrations of pyruvate as the substrate were critical for achieving active fixation.

Chemical evidence concerning the function of molybdenum in nitrogenase.

Abstract: NITROGENASE is a metalloenzyme containing molybdenum and iron, and both metals are considered to be directly involved in nitrogen fixation. The iron probably picks up the nitrogen molecule (dinitrogen) to form a dinitrogen complex, but the role of molybdenum is uncertain1. We now present a model which indicates the probable function of the molybdenum.

Nitrogen fixation in moulds and yeasts--a reappraisal.

Abstract: The ability to fix nitrogen of 10 strains of the yeasts Rhodotorula, Bullera and Torulopsis and 4 strains of Pullularia, all isolated from soils and some supplied by other investigators was examined using both the heavy nitrogen (15N2) and acetylene reduction techniques. Rigorous standards for aseptic culture, freedom from combined nitrogen and precision of analysis were maintained. No fixation was observed in any of the organisms and the ability of any eucaryote cell to fix nitrogen is doubted. Suggestions for the previous reports of fixation are made.

Nitrogen Fixation in Legume Root Nodules: Biochemical Studies with Soybean

Abstract: It is now clear from studies with soybean root nodules that the nitrogen fixing activity resides in the bacteroids which are the symbiotic form of the root nodule bacteria. These develop as a result of a complex series of changes in metabolism and structure which occur in the bacteria during the final stages of growth within membrane-enclosed vesicles in the host cytoplasm. Nitrogenase appears when these changes are complete. The primary product of nitrogen fixation is NH$\_{3}$, which in intact nodules, is rapidly transformed into $\alpha $-amino compounds which are used by the host plant. In suspensions of bacteroids and in cell-free extracts prepared from them, the reaction terminates in NH$\_{3}$, which is released into the medium. Free O$\_{2}$, which is required for the production of energy for nitrogen fixation by nodules and by bacteroid suspensions, also causes inactivation of the nitrogen fixing system and exerts important kinetic influences upon the reaction. Reducing power and energy for the reduction of N$\_{2}$ to NH$_{3}$ is provided by a photosynthetic product from the host in nodules; in bacteroid suspensions, a substrate such as succinate is required. In cell-free extracts, requirements for energy and reductant are met by ATP and dithionite. The natural reductant has not yet been identified. A schematic representation of various factors which affect nitrogen fixation in nodules, bacteroid suspensions and cell-free extracts is presented.

Nitrogen fixation in some anoxic lacustrine environments.

Abstract: Low rates of acetylene reduction to ethylene in water samples from two dystrophic lakes indicate the presence of nitrogenase and in situ nitrogen fixation. Highest rates were found in anoxic water from the aphotic zone. Environmental conditions in these lakes suggest the agents of fixation were bacteria.

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

Abstract: 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.

The relationship between nitrogen fixation and the production of HD from D2 by cell-free extracts of soya-bean nodule bacteroids.

Abstract: 1. Cell-free extracts prepared from soya-bean nodule bacteroids produced HD from D2 in the presence of dithionite, an ATP-generating system and nitrogen. 2. Crude extracts of bacteroids or of Azotobacter vinelandii showed some background D2 exchange when any one of these was omitted. 3. Partial purification of bacteroid extracts diminished this background activity and gave increased D2 exchange and nitrogen fixation. 4. Although increasing pN2 stimulated both reactions, the apparent Km (N2) for nitrogen fixation was much higher than the apparent Km (N2) for D2 exchange when partially purified bacteroid extracts were used. 5. Carbon monoxide was a competitive inhibitor of nitrogen fixation by partially purified bacteroid extracts, but D2 exchange was inhibited in a non-competitive fashion. 6. These results are discussed in relation to the possible existence of enzyme-bound intermediates of nitrogen fixation.

Mycorrhizal Nodules and Growth of Podocarpus in Nitrogen-poor Soil

Abstract: MOST genera with nodulated roots are legumes, and the nodules form after infection by nitrogen-fixing bacteria. Thirteen other genera of dicotyledons may also develop root nodules, which form only after infection, and in which nitrogen fixation has been proved or inferred, but in these the endophytes seem to be actinomycetes1,2. Among the gymnosperms, however, in Araucariaceae and Podocarpaceae, nodules develop without any stimulus from microorganisms3 although in nature they usually contain a coarse phycomycetous mycelium. This infection has been synthesized using Endogone spores4 and, like typical Endogone mycorrhizas, infected nodules sustain the growth of seedlings in phosphorus-deficient soils5. There is also a long standing belief that they fix nitrogen, supported recently by some experiments with 15 N (ref. 1). I have tested this hypothesis by attempting to grow seedlings of Podocarpus totara in a nitrogen-deficient soil.

Nitrogen Complexes as Models for Nitrogenase with Nitrogen on the Active Site

Abstract: NITROGEN gas reacts in mild conditions in protic media to give the nitrogen complexes [CoH(N2)(PPh3)3]1, and [Ru(NH3)5(N2)]X2 (X = mono anion)2, and related complexes, but the complexed nitrogen molecule has not yet been reduced to ammonia3. Nevertheless, the mechanism of nitrogen uptake in the formation of these complexes may represent the process by which nitrogen is bound in nitrogenase, and the reduction to ammonia could occur in nature by protonation of the complexed nitrogen with simultaneous influx of electrons from the metal. We have therefore studied quantitatively the reactions with strong acids of [CoH(N2)(PPh3)3]1, the substance formulated as [Co(N2)(PPh3)3]4, trans-[IrCl(N2)(PPh3)2]5, and [Ru(NH3)5-(N2)]X2 (X = Cl or BF4)6,7. If protonation occurred, some reduction of the nitrogen with simultaneous oxidation of the metal would be expected. We have found that in general there is oxidation of the metal by the acid, but it leads to nitrogen evolution, often together with hydrogen, and no ammonia.

Physical Environment and Symbiotic Nitrogen Fixation VI. Nitrogen Retention Within the Nodules of Trifolium Subterraneum L

Abstract: The effect of bacterial strain and root temperature on the retention of nitrogen in the root system of Trifolium Bubterraneum plants was re-examined. The root systems of plants nodulated by the moderately effective Rhizobium trifolii strain NA30 possessed a higher percentage nitrogen than those nodulated by the fully effective strain TAl, although the number of nodules formed by each strain was similar. The difference was due to a greater weight of nodule tissue on the NA30-nodulated plants, and also to a higher percentage nitrogen in the NA30 nodules; this latter effect was due to a higher concentration of non-protein nitrogen. The overall effect of these differences was to reduce the amount of nitrogen translocated to the shoots of the NA30 plants, in both absolute terms and as a proportion of the total amount of nitrogen fixed. Another difference between the two strains was the rate of nitrogen fixation per unit (dry weight or leghaemoglobin content) of nodule tissue.

Seasonal changes of photosynthetic bacteria and their products

Abstract: In our preceding papers the distribution (1, 2), ecological problems (3,4), and the products of photosynthetic bacteria (5–8) have been studied. It was also reported that the growth and nitrogen fixation of photosynthetic bacteria were accelerated remarkably in the system of symbiosis with other heterotrophic microorganisms like Azotobacter (9) and Bacillus megaterium (10) ; moreover the products generated by photosynthetic bacteria were used by plants (11) and animals in water (2, 4), directly or indirectly. It is noteworthy that in paddy soil, this bacterial cells have a beneficial effect on the development of grains (11).

Method of fixing nitrogen in the atmosphere and the soil

Abstract: METHOD AND APPARATUS FOR PRODUCING HEAT IN THE FORM OF AN ARC OF ELECTRICAL ENERGY IN THE PRESENCE OF ATMOSPHERE TO REPRODUCE THE PROCESS OF FIXATION OF NITROGEN IN THE ATMOSPHERE AND IN THE GROUND AND CONVERT ATMOSPHERIC NITROGEN TO NITRATE IN A FORM USABLE BY PLANTS TO ENCOURAGE GROWTH.

Physical Environment and Symbiotic Nitrogen Fixation VII. Effect of Fluctuating Root Temperature on Nitrogen Fixation

Abstract: The effect of exposing nodulated plants to daily periods of high, moderate, or low root temperatures was examined, using Trifolium 8ubterraneum and three strains of Rhizobium trifolii. With strains whose nitrogen fixation was severely retarded by continuous exposure to high root temperatures, the results from treatments involving exposure of 4, 8, 12, and 20 hr/day to 30°C and continuous illumination were consistent with the effect being on the rate of nitrogen fixation, without any permanent impairment to the symbiotic system. With a 12 hr/day light period, a daily 12-hr exposure to 30°C during the dark period reduced total nitrogen fixation as much as exposure to 30°C during the light period. This indicated that the rate of nitrogen fixation during normal dark periods could be as high as that during periods of illumination. Similar conclusions were drawn from the same type of experiments involving daily exposure to moderate (14 and 16°C) root temperatures.

The measurement of gains and losses of nitrogen in grazed pastures

Abstract: In a study of methods of measuring gains and losses o£ nitrogen in grazed pastures, the following aspects were investigated: (1) methods of measuring net nitrogen changes in large plots of grazed pastures by direct sampling, (2) the use of 15N as a tracer to estimate legume nitrogen fixation, and (3) the use of 15N to measure losses of nitrogen under field conditions (1) In grazed pasture plots 3 to 6 ac in area, there were large differences between pastures in the number of soil or plant samples needs for a chosen level of precision Using stratified random sampling, the estimated number of 18 in diameter soil cores needed for a least significant difference of 50 lb N/ac in the 0-6 in soil layer varied from 190 in a setaria pasture on a yellow podzolic soil to 1070 in a green panic pasture on a sedentary clay soil The number of 5 x 1 lk2 quadrats needed for a least significant difference of 10 lb N/ac in the plant component (cut at ground level) varied from 10 in a Townsville lucerne/spear grass pasture to 154 in a green panic pasture The number of soil or plant samples needed was not closely related to the nitrogen content of the respective components Statistically, the most efficient method of measuring nitrogen changes was to subdivide the plots into strata and repeatedly sample the same set of sampling sites within these strata, taking one sample per site on each occasion Calculations based on estimates of costs and variance components for different stages of sampling showed that the above method was also the most economical The precision of measurements of net nitrogen changes in two continuously grazed pastures was similar to that predicted from the sampling studies In one pasture, a fertilized Townsville lucerne/spear grass pasture at Rodd's Bay, Queensland, the soil/plant/animal system lost 55 lh N/ac (SE ± 18) during 2½ years in spite of an estimated addition of at least 18 lb N/ac by Townsville lucerne In the other pasture, a fertilized Siratro/grass pasture at Samford, Queensland, there was no change in the nitrogen content of the soil/plant/animal system (SE ± 17 lb N/ac) despite an estimated addition of approximately 45 lb N/ac by Siratro, It was thought that a large part of the compensating losses of nitrogen was due to transfer of nitrogen by cattle to camping areas in the pastures and volatilization of nitrogen from dung and urine (2) The 15N technique appeared to work quite well for the separation of uptake of soil nitrogen and symbiotic nitrogen fixation by legumes grown in pots, though the basic assumption that the legume and grass take up soil nitrogen and labelled fertilizer nitrogen in the same ratio has not yet been tested The partition of 15N uptake between grass and legume in microplots within large plots (several acres) of pasture was affected by the proportion of legume (on a dry matter basis) in the mixtures The partition of 15N uptake indicated that in pastures containing up to 20 per cent Townsville lucerne or up to 60 per cent Siratro, the legume obtained less than 20 per cent of the available soil nitrogen (3) Studies of the recovery of 15N from microplots in a pasture indicated that reliable measurements of 15N recovery in field experiments can be made for a reasonable cost of 15N and without excavating the entire soil mass from the microplots There was no evidence that the core sampling method gave biased results The method of using core sampling to measure recovery in unconfined microplots was discussed in detail Under conditions considered unfavourable for leaching or denitrification, 235 per cent (SE ±37) of the applied 15NO3-N was apparently lost from the soil/plant system (0-12 in soil depth) after ten months

Nitrogen Fixation and Availability

Abstract: D escribed here is an experiment in symbiotic plant growth which can be set up and maintained with a minimum of equipment The experiment is designed to show that wheat plants grown in a nitrogen deficient medium can obtain an adequate nitrogen supply from soybean plants whose roots were infected with the bacterium Rhizobium, a symbiotic nitrogen fixing bacteria The plants can be grown from seed and within 26 days the results are striking Wheat plants grown in a nitrogen deficient medium do not survive whereas wheat plants grown with soybean plants innoculated with Rhizobium look healthy and vigor-