Nitrogen Fixation in Continuous Culture with NH4Cl-Containing Media

N balance and ¹⁵N dilution were determined from growth of Azospirillum brasilense Sp7 and two unidentified gram-negative nitrogen-fixing microorganisms in continuous culture supplied with NH₄Cl. At the 1.1 and 2.2 mM NH₄Cl steady states (N-to-C ratios of 1:68 and 1:34, respectively), the organisms grew with NH₄Cl and N₂ as N sources simultaneously under carbon limitation. No ammonium could be detected in the supernatant of these cultures.     

Nitrogen Fixation by Rhodospirillum rubrum Grown in Nitrogen-limited Continuous Culture

Cell-free extracts of the photosynthetic bacterium Rhodospirillum rubrum were inconsistent in reducing N(2). An internally illuminated fermentor, designed for the continuous culture of this organism on N(2) under nitrogen-limited conditions, produced cells which yielded cell extracts with consistent activity for cell-free N(2) fixation. A nitrogen-limited continuous culture, supplied ammonia rather than N(2), gave cell-free extracts with even more active N(2) fixation. Extracts of cells grown in the fermentor with glutamate nitrogen as the limiting nutrient in continuous culture did not reduce N(2), but whole cells fixed (15)N-enriched N(2). The discovery that cells from ammonia and glutamate nitrogen-limited continuous cultures are capable of N(2) reduction suggests that R. rubrum cells produce the N(2)-reducing enzymes in response to conditions of nitrogen deficiency rather than in response to the presence of N(2). Examination of the effect of the pN(2) on N(2) reduction by cell-free preparations of R. rubrum indicated that the K(N(2)) is approximately 0.071 atm. Cell-free extracts from R. rubrum were tested for their ability to reduce substrates other than N(2).     

Nitrogen use efficiency (NUE) in rice links to NH4 + toxicity and futile NH4 + cycling in roots

Aims

Rice is known as an ammonium (NH₄ ⁺)-tolerant species. Nevertheless, rice can suffer NH₄ ⁺ toxicity, and excessive use of nitrogen (N) fertilizer has raised NH₄ ⁺ in many paddy soils to levels that reduce vegetative biomass and yield. Examining whether thresholds of NH₄ ⁺ toxicity in rice are related to nitrogen-use efficiency (NUE) is the aim of this study.

Methods

A high-NUE (Wuyunjing 23, W23) and a low-NUE (Guidan 4, GD) rice cultivar were cultivated hydroponically, and growth, root morphology, total N and NH₄ ⁺ concentration, root oxygen consumption, and transmembrane NH₄ ⁺ fluxes in the root meristem and elongation zones were determined.

Results

We show that W23 possesses greater capacity to resist NH₄ ⁺ toxicity, while GD is more susceptible. We furthermore show that tissue NH₄ ⁺ accumulation and futile NH₄ ⁺ cycling across the root-cell plasma membrane, previously linked to inhibited plant development under elevated NH₄ ⁺, are more pronounced in GD. NH₄ ⁺ efflux in the root elongation zone, measured by SIET, was nearly sevenfold greater in GD than in W23, and this was coupled to strongly stimulated root respiration. In both cultivars, root growth was affected more severely by high NH₄ ⁺ than shoot growth. High NH₄ ⁺ mainly inhibited the development of total root length and root area, while the formation of lateral roots was unaffected.

Conclusions

It is concluded that the larger degree of seedling growth inhibition in low- vs. high-NUE rice genotypes is associated with significantly enhanced NH₄ ⁺ cycling and tissue accumulation in the elongation zone of the root.     

pH、温度、NH4+和盐度对圆果雀稗内生固氮菌固氮特性的影响

采用乙炔还原法对从圆果雀稗(Paspalum orbiculare)根、茎中分离到的5株内生固氮菌(POJy11、POG12、POGy22、POGy612、POG4)的固氮特性进行了研究。结果表明,POGy612、POG4固氮的pH适应范围是6.0-10.0,其余3株菌为6.0-8.0;POGy22固氮的最适温度为35℃,其余4株菌为30℃;随着NH4 浓度和NaCl浓度的升高,菌株固氮活性总体呈下降趋势,其中POGy22的固氮活性受NH4 抑制最明显,NH4 浓度为1.0 mmol/L时,已无固氮活性,但POGy612在NH4 浓度为2.5 mmol/L时的固氮活性最高,达到1 045.0 nmol ml-1h-1,POJy11、POGy22在NaCl浓度为0.5%时的固氮活性最高,分别达到735.5 nmol ml-1h-1、703.0 nmol ml-1h-1。     

Continuous Culture Used for Media Optimization

A technique is described in which continuous culture is used for the optimization of media in terms of growth-supporting ability. The technique consists of identifying growth-limiting nutrients by observing the reaction of the continuous system to injection of suspected growth-limiting nutrients into the growth vessel.     

Nitrogen Fixation by Photosynthetic Bacteria in Lowland Rice Culture

Propanil (3′,4′-dichloropropionanilide) was a potent inhibitor of the nitrogenase activity of blue-green algae (cyanobacteria) in flooded soil, but the herbicide at comparable concentrations was not toxic to rice, protozoa, and nitrogen-fixing bacteria. Ethanol-amended flooded soils treated with propanil exhibited higher rates of nitrogenase activity than those not treated with the herbicide. The enhanced nitrogenase activity in propanil-treated soils was associated with a rise in the population of purple sulfur bacteria, especially of cells resembling Chromatium and Thiospirillum. By employing propanil and a means of excluding light from the floodwater to prevent the development of phototrophs during rice growth under lowland conditions, the relative activities of blue-green algae, photosynthetic bacteria, and the rhizosphere microflora were determined. The results suggest that the potential contribution of photosynthetic bacteria may be quite high.     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

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 Fixation (Acetylene Reduction) by Epiphytes of Freshwater Macrophytes

The involvement of epiphytic microorganisms in nitrogen fixation was investigated in a shallow freshwater pond near Ithaca, N.Y. The acetylene reduction technique was used to follow diel and seasonal cycles of nitrogen fixation by epiphytes of Myriophyllum spicatum. Acetylene-reducing activity was maximal between noon and 6 p.m., but substantial levels of activity relative to daytime rates continued through the night. Experiments with the seasonal course of activity showed a gradual decline during the autumn months and no activity in January or February. Activity commenced in May, with an abrupt increase to levels between 0.45 and 0.95 nmol of ethylene formed per mg (dry weight) of plant per h. Through most of the summer months, mean rates of acetylene reduction remained between 0.15 and 0.60 nmol/mg (dry weight) per h. It was calculated from diel and seasonal cycles that, in the pond areas studied, epiphytes were capable of adding from 7.5 to 12.5 μg of N per mg of plant per year to the pond. This amount is significant relative to the total amount of nitrogen incorporated into the plant. Blue-green algae (cyanobacteria), particularly Gloeotrichia, appeared to bear prime responsibility for nitrogen fixation, but photosynthetic bacteria of the genus Rhodopseudomonas were isolated from M. spicatum and shown to support high rates of acetylene reduction.     

Nitrogen Fixation (Acetylene Reduction) in a Salt Marsh Amended with Sewage Sludge and Organic Carbon and Nitrogen Compounds

Seasonal distribution of nitrogen fixation by Spartina alterniflora epiphytes and in surface and soil samples was investigated in a Georgia salt marsh which was amended with sewage sludge or with glucose and/or ammonium nitrate. There was no significant difference between the rates of fixation in the unamended and sewage sludge plots. Additional perturbation experiments suggested that nitrogen addition indirectly stimulates nitrogen fixation by enhancing Spartina production and root exudation. Glucose additions, on the other hand, suppressed nitrogen fixation on a long-term basis. It is suggested that the microbial population in the soil out-competed the plants for the available nitrogen and in turn suppressed plant production and possibly root exudation. A comparison of nitrogen fixation in clipped and unclipped Spartina plots substantiated the suggestion that root exudation probably supports nitrogen fixation. Fixation in the clipped plots was significantly lower (P < 0.05) than the rates in the unclipped plots.     

Nitrogen Fixation Associated with Sand Grain Root Sheaths (Rhizosheaths) of Certain Xeric Grasses

Nitrogen fixation (acetylene reduction) was found to be associated with sand grain root sheaths (rhizoseaths) occurring on the following xeric grasses: Oryzopsis hymenoides (Roem. and Shult.) Ricker, Agropyron dasystachyum (Hook.) Scrib., Stipa comata Trin. and Rupr., and Aristida purpurea Nutt. Acetylene reduction rates associated with whole plant specimens of these species varied from 515 to 920 nmol C₂H₄/(g dry wt.) × (6 days). Nitrogenase activity was shown to be associated with the rhizosheaths. Bacillus polymyxa-like nitrogen fixers were isolated from the rhizosheaths of each grass. The isolates reduced acetylene and assimilated ¹⁵N₂.     

Nitrogen Fixation in Wild Legumes

Isolations of the rhizobia from nodules of field plants of Ulexeuropaeus and Medicago lupulina have been examined for effectivenessin fixation of nitrogen by inoculating them into host plantsgrowing in a medium free of combined nitrogen. The results indicatethat vigorous fixation may be presumed to occur in associationwith field plants of the Ulex species, but that fixation inMedicago is probably decreased by the presence of ineffectiverhizobia in some nodules. The general importance of fixationin wild legumes is discussed, and it is pointed out that dueattention must be paid to non-legume genera with root nodules.These genera are locally abundant, and in former periods werestill more prominent in some regions of the world.     

Nitrogen balance sheet of (NH4)2SO4-gypsum pellets and mixtures and ureaformaldehyde applied to corn in pots of sand

Summary A comparison of ammonium sulphate added to sand pots in different ways and ureaformaldehyde as sources of N to corn plants was carried out. The results showed that nitrogen utilization by plants from ammonium sulphategypsum pellets was greater than its utilization when ammonium sulphate was mixed with gypsum or when the pellets were ground or from ureaformaldehyde. The leached nitrogen from the pellets, ammonium sulphate applied in 3 portions and ureaformaldehyde was not significantly different and was lower than other ammonium sulphate treatments. The nitrogen remaining in pots fertilized by ureaformaldehyde was much greater than the corresponding amount in the case of all ammonium sulphate treatments. Gaseous loss of nitrogen took place in all nitrogen treatments with the loss from ammonium sulphate-gypsum pellets being the lowest.Incubation in sand of ureaformaldehyde, urea, and ammonium sulphate was carried out to understand better the growth conditions of corn fertilized by ureaformaldehyde. In the case of ureaformaldehyde- and urea-sand systems, the pH increased, NO₂ ^– accumulated and considerable loss of nitrogen took place. The pH, the NO₂ ^– accumulation and the loss of N tended to decrease with gypsum increments. re]19720801     

Biological Nitrogen Fixation in Pachyrhizus ahipa (Wedd.) Parodi

The patterns of plant growth and N₂ fixation capability in Pachyrhizusahipa (Wedd) Parodi inoculated with Bradyrhizobium ‘PachyrhizusSpec 1’ strains (Lipha Tech) were investigated in a zero-Nculture system under greenhouse conditions The P ahipa plantis day-neutral with respect to reproductive development Competitionoccurred between the two storage organs (legume and tuber) andprevented high tuber yield in P ahipa The symbiotic effectivenessof the association was high, as the profuse nodulation providedthe inoculated plants with adequate amounts of N Nodules werepresent throughout the cycle of P ahipa The change in rate ofN₂ fixation (RNF) and relative growth rate (RGR) was almostparallel during ontogenesis The developmental pattern of N₂fixation activity revealed that 65% of total N₂ fixation occurredafter N began to accumulate in the reproductive (pod wall plusseed) tissue During pod filling allocation of N compounds tothe seeds exceeded N₂ fixation, the pod walls being the primarysource of redistributed N, followed by the leaves. Pachyrhizus ahipa (Wedd) Parodi, ahipa, tuber crop, dinitrogen fixation, dry matter, N partitioning, reproductive growth