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Influence of sewage discharge on nitrogen fixation and nitrogen flux from coral reefs in Kaneohe Bay, Hawaii.

Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N2 were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats.

Acetylene

Kinetic model of a determinate legume root nodule reveals plant metabolic characteristics for more efficient nitrogen fixation symbiosis.

While nitrogen fertilizers are widely used in agricultural production, their application incurs significant environmental and energetic costs. In contrast, some crops are less dependent on these fertilizers because they engage in symbioses with rhizobia, nitrogen-fixing bacteria that provide ammonium to the plant in exchange for carbon. However, the carbon cost associated with nitrogen fixation can negatively impact crop yields. Improving the efficiency of this metabolic process could alleviate this impact on crop productivity. Mathematical models can help us quantitatively explore metabolic behavior and identify potential targets for metabolic engineering. In this work, we developed a kinetic model of determinate root nodule metabolism, where this symbiotic exchange of carbon from the plant and nitrogen from the bacteria occurs. We used this model to evaluate how the predicted metabolic behavior differs between inefficient and efficient nodules, and to identify potential engineering targets for improving nitrogen fixation efficiency and rate. We show that the enzymes phosphoenolpyruvate carboxylase and pyruvate kinase have significant influence on the predicted rate and efficiency of nitrogen fixation, especially when their expression is varied in combination with oxidative Pentose Phosphate Pathway enzymes like glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. The model predicts that pairing a 3-fold decrease in glucose-6-phosphate dehydrogenase activity along with either a 3-fold increase in phosphoenolpyruvate carboxylase activity or decrease in pyruvate kinase activity could increase nitrogen fixation rate by 8.82% while improving nitrogen fixation efficiency by 10.99%.

Enzyme kinetics

Effect of ruthenium on nitrogen fixation by some nitrogen fixers.

The effect of ruthenium chloride in the culture media on the nitrogen-fixing ability of the three nitrogen fixers (unidentified species of Azotobacter, designated here as D3, B3, and B), isolated from Allahabad soil, was studied. It was observed that the nitrogen-fixing ability of the organisms is much increased in presence of 25-75 micro M concentration of ruthenium chloride in the culture media, while sugar consumption remains more or less steady. Also, if mg nitrogen fixed/g carbon consumed in two culture media with successive increasing concentrations of ruthenium chloride is compared by calculating the difference in increase of the amount of nitrogen fixed and carbon consumed in these two culture media, it was observed that high amounts of nitrogen are fixed by B3 and B between 25-50 micro M, and between 50-75 micro M concentration of ruthenium chloride by D3.

Azotobacter

Factors influencing dark nitrogen fixation in a blue-green alga.

Nitrogen-fixing activity declines first rapidly and then more gradually when Anabaenopsis circularis is transferred from light into dark conditions. The rate and duration of dark acetylene reduction (nitrogen fixation) depend upon conditions prevailing during the preceding light period. Factors (such as light intensity, CO2 concentration, and supply of glucose), which in the light affect photosynthesis and the accumulation of reserve carbon, have a profound effect on dark nitrogen fixation. Glucose greatly promotes nitrogen fixation in the light and supports prolonged nitrogenase activity in the dark. The results suggest that heterotrophic nitrogen fixation by blue-green algae in the field may be important both under light and dark conditions.

Acetylene

Regulation of nitrogen fixation in Rhizobium sp.

Regulation of nitrogen fixation by ammonium and glutamate was examined in Rhizobium sp. 32H1 growing in defined liquid media. Whereas nitrogenase synthesis in Klebsiella pneunoniae is normally completely repressed during growth on NH4+, nitrogenase activity was detected in cultures of Rhizobium sp. grown with excess NH4+. However, an "ammonium effect" on activity was invariably observed in cultures grown on NH4+ as sole nitrogen source; the nitrogenase activity was, depending on conditions, 14 to 36% of that of comparable glutamate-grown cultures. Glutamate inhibited utilization of exogenous NH4+ and, in one of two procedures described, glutamate partially alleviated the ammonium effect on nitrogenase activity. NH4+, apparently produced from N2, was excreted into the culture medium when growth was initiated on glutamate, but not when NH4+ was thesole source of fixed nitrogen for growth. These findings are discussed in relation to nitrogen fixation by Rhizobium bacteroids.

Ammonium Sulfate

Nitrogen fixation in the Rhode River estuary of Chesapeake Bay.

The distribution, seasonal variation, origin, and significance of biological nitrogen fixation has been determined for a Chesapeake Bay estuary using the acetylene reduction technique. Studies over a 15-month period have shown that nitrogen fixation occurs predominantly in the surface intertidal (marsh) and subtidal sediments. Negligible activity was found in surface waters. A marked seasonal variation in nitrogen fixation was observed for intertidal sediments incubated at a standard 20 degrees C. Average rates of about 37 and 12 ng N/g dry sediment per hour were observed in the late fall months of 1972 and 1973, respectively, and less than or equal to 5 ng N/g dry sediment per hour during other seaons. Peaks of activity were lowered considerably when samples were incubated at ambient temperatures (in situ). Activity in the subtidal sediments was low (less than or equal to 6.8 ng N/g dry sediment per hour but showed a similar (approximately twofold) seasonal variation in nitrogen fixation potential. Light-dark and substrate addition experiments suggest that heterotrophic bacteria are the principal agents for nitrogen fixation in sediments. Integrated estimates of nitrogen fixation in the estuary indicate that biological fixation probably accounts for less than 5% of the total influx of nitrogen into the system. Rates of activity in the intertidal sediments are insufficient to account for the high productivity of marine angiosperms found in the marsh.

Acetylene

[Nonsymbiotic nitrogen fixation in the podzolic soils of the Kola Peninsula].

Non-symbiotic nitrogen fixation was studied in virgin and cultivated podzol soils of the Kola Peninsula by the acetylene and isotope techniques. The activity of nitrogen fixation varied sharply in cultivated soils due to degradation of plant residues and the action of plant root secretion. No significant changes were observed in the activity of nitrogen fixation within the vegetative period in virgin soils. Less than 1 kg of nitrogen per hectare was accumulated in soils as a result of its non-symbiotic fixation during the vegetative periods of 1976--1977.

Aerobiosis

Involvement of the cytoplasmic membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids.

1. The nitrogen-fixing efficiency of freshly prepared suspensions of Rhizobium leguminosarum bacteroids from pea root nodules was considerably enhanced by addition of bovine serum albumin. Evidence was found that during preparation of bacteroids the cell membrane is exposed to the uncoupling effect of free fatty acids and to plant phospholipase D activity. Both effects could be counteracted by bovine serum albumin. 2. A technique was developed by which concentrations of free O2 and nitrogenase activity could be measured simultaneously under conditions of steady-state respiration. By means of this system it could be shown that in contrast to previous claims, high ATP/ADP ratios can be achieved in bacteroids even with a high concentration of O2 in the medium. 3. Nitrogen fixation was found to be controlled by the ATP/ADP ratio, the generation of reducing equivalents and the switch-off phenomenon. It was demonstrated that the generation of reducing equivalents for nitrogenase is regulated by the energized state and the integrity of the bacteroid cell membrane. The data indicate that the process of aerobic nitrogen fixation in R. leguminosarum bacteroids resembles that of Azotobacter vinelandii.

Adenosine Diphosphate

Intensity of nitrogen fixation in yellow lupine in presence of different doses of mineral nitrogen.

The effect of different doses of mineral nitrogen on the nitrogen-fixing activity of lupine nodules during vegetation was studied. It was shown that 0.5 of the dose of mineral nitrogen had an inhibitory effect only in the initial period of development of the nodules, whereas full and double doses of mineral nitrogen inhibited nitrogen fixation throughout the period of vegetation. Supplements of mineral nitrogen switch the lupine nutrition from the symbiotic to the autotrophic type, which is accompanied by a decrease in the nitrogen-fixing activity in the nodules and by an increase in the nitrate-reducing capacity of the roots and leaves.

Fabaceae

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

Ammonia

Nitrogen fixation by Spirillum sp. from rice roots.

Nitrogen fixation by Spirillum sp. obtained from the roots of rice plants grown at different levels of combined nitrogen was studied. The roots of rice plants exposed to low levels of combined nitrogen (20--40 kg N/ha) harboured Spirillum sp. possessing higher nitrogen-fixing efficiency as compared to the cultures from plants receiving 60--100 kg N/ha. Moreover, the nitrogen-fixing efficiency of these Spirillum spp. varied with age of the plant, irrespective of the dosage of combined nitrogen.

Fertilizers

Effects of some inorganic elements on nitrogen-fixation in blue-green algae and some ecological aspects of pollution.

Nitrogen-fixation by two species of Nostoc, one of them a lichen phycobiont, was generally stimulated by low concentrations of arsenic, cadmium, nickel, lead, palladium, and zinc. Higher concentrations (0.025 to 0.125 ppm) of arsenic, nickel, and palladium were also stimulatory; however, higher concentrations of cadmium, lead, and zinc tended to inhibit fixation. With the exception of palladium and zinc at low concentrations these six tested elements tended to inhibit nitrogen-fixation in Chlorogloea fritschii and Westiellopsis sp.

Arsenic

[Molecular nitrogen fixation in eutrophic Beloye Ozero].

The rate of production and mineralization of organic matter and the rate of fixation of molecular nitrogen were assayed in th eutrophic lake Beloye (near Moscow) at the end of summer stagnation (1976). The content of hydrogen sulphide reached 6.8 mg per litre which resulted in a change of the ecological environment in the lake. The fixation of molecular nitrogen in water layers was characterized by three maxima: 5.0 mcg N per litre per 4 hours (surface); 4.2 mcg N per litre per 4 hours (metalimnion) and 2.8 mcg N per litre per 4 hours (at bottom). The fixation of nitrogen in the trophogenous layer involved mainly the blue-green alga Anabaena. In the sulphide zone, molecular nitrogen was fixed by butyric and sulphate-reducing bacteria.

Cyanobacteria

Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation.

A series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneumoniae strain M5a1. The nif mutations were either located on plasmid pRD1 or on the K. pneumoniae chromosome. A total of 37 plasmid mutants and 28 chromosomal mutants were employed in complementation tests using the acetylene reduction technique. Most mutants could be assigned to one of seven nif cistrons: nifA, nifB, nifD, nifE, nifF, nifH, and nifK. Complementation analysis of two nif deletion mutants confirmed transductional evidence that these strains carry nifB-A-F deletions. One deletion mutant had, in contrast to previous transductional analysis, a functional nifK cistron and presumably is deleted for nifB-A-F-E. Examination of the biochemical phenotype of several mutants suggests that the nifA product has a regulatory function, and nifK, nifD and nifH are most probably the structural genes for nitrogenase.

Chromosomes, Bacterial

[Seasonal fluctuations in the potential soil capacity for nitrogen fixation].

The nitrogenase activity of soils to which no energy-rich substances were added is low. This can be attributed to a low content of easily available organic substances. The potential activity of nitrogen fixation of the soils under study is different and changes with seasons. The highest ability for potential nitrogen fixation is displayed by soddy-calcareous and continuous-cultivated grey forest soils. The potential activity of nitrogen fixation in these soils correlates with the bacterial number on agar. Such a correlation is not observed in grey forest soils.

Ecology

The effect of 2,4-dichlorophenoxyacetic acid on growth and nitrogen-fixation of blue-green alga Anabaenopsis raciborskii.

Sodium salt of 2,4-dichlorophenoxyacetic acid (80% active ingredient), commonly applied for the control of aquatic weeds, was used to observe its effect on the growth and nitrogen fixation of a heterocystous bloom forming blue-green alga Anabaenopsis raciborskii. A concentration of 10 microgram per ml of 2,4-D showed stimulation of growth and nitrogen fixation and these were almost unaffected in presence of its 100 microgram per ml in the medium. The alga could tolerate up to 800 microgram per ml in liquid culture media with and without nitrate nitrogen and up to 90 microgram per ml on to agar plates. Nitrogen fixation was inhibited in presence of its higher concentrations.

2,4-Dichlorophenoxyacetic Acid

[Nitrogen fixation in combined cultures of Lipomyces and bacteria].

Nitrogen fixation by pure cultures of soil yeasts belonging to the genus Lipomyces Lodder et Kreger van Rij was not found by the acetylene method on a medium containing microelements and yeast autolysate. In a binary culture of L. lipofer 133 and Pseudomonas sp. 5, a nitrogen fixing bacterium, the content of bound nitrogen was 3.6 times higher in aerobic conditions and 15 times higher in anaerobic conditions than its accumulation by individual bacterial culture.

Aerobiosis