Growth of Spirillum lipoferum at constant partial pressures of oxygen, and the properties of its nitrogenase in cell-free extracts.

Spirillum lipoferum, an N2-fixing organism, was grown at constant concentrations of dissolved O2. When supplied with NH4+ aerobically, its doubling time was 1 h; when it fixed N2 microaerophilically, its doubling time was 5-5 to 7 h and the optimal PO2 for growth was 0-005 to 0-007 atm. At its optimal PO2 for growth on N2, S. lipoferum assimilated 8 to 10 mg nitrogen/g carbon substrate used; its efficiency was less at higher PO2 levels. Nitrogenase in cell-free extracts required Mg2+ and Mn2+, and the Fe-protein was activated by Rhodospirillum rubrum activating factor. The nitrogenase had an optimal pH of 7-1 to 7-4 and an apparent Km for acetylene of 0-0036 atm. Extracts of S. lipoferum lost their nitrogenase activity on storage at -18 degrees C, and activity was restored by adding purified Fe-protein from other N2-fixing bacteria.     

Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp.

Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.     

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.     

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.     

Ecological distribution of Spirillum lipoferum Beijerinck.

A survey in various countries revealed that the N2-fixing Spirillum lipoferum Beijerinck is a very common root and soil inhabitant in the tropics. More than half of the grass root and soil samples collected in tropical countries (four African countries and Brazil) contained abundant S. lipoferum populations, while less than 10% of the samples collected in temperate South Brazil, Kenya, and the U.S.A. contained the organism. There is a pronounced vegetation effect. Panicum maximum seems the most favorable among the forage grasses, while few positive samples were found under virgin tropical forest. Legume roots contained less S. lipoferum than adjacent soils. More than 80% of the samples from cereal roots (maize, sorghum, wheat, and rye) grown in fields fertilized with PK and Mo, in Rio de Janeiro State, were positive. Maize and sorghum grown under similar conditions in Wisconsin contained less than 10% of positive samples, but when maize fields were inoculated 90% of the root samples contained S. lipoferum. Alluvial soils were more favorable than eroded hill soils. Occurrence in soil was strongly pH-dependent with a pH around 7, being optimal (correlation coefficient r = 0.90). Sporadic occurrence was observed even in soils with pH 4.8. Surface-sterilized P. maximum roots collected from soils with pH ranging from 4.8 to 7.2 contained high S. lipoferum numbers which did not correlate with soil pH (r = 0.41). Amendment with malate of acid soils was not very effective in increasing nitrogenase (N2-ase) activity, but in two soils with pH above 6.4, high N2-ase activity was obtained after 16 to 48 h of incubation. In two soils from a temperate climate region, inoculation with S. lipoferum increased N2-ase activity produced through malate amendment.     

Growth of a Bacterium Under a High-Pressure Oxy-Helium Atmosphere

Growth of a barotolerant marine organism, EP-4, in a glutamate medium equilibrated with an oxy-helium atmosphere at 500 atmospheres (atm; total pressure) (20°C) was compared with control cultures incubated at hydrostatic pressures of 1 and 500 atm. Relative to the 1-atm control culture, incubation of EP-4 at 500 atm in the absence of an atmosphere resulted in an approximately fivefold reduction in the growth rate and a significant but time variant reduction in the rate constants for the incorporation of substrate into cell material and respiration. Distinct from the pressurized control and separate from potential effects of dissolution of helium upon decompression of subsamples, exposure of the organism to high-pressure oxy-helium resulted in either a loss of viability of a large fraction of the cells or the arrest of growth for one-third of the experimental period. After these initial effects, however, the culture grew exponentially at a rate which was three times greater than the 500-atm control culture. The rate constant for the incorporation of substrate into cell material was also enhanced twofold in the presence of high-pressure oxy-helium. Dissolved oxygen was well controlled in all of the cultures, minimizing any potential toxic effects of this gas.     

N(2) fixation by bacteria associated with maize roots at a low partial o(2) pressure

Nitrogen fixation by bacteria associated with roots of intact maize plants was measured by exposing the roots to N(2) at a partial O(2) pressure (pO(2)) of 2 or 10 kPa. The plants were grown in a mixture of Weswood soil and sand and then transferred to plastic cylinders containing an N-free plant nutrient solution. The solution was sparged continuously with a mixture of air and N(2) at a pO(2) of 2 or 10 kPa. Acetylene reduction was measured after the roots were exposed to the low pO(2) overnight. The air-N(2) atmosphere in the cylinders was then replaced with an O(2)-He atmosphere at the same pO(2), and the roots were exposed to 20 kPa of N(2) for 20 to 22 h. Incorporation of N into the roots was 200 times greater at 2 kPa of O(2) than at 10 kPa of O(2). Adding l-malate (1 g of C liter) to the nutrient solution increased root-associated nitrogenase activity, producing a strong N label which could be traced into the shoots. Fixed N was detected in the shoots within 5 days after the plants were returned to unfertilized soil. In a similar experiment with undisturbed plants grown in fritted clay, movement of fixed N into the shoots was evident within 4 days after the roots were exposed to N(2) at 2 kPa of O(2). Inoculation with Azospirillum lipoferum yielded no significant differences in shoot dry weight, total nitrogen content, percent nitrogen, or N enrichment of plant tissues. Inoculated plants did exhibit greater root dry weight than uninoculated plants, however.     

Characterization of a nitrogen-fixing bacterial strain from the roots of Digitaria sanguinalis.

Rates of nitrogen fixation of 3 to 10 g of N2 fixed per hectare per day were associated with root systems of Digitaria sanguinalis. A Gram-negative motile aerobic bacterial strain that was capable of N2 fixation was isolated from a washed root sample of one of these plants. Optimal growth and N2 fixation occurred at a pH of about 6.5, a temperature of 30-37 degrees C, and at a pO2 of about 0.01 atm. Increased rates of N2 fixation resulted when this strain was grown in mixed cultures with aerobic or facultative bacteria. Observations of cellular and cultural morphology and results of biochemical and physiological studies indicate that the isolate may be related to the Azotobacteraceae but that it is not identical with any of the members of this family. The importance of N2 fixation by this isolate in nature is unknown.     

Carbon and ammonia metabolism of Spirillum lipoferum.

Intact cells and extracts from Spirillum lipoferum rapidly oxidized malate, succinate, lactate, and pyruvate. Glucose, galactose, fructose, acetate, and citrate did not increase the rate of O2 uptake by cells above the endogenous rate. Cells grown on NH+/4 oxidized the various substrates at about the same rate as did cells grown on N2. Added oxidized nicotinamide adenine dinucleotide generally enhanced O2 uptake by extracts supplied organic acids, whereas oxidized nicotinamide adenine dinucleotide phosphate had little effect. Nitrogenase synthesis repressed by growth of cells in the presence of NH+/4 was derepressed by methionine sulfoximine or methionine sulfone. The total glutamine synthetase activity from N2-grown cells was about eight times that from NH+/4-grown S. lipoferum; the response of glutamate dehydrogenase was the opposite. The total glutamate synthetase activity from N2-grown S. lipoferum was 1.4 to 2.6 times that from NH+/4-grown cells. The levels of poly-beta-hydroxybutyrate and beta-hydroxybutyrate dehydrogenase were elevated in cells grown on N2 as compared with those grown on NH+/4. Cell-free extracts capable of reducing C2H2 have been prepared; both Mg2+ and Mn2+ are required for good activity.     

Bicarbonate Requirement for Elimination of the Lag Period of Hydrogenomonas eutropha

Carbon dioxide and oxygen concentrations have a profound effect on the lag period of chemoautotrophically grown Hydrogenomonas eutropha. Minimum lag periods and high growth rates were obtained in shaken flask cultures with a prepared gas mixture containing 70% H(2), 20% O(2), and 10% CO(2). However, excessively long lag periods resulted when the same gas mixture was sparged through the culture. The lag period was shortened in sparged cultures by decreasing both the pO(2) and the pCO(2), indicating that gas medium equilibration had not occurred in shaken cultures. The lag period was completely eliminated at certain concentrations of O(2) and CO(2). The optimum pO(2) was 0.05 atm, but the optimum pCO(2) varied according to the pH of the medium and physiological age of the inoculum. At pH 6.4, the pCO(2) required to obtain immediate growth of exponential, postexponential, and stationary phase inocula at equal specific rates was 0.02, 0.05, and 0.16 atm, respectively. With each 0.3-unit increase in the pH of the medium, a 50% decrease in the CO(2) concentration was needed to permit growth to occur at the same rate. The pCO(2) changes required to compensate for the pH changes of the medium had the net effect of maintaining a constant bicarbonate ion concentration. Initial growth of H. eutropha was therefore indirectly related to pCO(2) and directly dependent upon a constant bicarbonate ion concentration.     

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).     

The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N2O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N2O (11 micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pond sediment

A Gram-negative bacterium, identified as Stenotrophomonas maltophilia by fatty acid analysis and 16S rRNA sequencing, was isolated from a seleniferous agricultural evaporation pond sediment collected in the Tulare Lake Drainage District, California. In cultures exposed to the atmosphere, the organism reduces selenate (SeO4(2-)) and selenite (SeO3(2-)) to red amorphous elemental selenium (Se degrees ) only upon reaching stationary phase, when O2 levels are less than 0.1 mg l(-1). In 48 h, S. maltophilia removed 81.2% and 99.8% of added SeO4(2-) and SeO3(2-) (initial concentration of 0.5 mM), respectively, from solution. Anaerobic growth experiments revealed that the organism was incapable of using SeO4(2-), SeO3(2-), SO4(2-) or NO3- as a terminal electron acceptor. Transmission electron microscopy of cultures spiked with either Se oxyanion were found to contain spherical extracellular deposits. Analysis of the deposits by energy-dispersive X-ray spectroscopy revealed that they consist of Se. Furthermore, S. maltophilia was active in producing volatile alkylselenides when in the presence of SeO4(2-) and SeO3(2-). The volatile products were positively identified as dimethyl selenide (DMSe), dimethyl selenenyl sulphide (DMSeS) and dimethyl diselenide (DMDSe) by gas chromatography-mass spectrometry. Our findings suggest that this bacterium may contribute to the biogeochemical cycling of Se in seleniferous evaporation pond sediments and waters. This organism may also be potentially useful in a bioremediation scheme designed to treat seleniferous agricultural wastewater.     

Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass. Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration. Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration. During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier. As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased. Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2. Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2. Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1. We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates.     

Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants

Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N₂-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc. A heavy inoculum of S. lipoferum grown on NH₄⁺ was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N₂. Conversion to N₂ fixation was prompt upon exhaustion of fixed nitrogen; growth on N₂ was most rapid at a pO₂ of 0.005 to 0.007 atm. Numbers of S. lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium. Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C₂H₂ are presumptive evidence that they are S. lipoferum. With most-probable-number tables the observations can be converted to numbers of S. lipoferum in the samples. The most-probable-number method indicated that numbers of S. lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30°C at a pO₂ initially adjusted to 0.01 atm.     

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Growth energetics of Clostridium sporogenes NCIB 8053: modulation by CO2

The effects of the partial pressure of carbon dioxide on the growth energetics of Clostridium sporogenes NCIB 8053 grown in chemostat culture were investigated in defined minimal media. Both the 'maintenance' requirements and the growth yield coefficients were dependent upon the partial pressure of carbon dioxide in otherwise glucose-limited cultures. Since growth yield coefficients decreased along with the apparent 'maintenance' requirements in essential amino acid/fatty acid medium when the partial pressure of carbon dioxide was increased above 0.5 atm, the occurrence of some type of metabolic uncoupling seemed likely. By contrast, when the organism was grown in amino acid complete medium both the maintenance requirements and the growth yield coefficients were increased when the partial pressure of carbon dioxide was raised above 0.5 atm partial pressure of carbon dioxide, suggesting an increased efficiency of growth. A futile cycle involving carbon dioxide is proposed as a factor contributing to the variable extent of free energy dissipation within this organism.