Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Denitrification, Acetylene Reduction, and Methane Metabolism in Lake Sediment Exposed to Acetylene

Samples of sediment from Lake St. George, Ontario, Canada, were incubated in the laboratory under an initially aerobic gas phase and under anaerobic conditions. In the absence of added nitrate (NO₃⁻) there was O₂-dependent production of nitrous oxide (N₂O), which was inhibited by acetylene (C₂H₂) and by nitrapyrin, suggesting that coupled nitrification-denitrification was responsible. Denitrification of added NO₃⁻ was almost as rapid under an aerobic gas phase as under anaerobic conditions. The N₂O that accumulated persisted in the presence of 0.4 atm of C₂H₂, but was gradually reduced by some sediment samples at lower C₂H₂ concentrations. Low rates of C₂H₂ reduction were observed in the dark, were maximal at 0.2 atm of C₂H₂, and were decreased in the presence of O₂, NO₃⁻, or both. High rates of light-dependent C₂H₂ reduction occurred under anaerobic conditions. Predictably, methane (CH₄) production, which occurred only under anaerobiosis, was delayed by added NO₃⁻ and inhibited by C₂H₂. Consumption of added CH₄ occurred only under aerobic conditions and was inhibited by C₂H₂.     

Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

Hydrogen Metabolism by Decomposing Cyanobacterial Aggregates in Big Soda Lake, Nevada

Hydrogen production by incubated cyanobacterial epiphytes occurred only in the dark, was stimulated by C₂H₂, and was inhibited by O₂. Addition of NO₃⁻ inhibited dark, anaerobic H₂ production, whereas the addition of NH₄⁺ inhibited N₂ fixation (C₂H₂ reduction) but not dark H₂ production. Aerobically incubated cyanobacterial aggregates consumed H₂, but light-incubated rates (3.6 μmol of H₂ g⁻¹ h⁻¹) were statistically equivalent to dark uptake rates (4.8 μmol of H₂ g⁻¹ h⁻¹), which were statistically equivalent to dark, anaerobic production rates (2.5 to 10 μmol of H₂ g⁻¹ h⁻¹). Production rates of H₂ were fourfold higher for aggregates in a more advanced stage of decomposition. Enrichment cultures of H₂-producing fermentative bacteria were recovered from freshly harvested, H₂-producing cyanobacterial aggregates. Hydrogen production in these cyanobacterial communities appears to be caused by the resident bacterial flora and not by the cyanobacteria. In situ areal estimates of dark H₂ production by submerged epiphytes (6.8 μmol of H₂ m⁻² h⁻¹) were much lower than rates of light-driven N₂ fixation by the epiphytic cyanobacteria (310 μmol of C₂H₄ m⁻² h⁻¹).     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).     

Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean

Since NO₃⁻ availability in the rooting medium seriously limits symbiotic N₂ fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO₃⁻ and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M₂ generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant⁻¹) than the Williams parent (187 nodules plant⁻¹) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO₃⁻; each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO₃⁻ than did Williams at 1.5 millimolar NO₃⁻. The NO₃⁻ tolerance did not appear to be related to an altered ability to take up or metabolize NO₃⁻, based on solution NO₃⁻ depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M₅ generation in all three mutants.     

Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean

Soybeans grown with 2 millimolar NO₃⁻, which optimized apparent N₂ fixation by Rhizobium symbionts, showed significantly different rates of apparent photosynthesis and C₂H₂ reduction during seedling development at two irradiances. Those physiological processes were lower for several weeks in plants grown at 1,500 microeinsteins per meter² per second than in those exposed to 700 microeinsteins per meter² per second. The irradiance-induced retardation was evident in short-term rates of apparent photosynthesis and N₂ fixation, as well as in measures of dry matter and total N accumulation. In spite of their previously inhibited development, plants grown at 1,500 microeinsteins per meter² per second were indistinguishable by day 28 from those exposed to 700 microeinsteins per meter² per second in terms of whole-shoot CO₂-exchange rate; by day 35 they were identical in terms of whole-plant C₂H₂-reduction rate. On day 38 there was no significant difference in dry weight or N content between treatments. Shifting plants between irradiance treatments on day 21 showed that the higher irradiance also had a short-term inhibitory effect on C₂H₂ reduction. The fact that 16 millimolar NO₃⁻ prevented the continuous exposure to 1,500 microeinsteins per meter² per second from inhibiting apparent photosynthesis suggested that seedlings grown on 2 millimolar NO₃⁻ with Rhizobium were N-limited. Although rates of apparent photosynthesis were similar by day 28, the additional week required to produce equal rates of apparent N₂ fixation between irradiance treatments showed that physiological adaptations of shoots, as well as photosynthesis per se, can affect root nodule activity.     

Charge Balance in NO(3)-Fed Soybean: Estimation of K and Carboxylate Recirculation

Soybeans (Glycine max L. Merr., cv Kingsoy) were grown on media containing NO₃⁻ or urea. The enrichments of shoots in K⁺, NO₃⁻, and total reduced N (N_r), relative to that in Ca²⁺, were compared to the ratios K⁺/Ca²⁺,NO₃⁻/Ca²⁺, and N_r/Ca²⁺ in the xylem saps, to estimate the cycling of K⁺, and N_r. The net production of carboxylates (R⁻) was estimated from the difference between the sums of the main cations and inorganic anions. The estimate for shoots was compared to the theoretical production of R⁻ associated with NO₃⁻ assimilation in these organs, and the difference was attributed to export of R⁻ to roots. The net exchange rates of H⁺ and OH⁻ between the medium and roots were monitored. The shoots were the site of more than 90% of total NO₃⁻ reduction, and N_r was cycling through the plants at a high rate. Alkalinization of the medium by NO₃⁻-fed plants was interrupted by stem girdling, and not restored by glucose addition to the medium. It was concluded that the majority of the base excreted in NO₃⁻ medium originated from R⁻ produced in the shoots, and transported to the roots together with K⁺. As expected, cycling of K⁺ and reduced N was favoured by NO₃⁻ nutrition as compared to urea nutrition.     

Calorimetry of nitrogenase-mediated reductions in detached soybean nodules

Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N₂ fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.

There was major heat output by nodules for all of the nitrogenase substrates tested (H⁺, N₂, N₂O, and C₂H₂) further establishing the in vivo energy inefficiency of biological N₂ fixation. Exposure to a short burst of 100% O₂ partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H⁺ reductions was 171 ± 6 kilocalories per mole H₂ evolved in an Ar-O₂ atmosphere, that for N₂ fixation was 784 ± 26 kilocalories per mole H₂ evolved and N₂ fixed, and that for C₂H₂ reduction was 250 ± 12 kilocalories/mole C₂H₄ formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e⁻ is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N₂ fixation cost of 9.5 grams glucose per gram N₂ fixed or 3.8 grams C per gram N₂, which is in close agreement with earlier calculations based on nodular CO₂ production.

     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Simultaneous Measurement of Denitrification and Nitrogen Fixation Using Isotope Pairing with Membrane Inlet Mass Spectrometry Analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N₂ gas species (²⁸N₂, ²⁹N₂, and ³⁰N₂) were monitored during incubation experiments after the addition of ¹⁵NO₃⁻. Formulas were developed to estimate the production (denitrification) and consumption (N₂ fixation) of N₂ gas from the fluxes of the different isotopic forms of N₂. Proportions of the three isotopic forms produced from ¹⁵NO₃⁻ and ¹⁴NO₃⁻ agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 μg-atoms of N m⁻² h⁻¹. They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N₂ production rates due to denitrification could be separated from high N₂ consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 μg-atoms of N m⁻² h⁻¹) were lower than nitrogen fixation rates (average, 60 μg-atoms of N m⁻² h⁻¹). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N₂ isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Root respiration associated with nitrate assimilation by cowpea

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using ¹⁵NO₃⁻, and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO₂ per plant per hour for growth and maintenance. Adding 10 millimolar NO₃⁻ to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO₃⁻ from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N₂ fixation.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Nitrogen Fixation Associated with the New Zealand Mangrove (Avicennia marina (Forsk.) Vierh. var. resinifera (Forst. f.) Bakh.)

Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and ¹⁵N₂ uptake. Nitrogenase activity (C₂H₂ reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = −0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 μmol of C₂H₄ m⁻² h⁻¹ under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by ¹⁵N₂ uptake ranged from 5.1 to 7.8 nmol of N₂ g (dry weight)⁻¹ h⁻¹, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ in summer, but values up to 1,500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ were obtained.     

Light-Limited Growth Rate Modulates Nitrate Inhibition of Dinitrogen Fixation in the Marine Unicellular Cyanobacterium Crocosphaera watsonii

Biological N₂ fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO₃ ⁻). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N₂ fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N₂-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N₂ fixation. When growth (µ) was limited by low light (µ = 0.23 d⁻¹), short-term experiments indicated that 0.4 µM NH₄ ⁺ reduced N₂-fixation by ∼90% relative to controls without added NH₄ ⁺. In fast-growing, high-light-acclimated cultures (µ = 0.68 d⁻¹), 2.0 µM NH₄ ⁺ was needed to achieve the same effect. In long-term exposures to NO₃ ⁻, inhibition of N₂ fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO₃ ⁻ did not inhibit N₂-fixation rates in comparison with cultures growing on N₂ alone. Instead NO₃ ⁻ supported even faster growth, indicating that the cellular assimilation rate of N₂ alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO₃ ⁻ did not support faster growth rates but instead inhibited N₂-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N₂ fixation by fixed N sources based on the growth rate as controlled by light.     

Modeling the C Economy of Anabaena flos-aquae: Estimates of Establishment, Maintenance, and Active Costs Associated with Growth on NH(3), NO(3), and N(2)

Steady state cultures of Anabaena flos-aquae were established over a wide range of phosphate-limited growth rates while N was supplied as either NH₃, NO₃⁻, or N₂ gas. At growth rates greater than 0.03 per hour, rates of gross and net carbon fixation were similar on all N sources. However, at lower growth rates (<0.03 per hour) in the NO₃⁻ and N₂ cultures, gross photosynthesis greatly exceeded net photosynthesis. The increase in photosynthetic O₂ evolution with growth rate was greatest when N requirements were met by NO₃⁻ and least when met by NH₃. These results were combined with previously reported measurements of cellular chemical composition, N assimilation, and acetylene reduction (Layzell, Turpin, Elrifi 1985 Plant Physiol 78: 739-745) to construct empirical models of carbon and energy flow for cultures grown at 30, 60, and 100% of their maximal growth rate on all N sources. The models suggested that over this growth range, 89 to 100% of photodriven electrons were allocated to biomass production in the NH₃ cells, whereas only 49 to 74% and 54 to 90% were partitioned to biomass in the NO₃⁻-and N₂-grown cells, respectively. The models were used to estimate the relative contribution of active, maintenance, and establishment costs associated with NO₃⁻ and N₂ assimilation over the entire range of growth rates. The models showed that the relative contribution of the component costs of N assimilation were growth rate dependent. At higher growth rates, the major costs for NO₃⁻ assimilation were the active costs, while in N₂-fixing cultures the major energetic requirements were those associated with heterocyst establishment and maintenance. It was concluded that compared with NO₃⁻ assimilation, N₂ fixation was energetically unfavorable due to the costs of heterocyst establishment and maintenance, rather than the active costs of N₂ assimilation.     

Root and nodule respiration in relation to acetylene reduction in intact nodulated peas

Inoculated pea plants (Pisum sativum L.) were grown with N-free nutrients in a controlled environment room and rates of respiratory CO₂ evolution and C₂H₂ reduction by the intact nodulated roots were determined. Experiments followed changes related to diurnal cycles, light and dark treatments, partial defoliation, aging of plants and NH₄NO₃ addition. In all experiments, changes in C₂H₂ reduction were associated with parallel changes in the respiration rate, although in all but the defoliation experiment there was a basal level of respiration which was independent of the rate of C₂H₂ reduction. In conditions which affected growth or plant size as well as C₂H₂ reduction, respiration changed by an average of 0.42 mg CO₂ (μmol C₂H₂ reduced)⁻¹. However, some treatments decreased C₂H₂ reduction without greatly changing the growth and in these conditions respiration was decreased by an average of 0.27 mg CO₂ (μmol C₂H₂ reduced)⁻¹. While this value may also include some respiration associated with other processes, it is proposed that it more closely estimates respiration directly associated with energy utilization for acetylene reduction; whereas the higher value includes respiration related to maintenance and growth processes as well.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.