Contribution of diazotrophy to nitrogen inputs supporting Karenia brevis blooms in the Gulf of Mexico

Blooms of Karenia brevis plague the West Florida Shelf (WFS) region in the Gulf of Mexico (GOM) where they exert harmful effects on aquatic biota and humans. Because productivity on the WFS is N limited, new N inputs into the region are thought to trigger blooms of K. brevis. Here we examine the potential for new N inputs via N₂ fixation by Trichodesmium and other diazotrophic plankton to contribute to the N demand of K. brevis. Because of possible methodological biases, we also compared N₂ fixation rates by cultured Trichodesmium using the ¹⁵N₂ bubble addition method and the ¹⁵N₂ saturated seawater. Both methods yielded identical results in 12 and 24 h incubations; however, there was more variability in rate estimates made using the bubble addition method. Pelagic N₂ fixation rates by other planktonic diazotrophs ranged from 0 to 13.6 nmol N L⁻¹ d⁻¹, comparable to or higher than rates observed in oligotrophic gyres. These rates should be considered conservative estimates because they were made using the bubble addition method. Integrating over our study area, we estimate that new inputs of N to the WFS via N₂ fixation are on the order of 0.011 Tmol N annually. Further, we measured directly the trophic transfer of recently fixed N₂ to co-occurring plankton that included K. brevis and found that up to 47% of N₂ fixed was transferred to non-diazotrophic plankton even in short (<6 h) incubations where N₂ fixation was likely underestimated.     

The Contamination of Commercial 15N2 Gas Stocks with 15N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen (¹⁵N) N₂ gas stocks with ¹⁵N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. ¹⁵N₂ gas is used to estimate N₂ fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled ¹⁵N₂ into organic matter. However, the microbial assimilation of bioavailable ¹⁵N-labeled N₂ gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N₂ fixation rates. ¹⁵N₂ gas procured from three major suppliers was analyzed for the presence of these ¹⁵N-contaminants. Substantial concentrations of ¹⁵N-contaminants were detected in four Sigma-Aldrich ¹⁵N₂ lecture bottles from two discrete batch syntheses. Per mole of ¹⁵N₂ gas, 34 to 1900 µmoles of ¹⁵N-ammonium, 1.8 to 420 µmoles of ¹⁵N-nitrate/nitrite, and ≥21 µmoles of ¹⁵N-nitrous oxide were detected. One ¹⁵N₂ lecture bottle from Campro Scientific contained ≥11 µmoles of ¹⁵N-nitrous oxide per mole of ¹⁵N₂ gas, and no detected ¹⁵N-nitrate/nitrite at the given experimental ¹⁵N₂ tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles ¹⁵N-nitrous oxide per mole ¹⁵N₂, and trace concentrations of ¹⁵N-ammonium and ¹⁵N-nitrate/nitrite. ¹⁵N₂ gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the ¹⁵N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N₂ fixation assays suggests that the degree of detected ¹⁵N-ammonium contamination could yield inferred N₂ fixation rates ranging from undetectable, <0.01 nmoles N L⁻¹ d⁻¹, to 530 nmoles N L⁻¹ d⁻¹, contingent on experimental conditions. These rates are comparable to, or greater than, N₂ fixation rates commonly detected in field assays. These results indicate that past reports of N₂ fixation should be interpreted with caution, and demonstrate that the purity of commercial ¹⁵N₂ gas must be ensured prior to use in future N₂ fixation rate determinations.     

Direct flux and 15N tracer methods for measuring denitrification in forest soils

Estimates of denitrification are one of the key uncertainties in the terrestrial nitrogen (N) cycle, primarily because reliable measurements of this highly variable process—especially the production of its terminal product (N₂)—are difficult to obtain. We evaluated the ability of gas-flow soil core and ¹⁵N tracer methods to provide reliable estimates of denitrification in forest soils. Our objectives were to: (1) describe and present typical results from new gas-flow soil core and in situ ¹⁵N tracer methods for measuring denitrification, (2) discuss factors that affect the relevance of these methods to actual in situ denitrification, and (3) compare denitrification estimates produced by the two methods for a series of sites in a northern hardwood forest ecosystem. Both methods were able to measure accumulations of N₂ over relatively short (2–5 h) incubations of either unamended or tracer-amended intact soils. Denitrification rates measured by the direct flux soil core method were very sensitive to incubation oxygen (O₂) concentration and decreased with increased O₂ levels. Denitrification rates measured by the in situ ¹⁵N tracer method were very sensitive to the ¹⁵N content of the nitrate (NO₃ ^?) pool undergoing denitrification, which limits the applicability of this method for quantifying denitrification in N-poor ecosystems. While its ability to provide accurate estimates of denitrification was limited, the ¹⁵N tracer method provided estimates of the short-term abiotic and biotic transformations of atmospheric N deposition to gas. Furthermore, results suggest that denitrification is higher and that N₂O:N₂ ratios are lower (<0.02) than previously thought in the northern hardwood forest and that short-term abiotic and biotic transformations of atmospheric N deposition to gas are significant in this ecosystem.     

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.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

Isotopic analysis of cyanobacterial nitrogen fixation associated with subarctic lichen and bryophyte species

Dinitrogen fixation by cyanobacteria is of particular importance for the nutrient economy of cold biomes, constituting the main pathway for new N supplies to tundra ecosystems. It is prevalent in cyanobacterial colonies on bryophytes and in obligate associations within cyanolichens. Recent studies, applying interspecific variation in plant functional traits to upscale species effects on ecosystems, have all but neglected cryptogams and their association with cyanobacteria. Here we looked for species-specific patterns that determine cryptogam-mediated rates of N₂ fixation in the Subarctic. We hypothesised a contrast in N₂ fixation rates (1) between the structurally and physiologically different lichens and bryophytes, and (2) within bryophytes based on their respective plant functional types. Throughout the survey we supplied ¹⁵N-labelled N₂ gas to quantify fixation rates for monospecific moss, liverwort and lichen turfs. We sampled fifteen species in a design that captures spatial and temporal variations during the growing season in Abisko region, Sweden. We measured N₂ fixation potential of each turf in a common environment and in its field sampling site, in order to embrace both comparativeness and realism. Cyanolichens and bryophytes differed significantly in their cyanobacterial N₂ fixation capacity, which was not driven by microhabitat characteristics, but rather by morphology and physiology. Cyanolichens were much more prominent fixers than bryophytes per unit dry weight, but not per unit area due to their low specific thallus weight. Mosses did not exhibit consistent differences in N₂ fixation rates across species and functional types. Liverworts did not fix detectable amounts of N₂. Despite the very high rates of N₂ fixation associated with cyanolichens, large cover of mosses per unit area at the landscape scale compensates for their lower fixation rates, thereby probably making them the primary regional atmospheric nitrogen sink.     

A NEW NITROGEN-FIXING NON-LEGUME: CHAMAEBATIA FOLIOLOSA (ROSACEAE)

Specimens of Chamaebatia foliolosa Benth. with nodule structures on their roots fix atmospheric nitrogen. The nodules are similar to those of other non-legumes in gross morphology and structure, containing hyphal strands, some with club-shaped vesicles at their ends. A fixation rate of 130 nmoles N₂ per g fresh weight per hr is reported by using ¹⁵N₂ as a tracer. Equivalent rates of acetylene reduction were observed.     

A test of a field‐based 15N–nitrous oxide pool dilution technique to measure gross N2O production in soil

Soils are both a major source and sink of nitrous oxide (N₂O), but the proportion of soil N₂O production released to the atmosphere (termed the N₂O yield) is poorly constrained due to the difficulty in measuring gross N₂O production. The quantification of gross N₂O fluxes would greatly improve our ability to predict N₂O dynamics across the soil‐atmosphere interface. We report a new approach, the ¹⁵N₂O pool dilution technique, to measure rates of gross N₂O production and consumption under laboratory and field conditions. In the laboratory, gross N₂O production and consumption compared well between the ¹⁵N₂O pool dilution and acetylene inhibition methods whereas the ¹⁵NO₃^? tracer method measured significantly higher rates. In the field, N₂O emissions were not significantly affected by increasing chamber headspace concentrations up to 100 ppb ¹⁵N₂O. The pool dilution model estimates of ¹⁴N₂O and ¹⁵N₂O concentrations as well as net N₂O fluxes fit observed data very well, suggesting that the technique yielded robust estimates of gross N₂O production. Estimated gross N₂O consumption rates were underestimated relative to rates calculated as the difference between gross and net N₂O production rates, possibly due to heterogeneous and/or inadequate tracer diffusion to deeper layers in the soil profile. Gross N₂O production rates were high, averaging 8.4 ± 3.2 mg N m^?2 day^?1, and were most strongly correlated to mineral nitrogen concentrations and denitrifying enzyme activity (R² = 0.73). Gross N₂O production rates varied spatially, with the highest rates in soils with the best drainage and the highest mineral N availability. Estimated and calculated N₂O consumption rates constrained the average N₂O yield from 0.70 to 0.84. Our results demonstrate that the ¹⁵N₂O pool dilution technique can provide well‐constrained estimates of N₂O yields and field rates of gross N₂O production correlated to soil characteristics, improving our understanding of terrestrial N₂O dynamics.     

Significant nonsymbiotic nitrogen fixation in Patagonian ombrotrophic bogs

Nitrogen (N) nutrition in pristine peatlands relies on the natural input of inorganic N through atmospheric deposition or biological dinitrogen (N₂) fixation. However, N₂ fixation and its significance for N cycling, plant productivity, and peat buildup are mostly associated with the presence of Sphagnum mosses. Here, we report high nonsymbiotic N₂‐fixation rates in two pristine Patagonian bogs with diversified vegetation and natural N deposition. Nonsymbiotic N₂ fixation was measured in samples from 0 to 10, 10 to 20, and 40 to 50 cm depth using the ¹⁵N₂ assay as well as the acetylene reduction assay (ARA). The ARA considerably underestimated N₂ fixation and can thus not be recommended for peatland studies. Based on the ¹⁵N₂ assay, high nonsymbiotic N₂‐fixation rates of 0.3–1.4 μmol N₂ g^?1 day^?1 were found down to 50 cm under micro‐oxic conditions (2 vol.%) in samples from plots covered by Sphagnum magellanicum or by vascular cushion plants, latter characterized by dense and deep aerenchyma roots. Peat N concentrations point to greater potential of nonsymbiotic N₂ fixation under cushion plants, likely because of the availability of easily decomposable organic compounds and oxic conditions in the rhizosphere. In the Sphagnum plots, high N₂ fixation below 10 cm depth rather reflects the potential during dry periods or low water level when oxygen penetrates the top peat layer and triggers peat mineralization. Natural abundance of the ¹⁵N isotope of live Sphagnum (5.6 δ‰) from 0 to 10 cm points to solely N uptake from atmospheric deposition and nonsymbiotic N₂ fixation. A mean ¹⁵N signature of ?0.7 δ‰ of peat from the cushion plant plots indicates additional N supply from N mineralization. Our findings suggest that nonsymbiotic N₂ fixation overcomes N deficiency in different vegetation communities and has great significance for N cycling and peat accumulation in pristine peatlands.     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.     

Nitrogen Transfer from Four Nitrogen-Fixer Associations to Plants and Soils

Nitrogen (N) fixation is the main source of ‘new’ N for N-limited ecosystems like subarctic and arctic tundra. This crucial ecosystem function is performed by a wide range of N₂ fixer (diazotroph) associations that could differ fundamentally in their timing and amount of N release to the soil. To assess the importance of different associative N₂ fixers for ecosystem N cycling, we tracked ¹⁵N-N₂ into four N₂-fixer associations (with a legume, lichen, free-living, moss) and into soil, microbial biomass and non-diazotroph-associated plants 3 days and 5 weeks after in situ labelling. In addition, we tracked ¹³C from ¹³CO₂ labelling to assess if N and C fixation are linked. Three days after labelling, half of the fixed ¹⁵N was recovered in the legume soils, indicating a fast release of fixed N₂. Within 5 weeks, the free-living N₂ fixers released two-thirds of the fixed ¹⁵N into the soil, whereas the lichen and moss retained the fixed ¹⁵N. Carbon and N₂ fixation were linked in the lichen shortly after labelling, in free-living N₂ fixers 5 weeks after labelling, and in the moss at both sampling times. The four investigated N₂-fixer associations released fixed N₂ at different rates into the soil, and non-diazotroph-associated plants have no access to ‘new’ N within several weeks after N₂ fixation. Although legumes and free-living N₂ fixers are immediate sources of ‘new’ N for N-limited tundra ecosystems, lichens and especially mosses, do not contribute to increase the N pool via N₂ fixation in the short term.     

Nitrogen fixation by subclover associations fertilized with sulfur

Summary The effect of S fertilization on symbiotic N₂ fixation was measured with the¹⁵N technique and the N difference method in a lysimeter study using Josephine loam (Typic Haploxurults). Nitrogen fixation by subclover (Trifolium subterraneum L.) was strongly enhanced by added S. The association of soft chess (Bromus mollis L.) or filaree (Erodium botrys (Cav.) Bertol.) with subclover increased the percentage of N in subclover that was fixed, with the results that N₂ fixation was increased beyond that due to the mere increase in subclover biomass. Nitrogen fixation estimates by¹⁵N dilution and N difference methods were highly correlated (r²=0.97), and S fertilizer did not result in any significant differences in N₂-fixation estimation by the two methods. Both methods were useful in distinguishing between soil N uptake and N₂ fixation where S applications produced highly significant increases in both uptake and fixation. Application of sulfur fertilizers to much annual rangeland has the potential to increase pasture productivity through enhanced N₂ fixation. Contribution of the University of California Hopland Field Station and Department of Agronomy and Range Science, Univ. of California, Davis, CA 95616.     

Co-Occurring Anammox,Denitrification, and Codenitrification in Agricultural Soils

Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N₂ gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N₂ in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N₂ production, molecular and ¹⁵N isotope analyses were conducted with soil samples collected at six different agricultural fields in the United States. Denitrifying and anammox bacterial abundances were measured based on quantitative PCR (qPCR) of nitrous oxide reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed spacer (ITS) of Fusarium oxysporum was quantified to estimate the abundance of codenitrifying and denitrifying fungi. ¹⁵N tracer incubation experiments with ¹⁵NO₃⁻ or ¹⁵NH₄⁺ addition were conducted to measure the N₂ production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N₂ production rates in soil samples. Denitrifying bacteria were found to be the most abundant, followed by F. oxysporum based on the qPCR assays. The potential denitrification rates by bacteria and fungi ranged from 4.118 to 42.121 nmol N₂-N g⁻¹ day⁻¹, while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N₂-N g⁻¹ day⁻¹. Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N₂ production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Nitrogen fixation in the High Arctic: a source of ‘new’ nitrogen?

Biological nitrogen (N₂) fixation performed by diazotrophs (N₂ fixing bacteria) is thought to be one of the main sources of plant available N in pristine ecosystems like arctic tundra. However, direct evidence of a transfer of fixed N₂ to non-diazotroph associated plants is lacking to date. Here, we present results from an in situ ¹⁵N–N₂ labelling study in the High Arctic. Three dominant vegetation types (organic crust composed of free-living cyanobacteria, mosses, cotton grass) were subjected to acetylene reduction assays (ARA) performed regularly throughout the growing season, as well as ¹⁵N–N₂ incubations. The ¹⁵N-label was followed into the dominant N₂ fixer associations, soil, soil microbial biomass and non-diazotroph associated plants three days and three weeks after labelling. Mosses contributed most to habitat N₂ fixation throughout the measuring campaigns, and N₂ fixation activity was highest at the beginning of the growing season in all plots. Fixed ¹⁵N–N₂ became quickly (within 3 days) available to non-diazotroph associated plants in all investigated vegetation types, proving that N₂ fixation is an actual source of available N in pristine ecosystems.     

Biological nitrogen fixation: A scientific perspective

The discoveries of Hellriegel and Wilfarth ended the period of controversy about the existence of biological N₂ fixation and launched a period featuring the agronomic application of the inoculation of legumes. Serious studies of the biochemistry of N₂ fixation started in the late 1920's, and defined some of the basic properties of the N₂-fixing system. Application of¹⁵N as a tracer gave definitive evidence for the role of ammonia as the key intermediate in biological N₂ fixation. It was demonstrated in the 1950's and 1960's that nitrogenase could reduce substrates other than N₂. With the achievement of consistent cell-free N₂ fixation it was possible to resolve the nitrogenase system into two proteins, electron donors, and ATP-hydrolyzing and regenerating systems. The sequence of electron transfer was established. Recently, studies of the genetics of the nitrogenase system have defined in detail how the system is assembled and controlled.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.

     

Nutrient Limitation to Nitrogen Fixation in Young Volcanic Sites

I used measures of ¹⁵N natural abundance and of nitrogenase activity (acetylene reduction) to examine whether the supply of non-N nutrients limits rates of N₂ fixation on young volcanic substrates in Hawaii. Leaves of the dominant tree (Metrosideros polymorpha, a nonfixer) were strongly depleted in ¹⁵N in control plots (–10.8 to –11.1⁰/₀₀). More than 5 y of repeated fertilization with P increased δ¹⁵N to –8.9 to –9.9⁰/₀₀, and the addition of all other essential plant nutrients (except N) together with P further increased ¹⁵N to –8.1 to –9.3⁰/₀₀. This pattern is consistent with enhanced N₂ fixation, because newly fixed N would have a δ¹⁵N near 0⁰/₀₀. Assays of nitrogenase activity in the experimental plots demonstrated that potential N fixation associated with nonvascular plants and with tree and fern litter were increased significantly by additions of P and by the combined nutrient treatment; when these were added together, the increase in nitrogenase activity was 6- to 11-fold over control plots. The supply of P and other weathering-derived nutrients constrains rates of N₂ fixation in these young volcanic sites and thereby contributes to the maintenance of N limitation to primary production and other ecosystem processes. Received 7 January 1999; accepted 3 May 1999.