Symbiotic nitrogen fixation in a tropical rainforest: 15N natural abundance measurements supported by experimental isotopic enrichment

* Leguminous trees are very common in the tropical rainforests of Guyana. Here, species-specific differences in N(2) fixation capability among nodulating legumes growing on different soils and a possible limitation of N(2) fixation by a relatively high nitrogen (N) and low phosphorus (P) availability in the forest were investigated. * Leaves of 17 nodulating species and 17 non-nodulating reference trees were sampled and their delta(15)N values measured. Estimates of N(2) fixation rates were calculated using the (15)N natural abundance method. Pot experiments were conducted on the effect of N and P availability on N(2) fixation using the (15)N-enriched isotope dilution method. * Nine species showed estimates of > 33% leaf N derived from N(2) fixation, while the others had low or undetectable N(2) fixation rates. High N and low P availability reduced N(2) fixation substantially. * The results suggest that a high N and low P availability in the forest limit N(2) fixation. At the forest ecosystem level, N(2) fixation was estimated at c. 6% of total N uptake by the tree community. We conclude that symbiotic N(2) fixation plays an important role in maintaining high amounts of soil available N in undisturbed forest.     

Soybean dry matter and N accumulation responses to flooding stress, N sources and hypoxia

Soybean (Glycine max [L.] Merr.) is generally considered sensitive to flooding stress. Data on relative sensitivities of biomass accumulation and N2 fixation to flooding stress, however, are limited. Additionally, it is not clear why plants dependent on N2 fixation appear to be more flood-sensitive than plants supplemented with inorganic N. This study evaluated the response to flooding and N source of biomass and N accumulation in various soybean genotypes. Soybean plants were grown in a potting mixture in a greenhouse and flooded for 21 d in degassed nutrient solution. An additional experiment evaluated root hypoxia by exposing roots of plants to a gas mixture supplying 1.5 kPa pO2. Dry matter and N were determined at various times following the initiation of flood or low O2 treatment. In all experiments, N2 fixation was more sensitive to flooding than was biomass accumulation. The decrease in N2 fixation occurred faster (within 7 d of flooding) than the decrease in biomass (within 14-21 d), and the decrease in N2 fixation was more pronounced than the decrease in biomass. Addition of nitrate decreased flood sensitivity relative to plants dependent on N2 fixation. Plant response to hypoxia was similar to flooding. Biomass of plants with roots exposed to 1.5 kPa pO2 was decreased by 34% when dependent on N2 fixation and 12% when supplemented with nitrate. Collectively, the data indicate that decreased soybean growth under flooding is a result of decreased N2 fixation and that supplementation of soybean plants with nitrate may improve their tolerance to flooding relative to those relying on N2 fixation.Keywords: Soybean, Glycine max, flooding stress, hypoxia, N source, nitrogen fixation.      

Legume species identity and soil nitrogen supply determine symbiotic nitrogen-fixation responses to elevated atmospheric [CO2

In nitrogen (N)-limited systems, the response of symbiotic N fixation to elevated atmospheric [CO2] may be an important determinant of ecosystem responses to this global change. Experimental tests of the effects of elevated [CO2] have not been consistent. Although rarely tested, differences among legume species and N supply may be important. In a field free-air CO2 enrichment (FACE) experiment, we determined, for four legume species, whether the effects of elevated atmospheric [CO2] on symbiotic N fixation depended on soil N availability or species identity. Natural abundance and pool-dilution 15N methods were used to estimate N fixation. Although N addition did, in general, decrease N fixation, contrary to theoretical predictions, elevated [CO2] did not universally increase N fixation. Rather, the effect of elevated [CO2] on N fixation was positive, neutral or negative, depending on the species and N addition. Our results suggest that legume species identity and N supply are critical factors in determining symbiotic N-fixation responses to increased atmospheric [CO2].     

Contemporaneous N(2) Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N(2) fixation associated with mats. The ability to contemporaneously conduct O(2)-sensitive N(2) fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O(2) evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N(2) fixation (nitrogenase activity), indicating potential inhibition of N(2) fixation by O(2) production. However, some degree of light-mediated N(2) fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of CO(2) fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) CO(2) fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N(2) fixation during illumination, with N(2) fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N(2) fixation.     

Emerging patterns of marine nitrogen fixation

Biological N(2) fixation is an important part of the marine nitrogen cycle as it provides a source of new nitrogen that can support biological carbon export and sequestration. Research in the past decade has focused on determining the patterns of distribution and abundance of diazotrophs, defining the environmental features leading to these patterns and characterizing the factors that constrain marine N(2) fixation overall. In this Review, we describe how variations in the deposition of iron from dust to different ocean basins affects the limiting nutrient for N(2) fixation and the distribution of different diazotrophic species. However, many questions remain about marine N(2) fixation, including the role of temperature, fixed nitrogen species, CO(2) and physical forcing in controlling N(2) fixation, as well as the potential for heterotrophic N(2) fixation.     

Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids

Decreased N2 fixation in soybean (Glycine max) L. Merr. during water deficits has been associated with increases in ureides and free amino acids in plant tissues, indicating a potential feedback inhibition by these compounds in response to drought. We evaluated concentrations of ureides and amino acids in leaf and nodule tissue and the concurrent change in N2 fixation in response to exogenous ureides and soil-water treatments for the cultivars Jackson and KS4895. Exogenous ureides applied to the soil and water-deficit treatments inhibited N2 fixation by 85% to 90%. Mn fertilization increased the apparent catabolism of ureides in leaves and hastened the recovery of N2 fixation following exogenous ureide application for both cultivars. Ureides and total free amino acids in leaves and nodules increased during water deficits and coincided with a decline in N2 fixation for both cultivars. N2 fixation recovered to 74% to 90% of control levels 2 d after rewatering drought-stressed plants, but leaf ureides and total nodule amino acids remained elevated in KS4895. Asparagine accounted for 82% of the increase in nodule amino acids relative to well-watered plants at 2 d after rewatering. These results indicate that leaf ureides and nodule asparagine do not feedback inhibit N2 fixation. Compounds whose increase and decrease in concentration mirrored the decline and recovery of N2 fixation included nodule ureides, nodule aspartate, and several amino acids in leaves, indicating that these are potential candidate molecules for feedback inhibition of N2 fixation.     

A simple model of feedback regulation for nitrate uptake and N2 fixation in contrasting phenotypes of white clover

A simple three equation model is proposed for the feedback regulation of nitrate uptake and N2 fixation, based on the concentration of the organic N substrate pool within the plant and two parameters denoting the N substrate concentrations at which half-maximal inhibition occurs. This model simulated three contrasting phenotypes of white clover (Trifolium repens L.) inbred lines with (1) normal rates of nitrate uptake and N2 fixation (NNU); (2) low rates of nitrate uptake (LNU); and (3) very low rates of N2 fixation (VLF). The LNU phenotype was simulated by a decrease in the value of the inhibition parameter for nitrate uptake and the VLF phenotype was simulated by a decrease in the value of the N2 fixation inhibition parameter. The model was tested against nitrate uptake data obtained from white clover plants growing in flowing nutrient culture. There was an accurate prediction of the increase in nitrate uptake caused by N2 fixation activity of the NNU and LNU inbred lines being interrupted by a switch in gas phase from air to Ar : O2. The model was also tested against data for nitrate uptake, N2 fixation and %N from fixation for the three inbred clover lines grown in flowing nutrient culture at 0, 5 or 20 mmol m(-3) N(3-). Again there was accurate prediction of nitrate uptake, although simulated values for N2 fixation were more variable. The simple model has potential use as a sub-routine in larger models of legume growth under field conditions.     

Understanding the variability in soybean nitrogen fixation across agroecosystems

Legume-based cropping systems have the potential to internally regulate N cycling due to the suppressive effect of soil N availability on biological nitrogen fixation. We used a gradient of endogenous soil N levels resulting from different management legacies and soil textures to investigate the effects of soil organic matter dynamics and N availability on soybean (Glycine max) N₂ fixation. Soybean N₂ fixation was estimated on 13 grain farm fields in central New York State by the ¹⁵N natural abundance method using a non-nodulating soybean reference. A range of soil N fractions were measured to span the continuum from labile to more recalcitrant N pools. Soybean reliance on N₂ fixation ranged from 36% to 82% and total N₂ fixed in aboveground biomass ranged from 40 to 224 kg N ha^?1. Soil N pools were consistently inversely correlated with % N from fixation and the correlation was statistically significant for inorganic N and occluded particulate organic matter N. However, we also found that soil N uptake by N₂-fixing soybeans relative to the non-nodulating isoline increased as soil N decreased, suggesting that N₂ fixation increased soil N scavenging in low fertility fields. We found weak evidence for internal regulation of N₂ fixation, because the inhibitory effects of soil N availability were secondary to the environmental and site characteristics, such as soil texture and corresponding soil characteristics that vary with texture, which affected soybean biomass, total N₂ fixation, and net N balance.     

15N discrimination and the sensitivity of nitrogen fixation to changes in dietary nitrogen in Reticulitermes flavipes (Isoptera: Rhinotermitidae)

Xylophagous termites possess symbiotic bacteria that fix atmospheric nitrogen (N(2)). Although symbiotic N(2) fixation is central to termite nutrition and ecologically important, it is energetically costly. Using stable isotopes, we tested the hypothesis that symbiotic N(2) fixation would decrease in workers of the eastern subterranean termite, Reticulitermes flavipes Kollar, which were exposed to high nitrogen diets. To calculate the isotope discrimination factor occurring as a result of digestion, Δ(dig), and which occurs as the result of N(2) fixation, Δ(fix), symbiotic N(2) fixation was inhibited via force feeding termites the antibiotic kanamycin. Antibiotic-treated termites and control (N(2)-fixing) termites were exposed to different concentrations of dietary N (0, 0.21, and 0.94% N), their (15)N signatures were obtained, and the percent nitrogen derived from the atmosphere within termite samples was calculated. As we hypothesized, symbiotic N(2) fixation rates were negatively correlated with dietary N, suggesting that high concentrations of dietary N suppressed symbiotic N(2) fixation in R. flavipes. A comparison of the (15)N isotope signatures of antibiotic-treated termites with their food sources demonstrated that Δ(dig) = 2.284‰, and a comparison of the (15)N isotope signatures of antibiotic-treated termites with control termites indicated that Δ(fix) = -1.238‰. These are the first estimates of Δ(dig) for R. flavipes, and the first estimate of Δ(fix) for any N(2)-fixing termite species.     

Stimulation of Symbiotic N2 Fixation in Trifolium repens L. under Elevated Atmospheric pCO2 in a Grassland Ecosystem

Symbiotic N2 fixation is one of the main processes that introduces N into terrestrial ecosystems. As such, it may be crucial for the sequestration of the extra C available in a world of continuously increasing atmospheric CO2 partial pressure (pCO2). The effect of elevated pCO2 (60 Pa) on symbiotic N2 fixation (15N-isotope dilution method) was investigated using Free-Air-CO2-Enrichment technology over a period of 3 years. Trifolium repens was cultivated either alone or together with Lolium perenne (a nonfixing reference crop) in mixed swards. Two different N fertilization levels and defoliation frequencies were applied. The total N yield increased consistently and the percentage of plant N derived from symbiotic N2 fixation increased significantly in T. repens under elevated pCO2. All additionally assimilated N was derived from symbiotic N2 fixation, not from the soil. In the mixtures exposed to elevated pCO2, an increased amount of symbiotically fixed N (+7.8, 8.2, and 6.2 g m-2 a-1 in 1993, 1994, and 1995, respectively) was introduced into the system. Increased N2 fixation is a competitive advantage for T. repens in mixed swards with pasture grasses and may be a crucial factor in maintaining the C:N ratio in the ecosystem as a whole.     

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 N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) 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 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) 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 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-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 N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

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.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr)

The relationship between ureide N and N₂ fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N₂ fixation in soybean and lima bean correlated with ureide N. In soybean, N₂ fixation, ureide N, acetylene reduction, and nodule mass were correlated when N₂ fixation was inhibited by applying KNO₃ solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N₂ fixation. Ureide N determinations in field studies with soybean correlated with N₂ fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N₂ fixation was estimated by ¹⁵N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N₂ fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N₂ fixation and ureide N. N₂ fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N₂ fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N₂ fixation.     

Oxygen-poor microzones as potential sites of microbial n(2) fixation in nitrogen-depleted aerobic marine waters

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N(2). Bioassays aimed at identifying environmental factors controlling the development and proliferation of N(2) fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N(2) fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N(2) fixation. In all cases bacterial N(2) fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O(2). Microelectrode O(2) profiles revealed that aggregates had lower internal O(2) tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O(2) was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N(2) fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N(2) fixation responses of these waters.     

Review article. Symbiotic N2 fixation response to drought

Symbiotic nitrogen fixation is highly sensitive to drought, which results in decreased N accumulation and yield of legume crops. The effects of drought stress on N2 fixation usually have been perceived as a consequence of straightforward physiological responses acting on nitrogenase activity and involving exclusively one of three mechanisms: carbon shortage, oxygen limitation, or feedback regulation by nitrogen accumulation. The sensitivity of the nodule water economy to the volumetric flow rate of the phloem into the nodule offers a common framework to understand each of these mechanism. As these processes are sensitive to volumetric phloem flow into the nodules, variations in phloem flow as a result of changes in turgor pressure in the leaves are likely to cause rapid changes in nodule activity. This could explain the special sensitivity of N2 fixation to drying soils. It seems likely that N feedback may be especially important in explaining the response mechanism in nodules. A number of studies have indicated that a nitrogenous signal(s), associated with N accumulation in the shoot and nodule, exists in legume plants so that N2 fixation is inhibited early in soil drying. The existence of genetic variation in N2 fixation response to water deficits among legume cultivars opens the possibility for enhancing N2 fixation tolerance to drought through selection and breeding.     

Denitrification and nitrogen fixation dynamics in the area surrounding an individual ghost shrimp (Neotrypaea californiensis) burrow system

Bioturbated sediments are thought of as areas of increased denitrification or fixed-nitrogen (N) loss; however, recent studies have suggested that not all N may be lost from these environments, with some N returning to the system via microbial dinitrogen (N(2)) fixation. We investigated denitrification and N(2) fixation in an intertidal lagoon (Catalina Harbor, CA), an environment characterized by bioturbation by thalassinidean shrimp (Neotrypaea californiensis). Field studies were combined with detailed measurements of denitrification and N(2) fixation surrounding a single ghost shrimp burrow system in a narrow aquarium (15 cm by 20 cm by 5 cm). Simultaneous measurements of both activities were performed on samples taken within a 1.5-cm grid for a two-dimensional illustration of their intensity and distribution. These findings were then compared with rate measurements performed on bulk environmental sediment samples collected from the lagoon. Results for the aquarium indicated that both denitrification and N(2) fixation have a patchy distribution surrounding the burrow, with no clear correlation to each other, sediment depth, or distance from the burrow. Field denitrification rates were, on average, lower in a bioturbated region than in a seemingly nonbioturbated region; however, replicates showed very high variability. A comparison of denitrification field results with previously reported N(2) fixation rates from the same lagoon showed that in the nonbioturbated region, depth-integrated (10 cm) denitrification rates were higher than integrated N(2) fixation rates (～9 to 50 times). In contrast, in the bioturbated sediments, depending on the year and bioturbation intensity, some (～6.2%) to all of the N lost via denitrification might be accounted for via N(2) fixation.     

Nitrogen fixation in soybean as influenced by cultivar and Rhizobium strain

Summary The¹⁵N-substratum labeling technique and other indirect methods were used to compare nitrogen (N₂) fixation in soybean varieties grown in the field in Greece and Romania. Significant variation in the amount (Ndfa) and proportion of N derived from fixation (% Ndfa) was found in different varieties. With 20 kg N/ha applied to soil, N₂ fixed ranged from 22 to 236 kg N/ha in Greece and from 17 to 132 kg N/ha in Romania. In general, varieties or treatments with higher dry matter yield supported greater fixation. Also, varieties with high Ndfa had high % Ndfa andvice versa. Breeding N₂-fixing legumes for high yields at low soil N levels therefore appears to be a reasonable strategy for enhancing N₂ fixation. Heavy applications of inorganic N fertilizer severely depressed N₂ fixation in two out of the three varieties used in Romania. One variety, F 74–412, however, derived slightly higher amounts of N₂ from fixation at 100 kg N/ha rate than when fertilized with 20 kg N/ha. In Greece, Chippewa, Williams and Amsoy-71 inoculated with a Nitragin inoculant fixed similar amounts of N₂ at both 20 and 100 kg N/ha fertilizer rates. However, when Chippewa and Williams were inoculated with amother, locally-isolated Rhizobium strain, N₂ fixation was substantially depressed at the higher N rate.     

A Simple, High-Precision, High-Sensitivity Tracer Assay for N(inf2) Fixation

We describe a simple, precise, and sensitive experimental protocol for direct measurement of N(inf2) fixation using the conversion of (sup15)N(inf2) to organic N. Our protocol greatly reduces the limit of detection for N(inf2) fixation by taking advantage of the high sensitivity of a modern, multiple-collector isotope ratio mass spectrometer. This instrument allowed measurement of N(inf2) fixation by natural assemblages of plankton in incubations lasting several hours in the presence of relatively low-level (ca. 10 atom%) tracer additions of (sup15)N(inf2) to the ambient pool of N(inf2). The sensitivity and precision of this tracer method are comparable to or better than those associated with the C(inf2)H(inf2) reduction assay. Data obtained in a series of experiments in the Gotland Basin of the Baltic Sea showed excellent agreement between (sup15)N(inf2) tracer and C(inf2)H(inf2) reduction measurements, with the largest discrepancies between the methods occurring at very low fixation rates. The ratio of C(inf2)H(inf2) reduced to N(inf2) fixed was 4.68 (plusmn) 0.11 (mean (plusmn) standard error, n = 39). In these experiments, the rate of C(inf2)H(inf2) reduction was relatively insensitive to assay volume. Our results, the first for planktonic diazotroph populations of the Baltic, confirm the validity of the C(inf2)H(inf2) reduction method as a quantitative measure of N(inf2) fixation in this system. Our (sup15)N(inf2) protocols are comparable to standard C(inf2)H(inf2) reduction procedures, which should promote use of direct (sup15)N(inf2) fixation measurements in other systems.     

Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests

Biological di-nitrogen fixation (N(2)) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N(2) fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N(2) conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N(2) fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N(2) fixation. Fixation is uniformly favored in surface organic soil horizons--a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N(2) fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought.