High Rates Of Nitrogen Fixation Research Articles

Anthropogenic impacts on nitrogen fixation rates between restored and natural Mediterranean salt marshes

AbstractTo test the effects of site and successional stage on nitrogen fixation rates in salt marshes of the Venice Lagoon, Italy, acetylene reduction assays were performed with Salicornia veneta‐ and Spartina townsendii‐vegetated sediments from three restored (6–14 years) and two natural marshes. Average nitrogen fixation (acetylene reduction) rates ranged from 31 to 343 μmol C2H4·m−2·h−1 among all marshes, with the greatest average rates being from one natural marsh (Tezze Fonde). These high rates are up to six times greater than those reported from Southern California Spartina marshes of similar Mediterranean climate, but substantially lower than those found in moister climates of the Atlantic US coast. Nitrogen fixation rates did not consistently vary between natural and restored marshes within a site (Fossei Est, Tezze Fonde, Cenesa) but were negatively related to assayed plant biomass within the acetylene reduction samples collected among all marshes. Highest nitrogen fixation rates were found at Tezze Fonde, the location closest to the city of Venice, in both natural and restored marshes, suggesting possible site‐specific impacts of anthropogenic stress on marsh succession.

Effect of Cyanophycin Metabolism in Recombinant Sinorhizobium (Ensifer) meliloti 1021 on the Symbiosis with Alfalfa (Medicago sativa)

The aim of this study was to investigate the effect of the synthesis and degradation of the nitrogen-rich polymer cyanophycin in rhizobia on symbiotic nitrogen fixation and crop yield in legumes. For this, cyanophycin synthetase from Anabaena sp. PCC7120 was expressed in the bacteroids of the symbiont S. meliloti 1021 either alone or together with an intracellular cyanophycinase from the same bacterium, either in the wild type or in a polyhydroxybutyratenegative (PHB-) mutant, and the effect on the growth of alfalfa host plants was studied. All strains induced the formation of nitrogen-fixing nodules in the host, but clear differences in various parameters were noticeable. Alfalfa plants infected with the wild type expressing only cyanophycin synthetase showed significantly lower shoot nitrogen contents and higher nitrogen fixation rates than plants inoculated with wild type, but the wild type phenotype was exceeded in S. meliloti 1021 expressing cyanophycinase in addition to cyanophycin synthetase. Growth of plants infected with the PHB- mutant expressing only cyanophycin synthetase was severely impaired in comparison to growth of plants infected with the PHB- mutant expressing both or neither of the two proteins. Transmission electron micrographs of sections of nodules induced by wild type S. meliloti 1021 producing cyanophycin synthetase and cyanophycinase showed that the rough endoplasmic reticulum and Golgi membranes were extended further in comparison to the nodules induced by the wild type, the PHB- mutant, or all other recombinant S. meliloti strains, indicating higher metabolic activities in these nodules.

The study of symbiotic traits – nodulation and activity of nitrogen fixation – in different cultivars of spring vetch (Vicia sativa L.)

Common vetch Vicia sativa L. is one of the major annual fodder legume crops in Russia. Like other legumes, it can enter into symbiosis with nodule bacteria of the genus Rhizobium and to fix molecular nitrogen from the air. This ability contributes to the enrichment of soils with nitrogen and the possibility of growing crops on poor soils. Vetch symbiotic genes have not yet been identified, so the breeding of this crop is confined to the assessment of breeding material with regard to nodulation and very rarely to the activity of nitrogen fixation measured by the acetylene method. This paper presents the results of studies of nodulation and nitrogen-fixing activity that we conducted with four spring vetch cultivars raised at the Siberian Research Institute for Plant Industry and Breeding. Two varieties of forage pea, namely Druzhnaya and traditional cv. Falenskaya 42, were taken as references. The activity of nitrogen fixation was assessed from nitrogenase activity, assayed by the acetylene method. The study revealed significant varietal differences in nodulation and nitrogen fixation, determined on a gas chromatograph Tsvet 500, Russia. The nitrogenase activity rates in vetch cultivars under study varied from 2811 to 6890 С2Н4 nmol/ plant/h. Two of them, Lena and Priobskaya 25, were prominent in high nodulation nitrogen fixation rates. Nodulation in the two reference pea cultivars was weaker than in vetch. However, their nitrogen fixation activities were higher.

High cell-specific rates of nitrogen and carbon fixation by the cyanobacterium Aphanizomenon sp. at low temperatures in the Baltic Sea.

Aphanizomenon is a widespread genus of nitrogen (N2)-fixing cyanobacteria in lakes and estuaries, accounting for a large fraction of the summer N2-fixation in the Baltic Sea. However, information about its cell-specific carbon (C)- and N2-fixation rates in the early growth season has not previously been reported. We combined various methods to study N2-fixation, photosynthesis and respiration in field-sampled Baltic Sea Aphanizomenon sp. during early summer at 10°C. Stable isotope incubations at in situ light intensities during 24 h combined with cell-specific secondary ion mass spectrometry showed an average net N2-fixation rate of 55 fmol N cell(-1) day(-1). Dark net N2-fixation rates over a course of 12 h were 20% of those measured in light. C-fixation, but not N2-fixation, was inhibited by high ambient light intensities during daytime. Consequently, the C:N fixation ratio varied substantially over the diel cycle. C- and N2-fixation rates were comparable to those reported for Aphanizomenon sp. in August at 19°C, using the same methods. High respiration rates (23% of gross photosynthesis) were measured with (14)C-incubations and O2-microsensors, and presumably reflect the energy needed for high N2-fixation rates. Hence, Aphanizomenon sp. is an important contributor to N2-fixation at low in situ temperatures in the early growth season.

Iron redox cycling and subsurface offshore transport in the eastern tropical South Pacific oxygen minimum zone

The distributions of dissolved iron (Fe), Fe(II) and Fe(III)-binding organic ligands were investigated in the upper 1000m of the eastern tropical South Pacific from January to March 2010, during an El Niño event. Dissolved Fe concentrations were exceedingly low in surface waters, showed minima near the chlorophyll maxima, and increased below that depth. High rates of nitrogen fixation have been inferred for this region from models, but our data suggest that surface Fe is frequently too low to support diazotrophs. Dissolved Fe and organic Fe(III) ligand concentrations at mid-depth were elevated in the nearshore stations, where virtually all dissolved Fe(III) was bound to these ligands. Subsurface Fe(II) maxima were observed in the secondary nitrite maxima of the oxygen minimum zone (OMZ), comprising 8 to 68% of dissolved Fe. Dissolved Fe concentrations displayed local maxima coinciding with the maxima in Fe(II) and nitrite. We propose that this zone, the most reducing part of the OMZ, plays an important role in Fe transport in the upper 400m of the water column.

The paradox of marine heterotrophic nitrogen fixation: abundances of heterotrophic diazotrophs do not account for nitrogen fixation rates in the Eastern Tropical South Pacific

Results of recent modelling efforts imply denitrification-influenced waters, such as those in the Eastern Tropical South Pacific (ETSP), may support high rates of biological nitrogen fixation (BNF), yet little is known about the N2 -fixing microbial community in this region. Our characterization of the ETSP diazotrophic community along a gradient from upwelling-influenced to oligotrophic waters did not detect cyanobacterial diazotrophs commonly found in other open ocean regions. Most of the nifH genes amplified by polymerase chain reaction (PCR) from DNA and RNA samples clustered with γ-proteobacterial nifH sequences, although a novel Trichodesmium phylotype was also recovered. Three quantitative PCR assays were developed to target γ-proteobacterial phylotypes, but all were found to be present at low abundances. An analysis of the expected BNF rates based on abundances and plausible cell-specific N2 fixation rates indicates that these γ-proteobacteria are unlikely to be responsible for previously reported BNF rates from corresponding samples. Therefore, the organisms responsible for the measured BNF rates remain poorly understood. Furthermore, there is little direct evidence, at this time, to support the hypothesis that heterotrophic N2 fixation contributes significantly to oceanic BNF rates based on our analysis of heterotrophic cell-specific N2 fixation rates required to explain BNF rates reported in previously published studies.

Rhizobium freirei sp. nov., a symbiont of Phaseolus vulgaris that is very effective at fixing nitrogen

Common bean (Phaseolus vulgaris L.) can establish symbiotic associations with several Rhizobium species; however, the effectiveness of most strains at fixing nitrogen under field conditions is very low. PRF 81(T) is a very effective strain, usually referred to as Rhizobium tropici and used successfully in thousands of doses of commercial inoculants for the common bean crop in Brazil; it has shown high rates of nitrogen fixation in all areas representative of the crop in the country. Here, we present results that indicate that PRF 81(T), although it belongs to the 'R. tropici group', which includes 10 Rhizobium species, R. tropici, R. leucaenae, R. lusitanum, R. multihospitium, R. miluonense, R. hainanense, R. calliandrae, R. mayense, R. jaguaris and R. rhizogenes, represents a novel species. Several morpho-physiological traits differentiated PRF 81(T) from related species. Differences were also confirmed in the analysis of rep-PCR (sharing less than 45 % similarity with the other species), MLSA with recA, atpD and rpoB genes, and DNA-DNA hybridization. The novel species, for which we propose the name Rhizobium freirei sp. nov., is able to establish effective root nodule symbioses with Phaseolus vulgaris, Leucaena leucocephala, Leucaena esculenta, Crotalaria juncea and Macroptilium atropurpureum. The type strain is PRF 81(T) ( = CNPSo 122(T) = SEMIA 4080(T) = IPR-Pv81(T) = WDCM 440(T)).

High rates of nitrogen fixation in equatorial upwelling region

Surface waters in upwelling regions of the ocean are generally rich in nutrients. Scientists had thought that these areas would have low rates of nitrogen fixation because diazotrophs—microbes that convert nitrogen gas from the atmosphere into usable forms, such as ammonia—could use the nutrients in the water directly instead of having to fix nitrogen gas. However, researchers recently recorded high rates of nitrogen fixation in an upwelling region in the equatorial Atlantic.

Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.

Nitrogen Fixation in Preserved, Reforested, Naturally Regenerated and Impaired Mangroves as an Indicator of Functional Restoration in Mangroves in an Arid Region of Mexico

Although several damaged mangrove ecosystems have been restored worldwide, so far, it has not been established whether a restored mangrove system regains all the functional properties of preserved mangroves. This study measured nitrogen fixation as an indicator of whether disturbed mangroves that were reforested or naturally regenerated fully recovered from this disturbance at a functional level. Rates of nitrogen fixation were measured for one year in impaired, preserved, reforested, and naturally regenerated mangroves dominated by the black mangrove (Avicennia germinans). There was no significant difference in rates of nitrogen fixation among preserved and adjacent reforested and naturally regenerated mangroves, but a significant reduction occurred in an impaired mangrove. Nitrogen fixation was mainly controlled by pH, salinity, and temperature. The highest rates of nitrogen fixation occurred in summer at pH values less than 6.4, whereas the impaired mangrove had higher pH and salinity and had very low nitrogen fixation activity. These results suggest that nitrogen fixation can be used as an ecological indicator of the success of reforestation and as a sensitive measure of perturbations in mangroves.

The effect of abiotic factors on asymbiotic nitrogen fixation in dead roots from the Pacific Northwest

High rates of asymbiotic nitrogen fixation have been observed in dead roots. However, little is known about how biotic and abiotic factors influence asymbiotic nitrogen fixation in dead roots. The objective of this study was to examine how nitrogen fixation in dead roots responded to three abiotic factors: temperature, oxygen concentration, and moisture content by measuring acetylene reduction rates in dead roots. For measuring the response to temperature and oxygen, we collected dead roots of Douglas-fir (Pseudotsuga menziesii) from one site in western Oregon, while the effect of moisture content was tested with samples from three species including Douglas-fir, western hemlock (Tsuga heterophylla), and Ponderosa pine (Pinus ponderosa) from three sites in western Oregon. We measured acetylene reduction rates at 5, 13, 20, 30, and 40°C and at oxygen concentrations of 0, 1, 2, 4, 8, and 21% in dead root samples that had been previously wetted. Unaltered root samples with different moisture contents were used to measure the effect of moisture content on acetylene reduction. The optimal temperature for acetylene reduction in roots was 20°C with an acetylene reduction rate of 9.6 nmol/g/d and a standard error of 3.2 nmol/g/d. Asymbiotic nitrogen fixation was greatly reduced above and below this temperature with acetylene reduction rates of 1.2 ± 0.6 nmol/g/d at 5°C and 0.2 ± 0.2 nmol/g/d at 40°C. The highest acetylene reduction rates (13.8 ± 4.2 nmol/g/d) occurred at an oxygen concentration of 2%. Rates dropped sharply under no oxygen condition (3.0 ± 2.8 nmol/g/d) and at an oxygen concentration of 21% (3.0 ± 0.8 nmol/g/d). Optimal acetylene reduction activity occurred above 60% root moisture content and activity was near zero below 60%. These abiotic factors have a similar influence on asymbiotic nitrogen fixation activity in other substrates like dead wood. In the Pacific Northwest, annual nitrogen fixation activity in dead roots tends to be primarily controlled by temperature while above-ground substrates like dead wood can have reduced activity during the dry summer months when lower moisture contents and higher oxygen concentrations can reduce activity.

Actinorhizal plant defence-related genes in response to symbiotic Frankia.

Actinorhizal plants have become increasingly important as climate changes threaten to remake the global landscape over the next decades. These plants are able to grow in nutrient-poor and disturbed soils, and are important elements in plant communities worldwide. Besides that, most actinorhizal plants are capable of high rates of nitrogen fixation due to their capacity to establish root nodule symbiosis with N2-fixing Frankia strains. Nodulation is a developmental process that requires a sequence of highly coordinated events. One of these mechanisms is the induction of defence-related events, whose precise role in a symbiotic interaction remains to be elucidated. This review summarises what is known about the induction of actinorhizal defence-related genes in response to symbiotic Frankia and their putative function during symbiosis.

Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root-nodule symbiosis with N2-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non-inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.

Retention of nitrogen, phosphorus and silicon in a large semi-arid riverine lake system

Lakes and reservoirs (impoundments) are often viewed as a sink for nutrients within the river continuum. To date, most studies on nutrient retention within impoundments are derived from the temperate climate zones of Europe and North America, only consider one nutrient, and are often short-term (1–2 years). Here, we present a long-term (17 year) data set and nutrient (nitrogen, phosphorus and silica) budget for two connected semi-arid lakes (the Lower Lakes) at the terminus of the River Murray, Australia. Most of the filterable reactive phosphorus and nitrate entering the lakes were retained (77 and 92%, respectively). Total phosphorus (TP) was also strongly retained (55% of the annual TP load on average) and the annual TP retention rates could be predicted as a function of the areal hydraulic loading rate (annual lake outflow/lake surface area). On average, there was a slight net retention (7%) of the annual total nitrogen (TN) load but a slight net export (6% of the load) of organic N. TN retention as function of the areal hydraulic loading rate was lower than expected from existing models, possibly because of high nitrogen fixation rates in the Lower Lakes. Silica was retained (39%) at similar rates to those observed in previous studies. There was also a marked increase in the TN:TP and TN:Si ratios within the lake (TN:TP~30 and TN:Si~0.67) compared to those entering (TN:TP~15, TN:Si~0.45), as a consequence of the relatively low net retention of nitrogen.

Herbivory by an invasive snail increases nitrogen fixation in a nitrogen-limited stream

Despite anthropogenic nitrogen contributions, nitrogen fixation contributes half of biosphere inputs but has rarely been quantified in streams. Herbivory controls algal biomass and productivity in streams, and we hypothesized that herbivory could also control nitrogen fixation. We released periphyton from herbivory in nitrogen-limited Polecat Creek, Wyoming, where heavy grazing by the invasive New Zealand mudsnail ( Potamopyrgus antipodarum ) dominates nitrogen cycling. One and two weeks after releasing periphyton, we found higher rates of nitrogen fixation on heavily grazed rocks (two-way analysis of variance (ANOVA), p = 0.012). Time elapsed after snail manipulation had no effect (two-way ANOVA, p = 0.24). Grazing changed periphyton composition by reducing the proportion of green algae and increasing the proportion of nitrogen-fixing diatoms (multivariate ANOVA, p = 0.001). Nitrogen fixation rates increased disproportionately to nitrogen-fixing algal cells, indicating that snails increased nitrogenase efficiency, probably by improving light and (or) nutrient availability to nitrogen fixers. We incorporated our nitrogen fixation rates into a published nitrogen budget for Polecat Creek and found that nitrogen flux into the periphyton was 50% higher when we included nitrogen fixation. Herbivory can increase nitrogen fixation in streams, and future studies should measure nitrogen fixation for a more thorough understanding of stream nitrogen cycling.

Interactive influence of phosphorus and iron on nitrogen fixation by soybean

Adequate supplies of phosphorus (P) and iron (Fe) to legumes have been shown to be crucial in obtaining high nitrogen fixation rates and growth. These responses are anticipated as a result of the high requirement for P in energy transfer processes in the nodule and for Fe as a constituent of nitrogenase and leghemoglobin. However, little attention has been given to documenting the response of nitrogen fixation rates resulting from concentrations of P and Fe that actually exist in nodules. In particular, an open question is whether there is an interaction between nodule P and Fe concentrations that maximize nitrogen fixation activity. This study was designed to induce various concentrations of P and Fe in the nodule and to measure the resultant nitrogen accumulation and nitrogen fixation rates. Plant nitrogen accumulation was linearly correlated with both nodule P and Fe concentration, and with total plant nitrogen fixation rate as measured by acetylene reduction rate. Therefore, total nitrogen fixation rate was also correlated with nodule P and Fe concentrations, but a higher linear correlation was obtained for Fe as compared to P concentration. Surprisingly, nodule ureide concentration, which is generally assumed to be a positive index of nitrogen fixation rate, was negatively correlated with nodule P and Fe concentrations. These results indicated that higher concentrations of P and Fe in the nodules not only stimulated higher nitrogen fixation rates, but were associated with an enhanced ability to export ureides from the nodules. Since there was a linear response to both P and Fe over the range of nodule concentrations induced in these experiments, no evidence for optimum interactive concentrations of these two elements in the nodules was obtained.

Composition of nifH in a wastewater treatment system reliant on N2 fixation

High levels of nitrogen fixation have been observed in the wastewaters of pulp and paper mills. In this study, we show that nitrogen fixation in a model pulp and paper wastewater treatment system is supported by a high density of nifH sequences that are of low diversity. Quantitative PCR revealed a ratio of nifH to 16S rDNA of 1.14 +/- 0.76 which shows that very high levels of the nifH gene were enriched to support the high rates of nitrogen fixation that occur in this wastewater. Changes in wastewater composition and dissolved oxygen levels did not affect the nifH levels and allowed stable wastewater treatment. The nifH sequences identified display a similar profile to those seen in forest soil environments where nifH sequences derived from alpha-proteobacteria and beta-proteobacteria are also prevalent.

Spatial and seasonal variations of Cyanobacteria and their nitrogen fixation rates in Sanya Bay, South China Sea

The nitrogen fixation rates of planktonic and intertidal benthic cyanobacteria were investigated in Sanya Bay from 2003 to 2005. Trichodesmium thiebautii was the dominant species of planktonic cyanobacteria during our study. Significant seasonal and spatial variations in Trichodesmium spp. abundance were observed (P < 0.01). The highest Trichodesmium concentrations occurred during intermonsoon periods and in the outer region of Sanya Bay (Outer Bay stations). At fixed station S03 the abundance of T. thiebautii ranged from 1.14 x 10(3) to 2060 x 10(3) trichomes m(-2), with an annual mean of 273 x 10(3) trichomes m(-2). The average nitrogen fixation rate per colony of T thiebautii was 0.27 nmol N h(-1) colony(-1) and it did not show any obvious seasonal variations. Nitrogen fixation by planktonic cyanobacteria was highest in the Outer Bay stations, where the estimated amount of new nitrogen introduced by Trichodesmium contributed 0.03 to 1.63% of the total primary production and up to 11.64% of the new production. Statistical results showed that significant seasonal and spatial variations of nitrogen fixation rates were found among the intertidal communities. The main benthic nitrogen-fixing cyanobacteria were identified as members of the genera Anabaena, Calothrix, Lyngbya, Nostoc and Oscillatoria. The highest nitrogen fixation rate was found in microbial mats and the lowest in reefs and rocky sediments. All the benthic communities studied presented their highest nitrogen fixation activity in summer, with an average nitrogen fixation rate of 33.31 mu mol N h(-1) m(-2), whereas the lowest nitrogen activity was detected in winter, with an average nitrogen fixation rate of 5.66 mu mol N h(-1) m(-2). A Pearson correlation analysis indicated that the nitrogen fixation rate of three types of intertidal communities was significantly positively correlated to seawater temperature (P < 0.05), whereas only the nitrogen fixation rate of the reefs and rock communities was significantly negatively correlated to seawater salinity (P < 0.05).

High rates of nitrogen fixation (acetylene reduction) on coral skeletons following bleaching mortality

Nubbins of the coral Acropora aspera were artificially bleached and nitrogen fixation ( acetylene reduction) rates were measured on the developing epilithic communities. Seasonal comparisons were made between corals that died in summer of heat stress and corals that died in winter from natural cold stress. Rates of acetylene reduction from artificially bleached corals peaked at 26.66 nmol cm(-2) h(-1) 2 weeks after summer mortality, while rates from natural winter mortality peaked at 18.07 nmol cm(-2) h(-1) 12 days after coral death. Comparative rates of acetylene reduction taken from live corals and coral rubble ranged between 0.56 and 1.16 nmol cm(-2) h(-1), and 0.15 and 12.77 nmol cm(-2) h(-1), respectively. N-2-fixation rates from dead corals were up to 30 times greater than those measured on live corals. The observed increase in N-2-fixation from dead corals may increase the availability of nitrogen for use in trophic processes within the reef for an extended period following the initial mortality event. If the spatial scale over which coral mortality has occurred in past thermal bleaching events is considered the ramifications of such an increase may be substantial.

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO2 exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.