Close Association of Azospirillum and Diazotrophic Rods with Different Root Zones of Kallar Grass

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloa fusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N₂-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by ¹⁵N₂ incorporation. On the rhizoplane we found 2.0 × 10⁷ diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCI treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 × 10⁷ cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 × 10⁴ nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1%, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Ecophysiological aspects of growth and nitrogen fixation inAzospirillum spp.

The nitrogenase activity ofAzospirillum spp. is efficiently regulated by environmental factors. InA. brasilense andA. lipoferum a rapid switch off of nitrogenase activity occurs after the addition of ammonium chloride. As in photosynthetic bacteria, a covalent modification of nitrogenase reductase (Fe-protein) is involved. InA. amazonense, a non-covalent mechanism causes only a partial inhibition of nitrogenase activity after ammonium chloride is added. In anaerobic conditions, nitrogenase reductase is also switched off by a covalent modification inA. brasilense andA. lipoferum. Short-time exposure ofAzospirillum to increased oxygen levels causes a partially reversible inhibition of nitrogenase activity, but no covalent modification is involved.Azospirillum spp. show variations in their oxygen tolerance. High levels of carotenoids confer a slightly improved oxygen tolerance. Certain amino acids (e. g. glutamate, aspartate, histidine and serine) affect growth and nitrogen fixation differently inAzospirillum spp. Amino acids may influence growth and nitrogen fixation ofAzospirillum in the association with plants.Azospirillum brasilense andA. halopraeferens are the more osmotolerant species. They utilize most amino acids poorly and accumulate glycine betaine, which also occurs in osmotically stressed grasses as a compatible solute to counteract osmotic stress. Nitrogen fixation is stimulated by glycine betaine and choline. Efficient iron acquisition is a prerequisite for competitive and aerotoleran growth and for high nitrogenase activity.Azospirillum halopraeferens andA. amazonense assimilate iron reasonably well, whereas growth of someA. brasilense andA. lipoferum strains is severely inhibited by iron limitation and by competition with foreign microbial iron chelators. However, growth of certain iron-limitedA. brasilense strains is stimulated by the phytosiderophore mugineic acid. Thus, various plant-derived substances may stimulate growth and nitrogen fixation ofAzospirillum.     

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO₂/C₂H₂ ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by ¹⁵N₂ incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.     

Phenotypic and molecular characterization of native Azospirillum strains from rice fields to improve crop productivity

Beneficial microorganisms have been considered as an important tool for crop improvement. Native isolates of Azospirillum spp. were obtained from the rhizospheres of different rice fields. Phenotypic, biochemical and molecular characterizations of these isolates led to the identification of six efficient strain of Azospirillum. PCR amplification of the nif genes (nifH, nifD and nifK) and protein profile of Azospirillum strains revealed inter-generic and inter-specific diversity among the strains. In vitro nitrogen fixation performance and the plant growth promotion activities, viz. siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH₃, phosphorus metabolism, ACC deaminase and iron tolerance were found to vary among the Azospirillum strains. The effect of Azospirillum formulations on growth of rice var. Khandagiri under field condition was evaluated, which revealed that the native formulation of Azospirillum of CRRI field (As6) was most effective to elevate endogenous nutrient content, and improved growth and better yield are the result. The 16S rRNA sequence revealed novelty of native Azospirillum lipoferum (As6) (JQ796078) in the NCBI database.     

Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O₂ (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Nitrogenase Activity (Acetylene Reduction) of Root-Associated, Cold-Climate Azospirillum, Enterobacter, Klebsiella, and Pseudomonas Species During Growth on Various Carbon Sources and at Various Partial Pressures of Oxygen

A comprehensive view of the diazotrophic bacterial flora of plants requires that attention be paid to the appropriate carbon and oxygen requirements during isolation of the bacteria. Twenty compounds (monosaccharides, disaccharides, polyols, and organic acids) were therefore examined as carbon and energy sources for nitrogenase activity in semisolid stab cultures at pO₂ values of 0.21, 0.02, and ≤0.002 with 12 strains of diazotrophic root-associated bacteria. With the facultatively anaerobic bacteria of the genera Klebsiella and Enterobacter, the best substrate was sucrose, followed by fructose and mannitol, whereas among the organic acids, only malic and fumaric acids supported any activity. With the obligately aerobic bacteria of the genera Azospirillum and Pseudomonas, disaccharides were not utilized for nitrogen fixation, but several organic acids were accepted in addition to monosaccharides and polyols; malate and glucose were the best substrates. The patterns of the carbon sources utilized for nitrogen fixation were coherent within the species, with the exception of one Klebsiella pneumoniae and one Enterobacter agglomerans strain, both isolated from the same individual grass plant, which were unable to utilize lactose. Anaerobic conditions (pO₂ value of ≤0.002) were required for maximum nitrogenase activity with the facultatively anaerobic bacteria, with the exception of one strain of E. agglomerans, which required atmospheric oxygen (pO₂ value of 0.21). Also, the obligately aerobic diazotrophs required atmospheric oxygen for maximum nitrogenase activity. The maximum specific nitrogenase activities (expressed as micromoles of C₂H₄ · milligram of bacterial protein⁻¹ · hour⁻¹) noted during the exponential growth phase of the bacteria were the following: 2.68 with Azospirillum lipoferum on malate, 2.41 with K. pneumoniae and 1.58 with E. agglomerans on sucrose, and 0.95 with Pseudomonas sp. on malate.     

Denitrification and nitrogen fixation by Azospirillum

A model system is described where Azospirillum and germinated wheat seeds were grown in association for a week and then assayed for nitrogen fixation (C₂H₂-reduction) and denitrification (N₂O-formation) activities. The association performed C₂H₂-reduction and N₂O-formation under microaerobic conditions. Both activities were measurable after already 3–5 h of incubation with substantial rates and were strictly dependent on the presence of both plants and bacteria. During the week of the growth of the association, the bacteria had lived exclusively from the carbon compounds supplied by the roots of the plants. C₂H₂-reduction activity by the association was more or less the same with all the Azospirillum brasilense strains, but lower with A. lipoferum and with the A. amazonense strains tested. Two nitrogenase negative mutants of Azospirillum brasilense showed virtually no activity in the association. C₂H₂-reduction activity was strongly dependent on the growth temperature of the association. Denitrification (N₂O-formation) was high also at higher temperatures and at pH-values in the medium around 7.8 but not at neutrality and was strictly dependent on nitrate. The Azospirillum strain used strongly determined the rate of the N₂O-formation in the association. It is suggested that Azospirillum may be beneficial to crops particularly under tropical conditions.Dedicated to Professor Dr. Gerhart Drews, Freiburg, on the occasion of his 60th birthday     

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

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

Aspects of nitrogen fixation and denitrification byAzospirillum

Summary Model experiments were performed to investigate the nitrogen fixation (C₂H₂ reduction) and denitrification (N₂O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C₂H₂ reduction or N₂O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C₂H₂ reduction and low N₂O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.     

Plant-bacteria interactions with special emphasis on the kallar grass association

Kallar grass is a highly salt-tolerant grass grown as a pioneer plant on alkaline, salt-affected soils in Pakistan. Nitrogen-fixing bacteria and kallar grass were found to be in close association, which was even root-zone specific: rhizoplane and endorhizosphere were colonized by two different populations. Among theAzospirillum isolates originating from the root surface, some were of a new species, now namedA. halopraeferens. To study plant-bacterium interactions, this natural kallar grass association was chosen. The possible role of bacterial chemotaxis and oxygen tolerance are discussed.     

Isolation and characterization of nitrogen-fixing bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis> from the soil of a <Emphasis Type="Italic">Sphagnum</Emphasis> peat bog

he presence of nitrogen-fixing bacteria of the genus Azospirillum in the soils of acidic raised Sphagnum bogs is revealed for the first time. Three Azospirillum strains, B2, B21, and B22, were isolated as a component of methane-oxidizing enrichment cultures, whereas attempts to isolate them directly from peat samples have failed. The results of comparative analysis of the nucleotide sequences of 16S rRNA genes, DNA-DNA hybridization, and the analysis of the sequences of the functional genes encoding nitrogenase and ribulose-1, 5-bisphosphate carboxylase reveal that all the newly obtained strains can be classified as Azospirillum lipoferum. Yet, unlike A. lipoferum, the isolates do not require biotin and utilize sucrose, inositol, and glycerol for growth. The cell morphology of strain B2 differs from that of the type strain and strains B21 and B22. The results obtained indicate the variability of morphological, physiological, and biochemical properties in closely related Azospirillum strains and suggest the existence of metabolic relationships between methanotrophic bacteria and the representatives of the genus Azospirillum under peat bog conditions.     

The effect of combined and separate inoculation of alfalfa plants with <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> on denitrification and nitrogen-fixing activities

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

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Radial gas diffusion from roots of rice (Oryza sativa L.) and Kallar grass (Leptochloa fusca L. Kunth), and effects of inoculation withAzospirillum brasilense Cd

Cortical root air space (aerenchyma) helps rice and Kallar grass to survive flooding conditions. The dependence of the oxygen concentration in the rhizosphere on the root aerenchyma volume, the plant age,-species and plant respiration is described. Additionally diffusional effects of different types of gases are evaluated. Inoculation of the rhizosphere with the micro-aerobically N₂-fixing microorganismAzospirillum brasilense Cd brought about an increased oxygen concentration in the rhizosphere by the factor 3.3 for rice and 5.3 for Kallar grass. This effect is thought to be due to enhanced root cell wall permeability probably caused by IAA-like phytohormones released by the bacteria.     

Azospirillum strains use phenolic compounds as intermediates for electron transfer under oxygen-limiting conditions

The effects of catechol, vanillic, caffeic (CAF), 2-hydroxyphenylacetic, 4-hydroxy- and 3,4-dihydroxybenzoic (3,4-DHBA) acids on the growth of a common rice rhizosphere inhabitant, Azospirillum lipoferum were studied. Two strains of this nonfermenting nitrogen-fixing bacterium were used: a motile strain (4B), and a nonmotile strain (4T). Under atmospheric conditions (pO₂ = 21 kPa), the growth of strain 4T was inhibited by catechol (0.1 mm) only. None of these compounds affected the growth of strain 413. Under 5 kPa O₂, no effect was observed on strain 413, whereas three of the six tested phenolics stimulated the growth of strain 4T; maximum effects were observed for 3,4-DHBA and CAF. As revealed by TLC and HPLC, under low oxygen, more new lipophilic compounds were formed from CAF by strain 4T, differing from CAF autooxydation products and from the products obtained under 21 kPa O₂. It was hypothesized that strain 4T had the ability to use an oxidized derivative of CAF as a terminal electron acceptor. This hypothesis was tested in experiments under nitrogen-fixing conditions, in the absence of oxygen, and in the presence of N₂O as a reoxidizing agent for CAF. Acetylene was used both as a substrate to measure nitrogenase activity (ARA) and to inhibit the biological transfer of electrons to N₂O. The addition of CAF in the presence of N₂O had the same effect on ARA rates as an addition of oxygen. It is concluded that the strain 4T of Azospirillum lipoferum is able to sustain some of its activities (e.g., N₂ fixation) using phenolics as alternative electron acceptors under low oxygen conditions.     

INTERACTION OF NITROGEN FIXATION AND PHOSPHORUS LIMITATION IN APHANIZOMENON FLOS-AQUAE (CYANOPHYCEAE)1

The effect of simultaneous nitrogen fixation and phosphorus limitation on the physiological adaptation and growth performance of Aphanizomenon flos-aquae (L.) Ralfs PCC 7905 was studied in continuous culture. In the absence of ammonia, N₂ fixation occurred and the maximum growth rate (as determined in diluted batch cultures) was lower. However, no distinction could be made between the steady-state N uptake rates (based on cellular N contents) of N₂-fixing cells and cells grown with ammonia. At the higher dilution rates, the residual P concentration increased with increasing dilution rate, more so under N₂-fixing conditions, compared to the cultures grown in the presence of ammonia. More generally, the yield of biomass per consumed P, as the biomass concentration itself, decreased with increasing dilution rate, and both were lower under N₂-fixing conditions. The restricted biomass production under N₂-fixing conditions suggests that reduction of N loading may benefit lake restoration projects. The influence of N₂-fixation on the severity of P limitation is discussed in terms of metabolic control analysis. From the increase of the residual P concentration on switching from ammonium to N₂-fixing conditions, it is deduced that under N₂-fixing and P-limited conditions, control of growth is shared by N and P metabolism.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.