Quantification and Removal of Some Contaminating Gases from Acetylene Used to Study Gas-Utilizing Enzymes and Microorganisms

Acetylene generated from various grades of calcium carbide and obtained from commercial- and purified-grade acetylene cylinders was shown to contain high concentrations of various contaminants. Dependent on the source of acetylene, these included, at maximal values, H₂ (0.023%), O₂ (0.779%), N₂ (3.78%), PH₃ (0.06%), CH₄ (0.073%), and acetone (1 to 10%). The concentration of the contaminants in cylinder acetylene was highly dependent on the extent of cylinder discharge. Several conventional methods used to partially purify cylinder acetylene were compared. A small-scale method for extensively purifying acetylene is described. An effect of acetylene quality on acetylene reduction assays conducted with purified nitrogenase from Azotobacter vinelandii was demonstrated.     

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.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria

Acetylene (0.1 atm) caused complete or almost complete inhibition of reduction of N₂O by whole cell suspensions of Pseudomonas perfectomarinus, P. aeruginosa and Micrococcus denitrificans. Acetylene did not inhibit reduction of NO₃^? or NO₂^? by these organisms. In the presence of acetylene there was stoichiometric conversion of NO₃^? or NO₂^? to N₂O with negligible subsequent reduction of the latter. In the absence of acetylene there was no or only transient accumulation of N₂O. The data are consistent with the view that N₂O is an obligatory intermediate in the reduction of NO₂^? to N₂ in all of the three organisms studied.     

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.     

Growth and N2 fixation in an Alnus hirsuta (Turcz.) var. sibirica stand in Japan

To estimate the N₂ fixation ability of the alder (Alnus hirsuta (Turcz.) var. sibirica), we examined the seasonal variation in nitrogenase activity of nodules using the acetylene reduction method in an 18-year-old stand naturally regenerated after disturbance by road construction in Japan. To evaluate the contribution of N₂ fixation to the nitrogen (N) economy in this alder stand, we also measured the phenology of the alder, the litterfall, the decomposition rate of the leaf litter, and N accumulation in the soil. The acetylene reduction activity per unit nodule mass (ARA) under field conditions appeared after bud break, peaked the maximum in midsummer after full expansion of the leaves, and disappeared after all leaves had fallen. There was no consistent correlation between ARA and tree size (dbh). The amount of N₂ fixed in this alder stand was estimated at 56.4 kg ha^?1 year^?1 when a theoretical molar ratio of 3 was used to convert the amount of reduced acetylene to the amount of fixed N₂. This amount of N₂ fixation corresponded to the 66.4% of N in the leaf litter produced in a year. These results suggested that N₂ fixation still contributed to the large portion of N economy in this alder stand.     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Direct Measurements of Steady-State Kinetics of Cyanobacterial N2 Uptake by Membrane-Leak Mass Spectrometry and Comparisons Between Nitrogen Fixation and Acetylene Reduction

A mass spectrometer with a membrane-covered inlet was used to measure nitrogen fixation by following changes in the concentration of dissolved N₂ in a stirred suspension of the cyanobacterium Anabaena variabilis in an open system. The results showed a good fit to Michaelis-Menten kinetics with a K_m for N₂ of 65 μM at 35°C, corresponding to 0.121 atmosphere of N₂. Corresponding values for the K_m for acetylene reduction were 385 μM (0.011 atmosphere at 35°C). Comparison of the values of V_max for N₂ uptake with those for the acetylene reduction assay under similar conditions gave an average value of 3.8 for the conversion factor between N₂ and C₂H₂ reduction. Reduction of protons to hydrogen was completely inhibited at sufficiently high concentrations of C₂H₂, but even at saturating N₂ concentrations, 1 mol of H₂ was produced for every mole of N₂ reduced. This explains the finding that the observed C₂H₂/N₂ ratio is higher than the value of 3 expected from the requirement for two electrons for acetylene reduction and six for nitrogen reduction. The results correlate well with a mechanism for N₂ reduction involving the equation: N₂ + 8H⁺ + 8e⁻ → 2NH₃ + H₂ which gives a conversion factor between C₂H₂ and N₂ of 4. It is proposed that, in general, 4 is a more appropriate value than 3 for the conversion factor.     

Production of Nitrous Oxide by Ammonia-Oxidizing Chemoautotrophic Microorganisms in Soil

Gas chromatographic studies showed that nitrous oxide was produced in each instance when sterilized (autoclaved) soil was incubated after treatment with ammonium sulfate and inoculation with pure cultures of ammonia-oxidizing chemoautotrophic microorganisms (strains of Nitrosomonas, Nitrosospira, and Nitrosolobus). Production of N₂O in ammonium-treated sterilized soil inoculated with Nitrosomonas europaea increased with the concentration of ammonium and the moisture content of the soil and was completely inhibited by both nitrapyrin and acetylene. Similar effects of nitrapyrin, acetylene, ammonium concentration, and soil moisture content were observed in studies of factors affecting N₂O production in nonsterile soil treated with ammonium sulfate. These observations support the conclusion that, at least under some conditions, most of the N₂O evolved from soils treated with ammonium or ammonium-producing fertilizers is generated by chemoautotrophic nitrifying microorganisms during oxidation of ammonium to nitrite.     

Factors affecting the reduction of acetylene by Rhizobium-soybean cell associations in vitro

The acetylene reduction assay was used to measure presumed N₂-reducing activity in Rhizobium-soybean cell associations in vitro. No acetylene reduction was observed in liquid suspensions of these organisms, but cells plated onto an agar medium from a liquid suspension of Rhizobium and soybean cells exhibited acetylene-dependent production of ethylene after 7 to 14 days. Aggregates of soybean cells 0.5 to 2.0 mm in diameter were required for this activity. Decreasing oxygen from 0.20 atm to 0.10, 0.04, or 0.00 atm completely inhibited acetylene reduction. The presence of 2,4-dichlorophenoxyacetic acid or kinetin increased endogenous ethylene production and inhibited acetylene-dependent ethylene production. Acetylene reduction was observed with three out of four strains of R. japonicum tested, and three rhizobial strains, which produce root nodules on cowpeas but not soybeans, formed an association capable of acetylene-dependent ethylene production.     

In Vivo Emission of Dinitrogen by Earthworms via Denitrifying Bacteria in the Gut

Earthworms emit the greenhouse gas nitrous oxide (N₂O), and ingested denitrifiers in the gut appear to be the main source of this N₂O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N₂), the end product of complete denitrification. When [¹⁵N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N₂ (and also labeled N₂O) under in vivo conditions; emission of N₂ by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N₂ per h per g (fresh weight), respectively. Isolated gut contents also produced [¹⁵N]nitrate-derived N₂ and N₂O under anoxic conditions. N₂ is formed by N₂O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [¹⁵N]nitrate-derived N₂ by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N₂O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N₂ (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N₂ per h per g (fresh weight), respectively; these emission rates approximated that of N₂O. These collective observations indicate that (i) earthworms emit N₂ concomitant with the emission of N₂O via the in situ activity of denitrifying bacteria in the gut and (ii) N₂O is quantitatively an important denitrification-derived end product under in situ conditions.     

Enumeration and Relative Importance of Acetylene-Reducing (Nitrogen-Fixing) Bacteria in a Delaware Salt Marsh

Three groups of N₂-fixing bacteria were enumerated from the top 1 cm of the surface in four vegetational areas in a Delaware salt marsh. The results over the 9-month sampling period showed that there were no discernible seasonal patterns for any of the groups enumerated (Azotobacter sp., Clostridium sp., and Desulfovibrio sp.). Azotobacter sp. was present in numbers of 10⁷ per g of dry mud, whereas the two anaerobic fixers were present in much lower numbers (10³ to 10⁴ per g of dry mud). There were no differences in the numbers of each group among the different vegetational areas, indicating that there was a heterogeneous population of N₂ fixers present. Additional studies indicate that the activity of sulfate reducers (Desulfovibrio sp.) may account for as much as 50% of the total observed acetylene reduction activity. Oxygen was found to exert little effect on the observed acetylene reduction activity, indicating that stable aerobic and anaerobic microenvironments exist in the surface layer of marsh sediments.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules

I have measured acetylene diffusion through plant tissues including nodules from several species of legume—vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O₂ where there is no N₂ reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

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.     

Evidence for Involvement of Gut-Associated Denitrifying Bacteria in Emission of Nitrous Oxide (N2O) by Earthworms Obtained from Garden and Forest Soils

Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N₂O)-emitting garden soils emitted 0.14 to 0.87 nmol of N₂O h⁻¹ g (fresh weight)⁻¹ under in vivo conditions. L. rubellus obtained from N₂O-emitting forest soil also emitted N₂O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878–1882, 1997). In contrast, commercially obtained Lumbricus terrestris did not emit N₂O; however, such worms emitted N₂O when they were fed (i.e., preincubated in) garden soils. A. caliginosa, L. rubellus, and O. lacteum substantially increased the rates of N₂O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N₂O emission by earthworms accounted for approximately 33% of the N₂O emitted by garden soils. In vivo emission of N₂O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N₂O. In the presence of nitrate, acetylene increased the N₂O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N₂O. In vivo emission of N₂O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N₂O emission of dissected earthworm gut sections were substantially higher than the rates of N₂O emission of dissected worms lacking gut sections, indicating that N₂O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N₂O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N₂O-emitting earthworms produced N₂O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N₂O by earthworms and contribute to the N₂O that is emitted from certain terrestrial ecosystems.     

Prevalence of nitrous oxide reducing capacity in denitrifiers from a variety of habitats

Summary A total of 81 strains isolated by T. N. Gamble from soils from eight countries, fresh water lake sediments and nitrified poultry manure were examined for their ability to grow on N₂O as their electron acceptor, as well as for their tendency to produce N₂O from NO ₃ ⁻ in the absence and presence of acetylene. Seventy-seven of the 81 strains were confirmed as denitrifiers. Fifty-nine of the 77 strains grew on N₂O, while 12 strains produced N₂O but could not utilize it. Six strains reduced NO ₃ ⁻ to N₂ but could not grow on N₂O, suggesting that even if N₂O is always an intermediate product of denitrification, it is not always a freely diffusible intermediate. The organisms, however, would consume N₂O that accumulated early in growth and accumulated N₂O in the presence of acetylene. Thus the total number of N₂O users was 65 strains or 83% of the total tested. This implies that the N₂O reducing capacity of denitrifiers occur widely in nature. A high proportion ofPseudomonas fluorescens biotype II reduced N₂O. The accumulation of N₂O from NO ₃ ⁻ in the presence of acetylene provides strong evidence that N₂O is generally an intermediate in denitrification as well as provides additional support for the usefulness of this chemical as a general inhibitor of N₂O reduction.     

Acetylene reduction (nitrogen fixation) and metabolic activities of soybean having various leaf and nodule water potentials

An apparatus was designed that permitted acetylene reduction (N₂ fixation) by root nodules to be measured in situ simultaneously with net photosynthesis, dark respiration, and transpiration of the shoot in soybean plants (Glycine max [L.] Merr. var. Beeson). Tests showed that acetylene reduction was linear with time for at least 5 hours, except for the first 30 to 60 minutes. Endogenous ethylene production did not affect the measurements. Successive determinations of acetylene reduction could be made without apparent aftereffects on the plant.     

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.