Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

Plant and Soil - To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for...     

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C₂H₂ at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C₂H₂ at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N₂ fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C₂H₂ than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N₂-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them. Only Bacillus strain C-11-25 has potential to benefit both temperate and tropical crops because it reduced significant acetylene over a wide temperature range.     

Root-associated n(2) fixation (acetylene reduction) by enterobacteriaceae and azospirillum strains in cold-climate spodosols

N(2) fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C(2)H(2) reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C(2)H(4).g (dry weight). h when incubated at pO(2) 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C(2)H(4). g (dry weight).h without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N(2)-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Dinitrogen fixation — acetylene reduction in soybeans during the reproductive growth period

In a greenhouse pot oulture experiment, a dinitrogen (N2) fixing — acetylene reduction activity profile was examined in detail as affected by plant age. Total [μmol C2H4 root-1 h-1] and speoifio nitrogenase [nmol C2H4 (mg nodule d. wt.)-1 min-1] activities peaked 63 days after sowing, near the end of flowering. The nitrogenase activities, nodule dry matter accumulation, top dry matter accumulation, and total nitrogen yield in the top dry matter were found to be highly correlated.     

Nitrogen fixation (acetylene reduction) inAquaspirillum magnetotacticum

Aquaspirillum magnetotacticum strain MS-1 and two nonmagnetic mutants derived from it reduced C₂H₂ microaerobically but not anaerobically even with NO₃ ^?. This organism apparently is not capable of NO₃ ^?-dependent nitrogen fixation. Cells ofA. magnetotacticum reduced C₂H₂ at rates comparable to those ofAzospirillum lipoferum grown under similar conditions, but much lower than that ofAzotobacter vinelandii grown aerobically. Cells ofA. magnetotacticum in anaerobic cultures lacking NO₃ ^? did not reduce C₂H₂ until O₂ was introduced. Optimum rates of C₂H₂ reduction byA. magnetotacticum were obtained at 200 Pa O₂. C₂H₂ reduction was inhibited by more than 1 kPa O₂ or 0.2 mM NO₃ ^? or NH₄ ⁺. These results suggest thatA. magnetotacticum fixes N₂ only under microaerobic, N-limited conditions.     

Nitrogen fixation (acetylene reduction) on a coral reef

Nitrogen fixation rates associated with various substrates on a fringing reef at Eilat, Red Sea, were estimated by in situ acetylene reduction. High rates of acctylene reduction were associated with bare substrates, such as sand and dead coral skeletons. Low rates of acetylene reduction were associated with substrates covered by macroalgae or living coral tissue. Estimates of nitrogen fixation in various reef zones, based on these measurements, indicate that the sand-covered lagoon is responsible for more than 70% of the fixation in the reef. Consequently, the lagoon may serve as an important source of nitrogen for the coral reef community.     

Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ

Diurnal variation in algal nitrogen fixation was studied in Lake Mendota, Wisconsin, during the summers of 1971 to 1973. Approximately two-thirds of the daily acetylene reduction in the surface decimeter occurred before noon. The decline in acetylene reduction (nmoles/liter·hr) near midday was partially because the algae relocated themselves at greater depths. However, acetylene reducing activity (nmoles per A₆₆₃ unit chlorophyll a per hour) also decreased as midday approached. Occasionally algae would resurface near the end of the day. On average, acetylene reduction (nmoles per liter per hour) was maximum at about 0900 Central standard time in the top decimeter, and acetylene reduction between 0830 and 0930 Central standard time represented 13% of the total daily acetylene reduction. Furthermore, acetylene reduction in the top decimeter, on average, represented 3.6% of the total acetylene reduction in the column. Calculation of the contribution by nitrogen fixation to a lake's fixed nitrogen budget is discussed.     

Field measurement of symbiotic nitrogen fixation in an established lucerne ley using15N and an acetylene reduction method

Summary Lucerne is an important forage legume in the south and south-east of Sweden on well-drained soils. However, data is lacking on the apparent amount of nitrogen derived through N₂ fixation by field-grown lucerne. This report provides basic information on the subject. The experiment was performed in a lucerne ley grown 40 km north of Uppsala. The input of nitrogen through fixation to the above-ground plant material of an established lucerne (Medicago sativa L.) ley was estimate by¹⁵N methodology during two successive years. The amount of fixed N was 242 kg N ha^–1 in 1982 and 319 kg N ha^–1 in 1983. The proportion of N derived from the atmosphere (%Ndfa) was 70% and 80% for the two years respectively. The first harvest in both years contained a lower proportion fixed N. Both N₂ fixation and dry matter production were enhanced during the second year, particularly in the first harvest. The Ndfa was 61% in the first harvest in 1982, compared to 72% Ndfa during the same period in 1983. This demonstrates the strong influence of environment on both dry matter production and N₂ fixation capacity of the lucerne.In addition anin situ acetylene reduction assay was used in 1982 to measure the seasonal distribution of the N₂ fixation and in 1983 to study the effect of soil moisture on the N₂ fixation process. The seasonal pattern showed great dependence on physiological development and harvest pattern of the lucerne ley. The maximum rate of N₂ fixation occurred at the bud or early flower stage of growth and was followed by a rapid decline as flowering proceeded. After harvest the nitrogenase activity markedly decreased and remained low during at least two weeks until regrowth of new shoots began. Irrigation doubled the nitrogenase activity of the lucerne in late summer 1983, when soil moisture content in the top soil was near wilting point. No changes in nitrogenase activity did occur in response to watering earlier during the summer, when the soil matric potential was around –0.30 MPa.     

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.     

Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C(2)H(4)/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C(2)H(2)) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.Higher rates of nitrogenase activity (15 to 53 nmol of C(2)H(4)/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germinans (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 mug of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation.     

15N2 incorporation and acetylene reduction by Azospirillum isolated from rice roots and soils

Summary Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by¹⁵N₂ incorporation and C₂H₂ reduction. C₂H₂ reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in¹⁵N₂ incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N₂-fixing ability and that plant genotype is of importance for optimal associations.     

Nitrogen fixation (acetylene reduction) and cross inoculation in 12 Prosopis (mesquite) species

The leguminous tree mesquite (Prosopis spp) exists on millions of hectares of semi-arid regions of the world. No whole plant acetylene reductions for mesquite have been reported in the literature and nodulation has only been reported for three of the forty-four species. We report greenhouse studies in which 12Prosopis species representing African and North and South American germplasm (1) became nodulated when inoculated with rhizobia strain isolated from a North American mesquite, (2) grew on a nitrogen free nutrient media, (3) reduced acetylene to ethylene, and (4) had a positive significant correlation between the acetylene reduction rates and above ground dry matter. The capability of mesquite to fix nitrogen must now be considered firmly established.     

Acetylene reduction,H2 evolution and 15N2 fixation in the Alnus incana-Frankia symbiosis

Acetylene reduction, ¹⁵N₂ reduction and H₂ evolution were measured in root systems of intact plants of grey alder (Alnus incana (L.) Moench) in symbiosis with Frankia. The ratios of C₂H₂: ¹⁵N₂ were compared with C₂H₂:N₂ ratios calculated from C₂H₂ reduction and H₂ evolution, and with C₂H₂:N₂ ratios calculated from accumulated C₂H₄ production and nitrogen content. It was possible to calculate C₂H₂:N₂ ratios from C₂H₂ reduction and H₂ evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched ¹⁵N could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H₂ evolution in combination with C₂H₂ reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.     

Diel interactions of oxygenic photosynthesis and n(2) fixation (acetylene reduction) in a marine microbial mat community

Diel variations in N(2) fixation (acetylene reduction), CO(2) fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO(2) fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 muM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N(2)-fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO(2) fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N(2) fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.     

Dinitrogen fixation (Acetylene reduction) and nitrogen accumulation in peas cultivated under the standard growth conditions

InPisum sativum cultivated under standard growth conditions the extent of N₂ fixation with time estimated by the acetylene reduction assay (PN₂F) and rates of the actual nitrogen accumulation of plant biomass (ANA) were calculated from six independent growth experiments. In the plants inoculated with indigenous soilRhizobium populations and cultivated on 0.63 mmol/L nitrate level the percentage PN₂F:ANA ratios ranged from 25.7 to 61.5%. In peas inoculated with the inoculant strain the PN₂F:ANA ratios were markedly higher, ranging from 59.8 to 65.1%. The plants cultivated on N-free nutrient solutions showed both PN₂F:ANA and C₂H₄N₂ ratios to be somewhat higher compared with the 0.63 mmol/L nitrate cultivated plants.