Problems of the Acetylene Reduction Technique Applied to Water-Saturated Paddy Soils

The acetylene reduction assay for the measurement of N₂ fixation in a water-saturated paddy soil is limited by the slow diffusion of acetylene and ethylene. In laboratory incubation tests, vigorous shaking after the assay period is needed to release ethylene into the gas within the assay vials. Shaking prior to the incubation is also effective for dissolving acetylene in the water-saturated soil. However, a water-saturated soil depth of less than 10 mm during incubation is recommended. In field assays, some amounts of ethylene remain in the water-saturated soil phase of the acetylene reduction assay chamber, but stirring the water-saturated soil before sampling reduces the amount of ethylene remaining in soil. Evidence of a downward movement of acetylene and an upward movement of ethylene through rice plants was obtained. Because of the rapid transfer of acetylene to rice plant roots, an in situ acetylene reduction assay covering a rice hill is likely to detect nitrogen fixation in the proximity of roots where acetylene is easily accessible. Acetylene introduction to the water-saturated soil phase prior to assay did not greatly increase the acetylene reduction rate. Carbon dioxide enrichment in the assay chamber did not enhance nitrogen fixation in a paddy including rice and algae during a 1-day cycle.     

Respiration and nitrogen fixation in nodulated roots of soya bean and pea

Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules.     

Diurnal and seasonal patterns of nitrogen fixation in analnus hirsuta plantation of central Korea

We used an acetylene reduction assay to measure rates of nitrogen fixation on a 38-year-oldAlnus hirsuta plantation in central Korea. The diurnal pattern of acetylene reduction changed significantly during May, August, and October, typically varying by 3-fold throughout the course of the day. Maximum rates occurred at 3 p.m. in May and October, but at 6 p.m. in August. Increasing trends were evident during the early growing season, with sustained high rates from mid-May through late September; July had the highest rates, averaging 7.2 μmole g^-1 dry nodule h^-1. The average nodule biomass for this plantation was 220 kg ha ’. Rates of acetylene reduction were related to soil temperature, but not to soil moisture content. Combining these nodule biomass calculations with seasonal average acetylene reduction rates yielded an estimate of current annual nitrogen fixation of 60 kg N ha^-1 for the plantation. This rate of annual nitrogen addition was very large in relation to the yearly nitrogen requirements of coniferous and deciduous forests in central Korea.     

Nitrogen cycling in an Antarctic ecosystem 2. Estimation of the amount of nitrogen fixation in a moss community on East Ongul Island

Nitrogen fixation by cyanobacteria in a moss community on East Ongul Island (69°00'S 39°35'E), Antarctica was investigated using the acetylene reduction method. The mean acetylene reduction rate at 10°C and 200 μE·m⁻²·s⁻¹ photosynthetically active radiation was 7.12 nmol C₂H₄ per square centimeter of moss community per hour. The effects of temperature, radiation, desiccation and rehydration on the acetylene reduction rates were examined. A simple predictive model was constructed in order to estimate the amount of nitrogen fixed in the field. Using this model, the daily amount of nitrogen fixation was calculated from microclimatic data (temperature and radiation) measured in the experimental field at Syowa Station on East Ongul Island between 1983 and 1984. The cumulative amount of nitrogen fixation in the growing season during this period was estimated to be 329 mg N per square meter of moss community. It is suggested that nitrogen fixation by cyanobacteria in the moss community is important as a nitrogen source for the community growth on East Ongul Island.     

Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Effects of Temperature on Growth and Nitrogen Fixation by Swards of Subterranean Clover

Subterranean clover (Trifolium subterraneum L.) cv. ‘Woogenellup’ swards were grown at 10, 15, 20 and 25 Cwith a 12 h photoperiod of 500 or 1000 µmol m^–2s^–1 [low and high photosynthetic photon flux density (PPFD)].Nitrogen-fixing swards received nutrient solution lacking combinednitrogen while control swards received a complete nutrient solution.Growth was measured by infra-red analysis of carbon dioxideexchange and by accumulation of dry matter. Swards were harvestedat intervals between 95 and 570 g d. wt m^–2 for estimationof nitrogenase activity by acetylene reduction and hydrogenevolution assays. Nitrogen fixation was also measured by increasein organic nitrogen. The growth rate was highest at 10 C at low PPFD, and at 10–15C at high PPFD. Nitrogen-fixing swards grew slower than thosereceiving combined nitrogen. Nitrogen fixation measured by increasein organic nitrogen responded similarly to the growth rate,as did acetylene reduction between 10 and 20 C. At 25 C therelationship between acetylene reduction and nitrogen fixationwas distrupted. The difference between the rates of acetylenereduction and hydrogen evolution, theoretically proportionalto nitrogen fixation, was not a reliable indicator of nitrogenfixation because hydrogen uptake developed. Trifolium subterraneum L, subterranean clover, growth, nitrogen fixation, temperature, acetylene reduction     

Comparison of common bean (Phaseolus vulgaris L.) genotypes for nitrogen fixation tolerance to soil drying

Aims

Common bean is a major source of protein for many people worldwide. However, the crop is often subjected to drought conditions and its advantage in undertaking symbiotic nitrogen fixation can be severely decreased. The primary objective of this study was to compare the resistance of nitrogen fixation of 12 selected genotypes to soil drying.

Methods

Twelve common bean genotypes of diverse genetic background were compared. Plants were grown in pots and subjected to soil drying over about 2 weeks. Nitrogen fixation was measured daily using a flow-through acetylene reduction technique. The plants were exposed to acetylene for only a short time period allowing repeated measures. The acetylene reduction rate of plants on drying soil was normalized against the rates measured for well-watered plants.

Results

Substantial variability was identified among genotypes in the threshold soil water content at which nitrogen fixation was observed to decrease. Genotypes SER 16, SXB 412, NCB 226, and Calima were found to have the greatest delay in their decrease in nitrogen fixation rates based on soil water content. These four genotypes expressed substantial tolerance of nitrogen fixation to soil drying. These experiments also resulted in data on the threshold soil water contents at which transpiration rates decreased. A decrease in transpiration rates at high soil water contents is potentially advantageous since it allows soil water conservation for use as the severity of the drought increases. There was a general trend of those genotypes with sustained nitrogen fixation rates to low soil water contents also expressing decreased transpiration rates at high soil water contents.

Conclusions

This study identified genetic variation among common bean genotypes in their response of nitrogen fixation and transpiration to soil drying. Five genotypes (SER 16, SXB 412, NCB 226, Calima, and SEA 5) expressed the desired traits for water-limited conditions, which might be exploited in breeding efforts.     

Further Errors in the Acetylene Reduction Assay: Effects of Plant Disturbance

A flow-through gas system was used to study the effects of disturbanceon nitrogenase (acetylene reduction) activity of nodulated rootsystems of soyabean (Glycine max) and white clover (Trifoliumrepens). Detopping plus removal of the rooting medium (by shaking)produced a substantial decrease in maximum nitrogenase activity.This response is due to a reduction in oxygen flux to the bacteroidscaused by an increase in the oxygen diffusion resistance ofthe nodule. The decrease in maximum nitrogenase activity wasmuch smaller for roots subjected to detopping only. Thus, theeffect of root shaking is more important than that of shootremoval. The effect of detopping plus root shaking on nitrogenase activityoccurred whether the plants were equilibrated and assayed at25°C or 15°C. However, the effect of disturbance onthe oxygen diffusion resistance of the nodules, and thus onnitrogenase activity, was greater at the higher temperature.At the lower temperature the oxygen diffusion resistance ofthe nodules had already been increased in response to the reducedrequirement for oxygen. These nodules were less susceptibleto the effects of disturbance. Thus, comparisons of the effectsof equilibration temperature on nitrogenase activity produceddifferent results depending on whether intact or disturbed systemswere used. With intact systems activity was lower at the lowertemperature but with detopped/shaken roots the lowest activityoccurred at the higher temperature. It is concluded that the use of detopped/shaken roots can producesubstantial errors in the acetylene reduction assay, which makesthe assay invalid even when used for comparative purposes. However,comparisons with rates of ¹⁵N₂ fixation and H₂ production showthat accurate measurements of nitrogenase activity can be obtainedfrom maximum rates of acetylene reduction by intact plants ina flow-through gas system. The continued use of assay proceduresin which cumulated ethylene production from disturbed systemsis measured in closed vessels must be questioned. Key words: Nodules, acetylene, nitrogenase activity     

Algal and bacterial non-symbiotic nitrogen fixation in paddy soils

Summary The nitrogen fixing activity of three Ivory Coast soils was tested in the laboratory by the acetylene reduction assay and the Kjeldahl method. Nitrogen fixation due to algae was estimated to be of the order of 4 to 8 (acetylene method) and 7 μg N per g soil per day (Kjeldahl method). Nitrogen fixation due to bacterial activity in the rice rhizosphere was estimated to be of the order of 2 to 5 (acetylene method) and 1 to 3 μg N per g soil per day (Kjeldahl method). These results emphasize the importance of the bacterial nitrogen fixation in the rhizosphere which had been hitherto overlooked. Comparison of acetylene method and Kjeldahl method results shows discrepancies the origin of which has been discussed. Time course of acetylene reduction by rhizosphere soils exhibits a lag phase which may be attributed to Postgate's switch off — switch on process.     

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.Addition of sodium molybdate, a specific inhibitor of sulphate reduction inhibited acetylene reduction activity in Z. noltii colonised sediments by > 80% as measured by both slurry and whole core techniques irrespective of the light regime, throughout the year inferring that sulphate reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. A mutualistic relationship between Z. noltii and nitrogen fixing SRB in the rhizosphere, based on the exchange of organic carbon and fixed nitrogen is proposed. In uncolonised sediments sodium molybdate initially severely inhibited acetylene reduction rates, but the level of this inhibition declined over the course of the year. These data indicate that the nitrogen fixing SRB associated with the Zostera roots and rhizomes were progressively replaced by an aerobic population of nitrogen fixers associated with the decomposition of this recalcitrant high C:N ratio organic matter.Acetylene and sulphate reduction rates in the seagrass beds showed distinct summer maxima which correlated with a reduced availability of NH ₄ ⁺ in the sediment and the growth cycle of Z. noltii in the Bassin. Overall, these data indicate that acetylene reduction (nitrogen fixation) activity in the rhizosphere of Z. noltii was regulated both by release of organic carbon from the plant roots and maintenance of low ammonium concentrations in the root zone due to efficient ammonium assimilation.Nitrogen fixation rates determined from acetylene reduction rates measured by the whole core technique ranged from 0.1 to 7.3 mg N m^–2 d^–1 in the Z. noltii beds and between 0.02 and 3.7 mg N m^–2 d^–1 in uncolonised sediments, dependent upon the season. Nitrogen fixation in the rhizosphere of Z. noltii was calculated to contribute between 0.4 and 1.1 g N m^–2 y^–1 or between 6.3 and 12% of the annual fixed nitrogen requirement of the plants. Heterotrophic nitrogen fixation therefore represents a substantial local input of fixed nitrogen to the sediments of this shallow coastal lagoon and contributes to the overall productivity of Z. noltii in this ecosystem.     

Soybean nodule gas permeability,nitrogen fixation and dirunal cycles in soil temperature

While diurnal cycles in nitrogen fixation rates are sometimes assumed to result from diurnal variation in photosynthetically active radiation, contradicting evidence exists that indicate soil temperature is the primary environmental influence. These studies assessed the significance of temperature on soybean nitrogen fixation under field conditions. Two groups of intact field-grown soybean plants, one at ambient and the other exposed to a 10°C diurnal variation in soil temperature, were nondestructively assayed for acetylene reduction rates. Activity was closely associated with soil temperature (R²=0.85), even when temperature was 12 h out of phase with ambient. Data were also obtained to determine if the effects of rhizosphere temperature on nitrogen fixation are mediated through an effect on the nodule oxygen permeability. Nodule oxygen permeability of intact, aeroponically grown soybean was closely correlated with the diurnal changes in temperature (R²=0.90).     

Control of photosynthesis during nitrogen depletion and recovery in a non-nitrogen-fixing cyanobacterium

When cells of Synechocystis strain PCC 6308 are starved for nitrogen, the amount of stored carbohydrate increases, the phycocyanin to chlorophyll a ratio decreases, and the rates of oxygen evolution and of carbon dioxide fixation decrease. When nitrate-nitrogen is replenished, the amount of carbohydrate decreases, the rate of oxygen evolution increases immediately, preceeding the increase in phycocyanin or carbon dioxide fixation. The rate of respiration first increases and then decreases upon nitrogen addition. Nitrogen-starved cells show no variable fluorescence; variable fluorescence recovered in parallel with oxygen evolution. This suggests that photosystem II is inactive in nitrogen depleted cells and not blocked by a build up of metabolic endproducts. Since carbon dioxide fixation does not increase until two to four hours after nitrate is replenished to nitrogen starved cells, it is suggested that reducing power may first be needed within the cell for some other process than photosynthesis, such as nitrate reduction.     

Identification of the Sources of Energy for Nitrogen Fixation and Physiological Characterization of Nitrogen-Fixing Members of a Marine Microbial Mat Community

Experimental manipulations of a microbial mat community were performed to determine sources of energy and reductant used for nitrogen fixation and to physiologically characterize the responsible diazotrophs. The dominant photolithotrophic members of this community were nonheterocystous cyanobacteria, but other potential nitrogen-fixing microorganisms were also present. Pronounced diel variability in rates of acetylene reduction was observed, with nighttime rates a factor of three to four higher than daytime rates. Acetylene reduction measured at night was dependent upon the occurrence of oxygenic photosynthesis the preceding day; mats incubated in the dark during the daytime reduced acetylene at rates comparable to those of light-incubated mats but were not able to reduce acetylene at the normally high rates the following night. The addition of various exogenous carbon compounds to these dark-incubated mats did not elicit nighttime acetylene reduction. Nighttime acetylene reduction apparently proceeds under anoxic conditions in these mats; the highest rates of acetylene reduction occur late at night. Additions of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of oxygenic photosynthesis) to mats resulted in a pronounced stimulation of acetylene reduction during the day, but acetylene reduction the next night proceeded at greatly reduced rates (relative to untreated mats). This daytime stimulation, under the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced anoxic conditions in the experimentally treated mats, was light dependent. These results suggest that nitrogen fixation in these mats may be attributed to the activities of nonheterocystous cyanobacteria utilizing storage products of oxygenic photosynthesis under anoxic conditions at night.     

Phosphorus fertilization and tillage effect on dinitrogen fixation in soybeans

Summary Tillage has been shown to affect the uptake of phosphorus (P) and yield of soybeans, [Glycine max (L.) Merr.], but there is little information concerning the effects of P fertilization on nitrogen (N₂) fixation in soybeans under no-tillage. Two field experiments were conducted in 1980 and 1981 to determine the effects of soil P on N₂ fixation under no-tillage and to study the interaction of P fertilization and tillage of N₂ fixation, nutrient uptake, and yield of soybeans. In Exp. I, P was applied in 1977 at five rates up to 384 kg P ha⁻¹ and the effects of residual soil P were evaluated in 1980 and 1981 under no-tillage management. Nitrogen fixation rates, as measured by acetylene reduction assay, were significantly affected by soil P in Exp. I, but the assay proved to be a poor technique for estimating total plant N in these tests. Acetylene reduction rates and plant P increased rapidly as soil P increased from 2 to 20 mg kg⁻¹, with little additional increase above 20 mg P kg⁻¹. In Exp. II, rates (0, 32, 64, and 128 kg P ha⁻¹) and time (fall, spring and fall plus spring) of P application were compared under conventional tillage and no tillage. However, plant P increased with increasing levels of applied P. Applied P had no affect on acetylene reduction rates but rates were greater for no-tillage than conventional tillage at the V9 and R5 stages of growth in 1981. Plant uptake of P was more efficient under no-tillage than under conventional tillage in 1980 and 1981. Application of 64 kg P ha⁻¹ under no-tillage resulted in equivalent plant P levels as the 128 kg P ha⁻¹ applied under conventional tillage.     

Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei

The ability of the benthic cyanobacterium Lyngbya wollei to fix nitrogen was studied using field samples and axenic cultures. L. wollei was collected and isolated from Lake Okeechobee, Florida, where it forms extensive mats. Rates of acetylene reduction up to 39.1 nmol mg dry wt⁻¹ h⁻¹ were observed for field samples. The maximum observed rate of acetylene reduction in axenic laboratory cultures was 200 nmol mg dry wt⁻¹ h⁻¹. Aerobic conditions limited nitrogen fixation activity, but dark/light cycles promoted the development of activity. Reduced oxygen levels appeared to be required for the development of significant levels of nitrogenase activity. The level of irradiance also had a significant impact on the level of activity. The potential significance of nitrogen fixation to Lyngbya production is discussed.     

Nitrogen Fixation in an Establishing Alfalfa (Medicago sativa L.) Ley in Sweden, Estimated by Three Different Methods

In an investigation of the ability of alfalfa to fix nitrogen under field conditions in Scandinavia, N₂ fixation during the establishment year ranged between 7.85 and 10.37 g of N m⁻², depending on the method used. The methods used were an in situ acetylene reduction method, a ¹⁵N isotope dilution method using two reference crops, and a total-N difference method. The dynamics of nitrogenase activity in relation to plant development was studied by using the acetylene reduction method. Also, the diurnal variation in N₂ fixation at the field site was studied with the acetylene reduction method; no diurnal change was detected, which is explained by the fact that the nodules within the soil were protected against short-term fluctuations in temperature. The significant amount of nitrogen fixed by alfalfa during its first year even at northern latitudes suggests that this crop offers an alternative to conventional field management of heavily fertilized nonlegume leys.     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

The Physiology and Nitrogen-fixing Capability of Aquatically and Terrestrially Grown Neptunia plena: The Importance of Nodule Oxygen Supply

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aeratedwater culture or vermiculite. Growth, nodulation, nitrogen fixationand nodule physiology were investigated. Over an 80-d period,plants grew and fixed nitrogen and carbon equally well in bothrooting media, although distribution of growth between plantparts varied. Total nodule dry weights and volumes were similarbut vermiculite-grown plants had three times as many (smaller)nodules than those grown in water. Oxygen diffusion resistanceof nodules exposed to 21% oxygen and 10% acetylene did not differsignificantly. Both treatments showed similar declines in rootrespiration and acetylene reduction activity (approx. 10%) whenroot systems were exposed to stepped decreases and increasesin rhizosphere oxygen concentration. However, nitrogenase activityof aquatically grown plants was irreversibly inhibited by rapidexposure of nodules to ambient air, whereas vermiculite-grownplants were unaffected. Aeration of water-cultured N. plenareduced stem length (but not mass) and number of nodules perplant. The concentration of nitrogen fixation by 163%. PossibleO₂ transport pathways from the shoot atmosphere to roots andnodules are discussed. Aquatic legume, diffusion resistance, Neptunia plena, nitrogen fixation, oxygen, root nodules     

Physiological studies on nitrogen fixation in the blue-green alga anabaena cylindrica

Summary Short-term manometric experiments with bacteria-free cultures of Anabaena cylindrica showed that the close dependency of nitrogen fixation upon photosynthesis could be temporarily eliminated in nitrogen-starved cells. Initial rates of nitrogen uptake by these cells in the absence of carbon dioxide were equally rapid in the light and dark, decreasing and finally ceasing after two hours. Continued steady nitrogen uptake was only maintained for long periods in the presence of carbon dioxide in the light. In the dark, nitrogen uptake was accompanied by carbon dioxide evolution.More oxygen was evolved in the light by cells fixing nitrogen than by those incubated under argon. This additional oxygen evolution could be accounted for by extra carbon dioxide fixation in the presence of nitrogen.Of a number of organic compounds tested, only sodium pyruvate stimulated nitrogen fixation. This stimulation was achieved both in the light and dark and in the presence and absence of carbon dioxide, showing that the role of pyruvate was other than acting as a carbon skeleton.Three metabolic inhibitors, cyanide and chlorpromazine (chiefly respiratory) and phenylurethane (photosynthetic) differentially inhibited photosynthesis and nitrogen fixation. The latter inhibitor had a more marked effect on photosynthesis while the two chiefly respiratory inhibitors had a stronger effect on nitrogen fixation.     

In situ acetylene-ethylene assay of biological nitrogen fixation in lowland rice soils

Summary An in situ device for assaying biological nitrogen fixation in flooded rice soils, using the acetylene reduction method, was developed. Diurnal variations in acetylene reduction by an inoculated field plot and by laboratory-grown cultures of nitrogen-fixing algae showed a prominent single-peak pattern of nitrogenase activity. The peak occurred at mid-day for laboratory-grown algae and at late afternoon for the algae grown in the field plot. Some nitrogenase activity was noted during the night. Acetylene reduction studies in rice fields of Albay province, Philippines, showed an estimated fixation of 18.5 to 33.3 kg N/ha each cropping season for the fields of Puro soil and 2.3 to 5.7 kg N/ha each cropping season for the fields of Santo Domingo soil. re]19751202