Influence of physical treatments on acetylene reduction (nitrogen fixation) in soils

Abstract

The effect of physical treatments such as drying, freezing, and heating on acetylene (C₂H₂) reduction (nitrogen fixation) in 3 types of soils was measured. Most treatments significantly affected ethylene (C₂H₄) production in the sandy loam. None of the treatments suppressed C₂H₄ formation in the sandy clay loam before 2 days. Muck soil with treatments of air‐drying, freeze‐drying, and freezing did not show different patterns in C₂H₄ production. However, auto claving and oven‐drying resulted in a pronounced increase in formation of C₂H₄. With exception of the heat treatments, no significant decreases in numbers of non‐symbiotic nitrogen‐fixers in soils were observed.     

Evaluation of acetylene reduction by excised roots for the determination of nitrogen fixation in grasses

The determination of N₂-fixation in grasses by the excised root assay is evaluated. Prolonged “pre-incubation” of excised sorghum roots caused the development of increased rates of N₂-ase activity. The results indicated that damage of N₂-ase by O₂ is unlikely to cause a prolonged delay before C₂H₂ reduction. Washing the collected roots with distilled water to prevent dessication and “pre-incubating” them at 30°C caused proliferation of the N₂-fixing microflora and synthesis of N₂-ase. Growth of the N₂-fixing microflora also occurred under C₂H₂ during the inactive period. The length of time before N₂-ase activity commenced was probably related to the initial concentration of available combined N. The results indicate that C₂H₂ reduction measurements with the excised grass roots tested overestimate the rates of N₂-fixation. The possible causes for the prolonged period before detection of N₂-ase activity by excised grass roots are discussed.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.     

An assessment of non-symbiotic nitrogen fixation in some Nigerian soils by the acetylene reduction technique

Short-term incubation studies of non-symbiotic nitrogen fixation using acetylene as the substrate for nitrogenase were carried out on six soil samples collected from various locations in the Western State of Nigeria, and on one soil sample collected from Wisconsin, U.S.A. Unamended soils reduced a very small amount of acetylene but with added organic materials (5% organic matter, 2% glucose or sucrose) acetylene reduction became substantial and in one case was more than 30,000 times that in the unamended soil. In general, more acetylene was reduced in anaerobic than in aerobic soils. Two of the soils were tested for suppression of acetylene reduction by NH₄⁺-N. With the addition of up to 100 ppm nitrogen as (NH₄)₂SO₄ to the soils incubated with sucrose, no depression of acetylene reduction was observed in one soil while it was reduced by about half in the other.     

Field measurements of symbiotic nitrogen fixation in an established pasture using acetylene reduction and a 15N method

The C₂H₂ reduction (A.R.) assay was investigated for quantitative measurement of symbiotic N₂ fixation in established legume-based pastures under field conditions.It was found that the rate of C₂H₄ production was relatively constant for approx 6 h. For an accurate estimation of the N₂ fixing activity, many field samples are required to overcome the errors due to the inherent spatial distribution of white clover (Trifolium repens L.) within the pasture. Furthermore, it is necessary to physically integrate the day-to-day and diurnal variations in the N₂ fixing activity of the legume to obtain a reliable estimate of the rate of C₂H₄ production and hence symbiotic N₂ fixation.The A.R. assay and an ¹⁵N technique were compared for measuring symbiotic N₂ fixation in established pastures. A 3 h incubation in the A.R. assay gave the best estimate of symbiotic N₂ fixation relative to the ¹⁵N technique. The 1 h incubation over-estimated and the 6 h incubation under-estimated the rate of symbiotic N₂ fixation.     

Oxygen inhibition of acetylene reduction (nitrogen fixation) in soil: Effect of glucose and oxygen concentrations

Sandy loam soil was amended with different concentrations of glucose and was incubated at different pO₂ levels. Under many conditions of incubation time and treatment, N₂ ase activity as determined by 1-h aerobic C₂H₂ reduction assay (flushed with Ar:O₂, 4:1 before assay) was significantly less than that determined by means of ambient assay (carried out at the pO₂ of incubation without flushing with Ar:O₂, 4:1 before assay). The difference between the N₂ase activity in aerobic assay and that in ambient assay increased with decreasing glucose and O₂ concentrations imposed during incubation. The inhition in aerobic assays was analogous to O₂-induced shut-off of N₂ase and amounted to 75 per cent inhibition after incubation at 0.06 atm pO₂ of samples amended with 0.75% glucose (w/w). Similar O₂ inhibition was observed after amendment with mannitol and with lactate. Times of incubation were chosen such that development of anaerobic N₂ase activity was either absent or too low to account for the observed effects of O₂ during assay. It was shown that 0.05 atm pC₂H₂ was adequate for routine 1-h assays of the soil system employed. Individual soil samples could be subjected to repeated 1-h assays (with removal of C₂H₂ and C₂H₄ by evacuation after each assay) thus avoiding side-effects of long exposure to C₂H₂.     

Conversion factor between acetylene reduction and nitrogen fixation in soil: Effect of water content and nitrogenase activity

The conversion factor between C₂H₂ reduction and N₂ fixation was studied in two soils. In one study small cores from (he two soils were drained to three water tensions: 0.20, 1.17 and 4.89 kPa. At each tension the N₂ase activity was measured with both O.1 aim ¹⁵N₂ and 0.1 aim C₂H₂. The conversion factor was different for the two soils. 1.0 and 3.1. respectively. The water content had no influence on the value of the conversion factor in this first study, in which the fixation corresponded to about 1mg N m^?2 day ^?1 at the depth 0–3 cm.In another study glucose was added to one of the soils to enhance the N₂ase activity. The activity was measured at 75 and 100% water saturation with both 0.9 atm ¹⁵N₂ and 0.1 atm C₂ H₂. At the lower water content the conversion factor was 2.6 and at water saturation the factor was 15.7. The fixation rates were high in this study. 98 mg N m^?2 day^?1 at the lower water content and 42 mg at water saturation.By theoretical calculations it was shown that the concentration of dissolved N₂ restricted the rate of fixation in the water-saturated samples of the second study, thus giving the high conversion factor. The critical level of N₂asc activity in water-saturated soil, above which the actual C₂H₂ to N₂-ratio will be higher than usual, was estimated to about 10mg Nm ²day_?1, under the experimental conditions used in these studies.     

Efficiency of acetylene reduction (nitrogen fixation) in soil: Effect of type and concentration of available carbohydrate

Sandy loam field soil and Acer saccharum (maple) forest soil were amended with different concentrations of glucose and mannitol and incubated at different pO₂ levels. Nitrogenase activity was determined by repeated 1-h C₂H₂ reduction assays performed at the ambient pO₂ of incubation. Calculated efficiencies of N₂ fixation increased with increasing anaerobiosis and with decreasing added carbohydrate concentration. Efficiencies up to 30 mg N₂ fixed per gram of glucose consumed were obtained under anaerobic conditions in the presence of 0.25% (w/w) glucose. Evidence suggested that low aerobic efficiencies were caused by intense competition for carbohydrate and by lower pH values attained. High concentrations (up to 3.0% w/w) of glucose under aerobic conditions suppressed the development of N₂ase activity. Mannitol supported N₂ase activity the development of which was very much delayed under aerobic conditions but little delayed under anaerobic conditions.     

Nodulation pattern and acetylene reduction (nitrogen fixation) activity of some highland and lowland Acacia species of Ethiopia

 Endosymbionts from the Ethiopian highland acacia species Acacia abyssinica, A. negrii and A. etbaica, and the lowland species A. nilotica, A. prasinata, A.senegal, A. seyal, A. tortilis and Faidherbia (Acacia) albida were isolated and characterized. Seven tree species were found to be nodulated by species of both Rhizobium and Bradyrhizobium. F. (Acacia) albida and A. senegal were nodulated by only Bradyrhizobium or Rhizobium, respectively. In A. abyssinica, both genera were isolated from the same nodule, whereas in A. nilotica and A. tortilis, both strains were isolated from different nodules of the same plant. The nitrogen fixation (acetylene reduction) activities varied considerably and showed no correlation with the nitrogen content of the plant. Highland species were as effective as lowland plants, thus demonstrating good potential for soil reclamation. The endosymbionts isolated proved rather promiscuous, efficiently nodulating other Acacia spp. and some tropical grain legumes, but did not nodulate temperate legumes. Received: 7 August 1997     

Seasonal fluctuations in nitrogen fixation (acetylene reduction) by free-living bacteria in soils contaminated with cadmium, lead and zinc

Acetylene reduction by non-symbiotic, heterotrophic micro-organisms in a range of soils containing different concentrations of heavy metals was determined using intact soil cores. The suitability of this method for the soils used in this investigation was established. Samples were collected seasonally, and were incubated under standard conditions (darkness: 15°). Mean values of metal concentrations in the soil (μg g^?1) were: Cd: 1–200; Pb: 60–8000; Zn: 70–26000, Cu: 20–40. Rates of acetylene reduction were generally low, from 2800 to 50000 nmol C₂H₄, m^?2 day^?1. Assuming a 3:1 ratio of C₂H₂ reduction to N₂ fixation, this represents a rate of 0.3 to 5.0 g N fixed ha^?1 day^?1 in the surface 150 mm of soil. No consistent effect of heavy metal concentration was found. The most important factors determining activity were soil moisture content and possibly inorganic nitrogen concentration. It thus appears that the bacteria in polluted soils are capable of adapting to potentially toxic concentrations of heavy metals, or that these metals are present in the soils tested in unavailable or non-toxic forms.     

An improved nitrogen difference method for estimating biological nitrogen fixation in legume-based intercropping systems

The nitrogen difference method (NDM) for quantifying N₂ fixation, based on the same amount of soil N exploited by N₂-fixing and non-N₂-fixing plant, may not be suitable to plants with different root traits. We tested the reliability of NDM in legume-based intercropping systems by two field experiments in Northwest China. In experiment 1, faba bean (Vicia faba), pea (Pisum sativum), and soybean (Glycine max) grew solely or intercropped with maize (Zea mays) with two N application rates (0, 225 kg ha^?1). The biomass of faba bean, pea, and maize was significantly increased, whereas that of soybean was decreased when intercropped than solely grown. Aggressivity analyses demonstrate greater N competition ability of faba bean and pea, but not soybean, than maize. An improved NDM (INDM) could mitigate these effects: $ {N_{{\text{fix - int}}}} = \left[ {{N_{{\text{leg - int}}}} + \frac{{1 - x}}{x}{N_{{\text{ref - int}}}} - \frac{{{N_{{\text{ref - sole}}}}}}{x}} \right] + \left[ {{\text{soil}}{N_{{\text{leg - int}}}} + \frac{{1 - x}}{x}{\text{soil}}{N_{{\text{ref - int}}}} - \frac{{{\text{soil}}{N_{{\text{ref - sole}}}}}}{x}} \right] $ , where x and 1???x are planting area of legume and non-legume in the intercropping system. Compared to traditional NDM (TNDM, $ {N_{{\text{fix - int}}}} = \left[ {{N_{{\text{leg - int}}}} - {N_{{\text{ref - sole}}}}} \right] + \left[ {{\text{soil}}{N_{{\text{leg - int}}}} - {\text{soil}}{N_{{\text{ref - sole}}}}} \right] $ ), %N _dfa (N derived from air) by INDM was decreased by 54.3% and 39.8% for faba bean, 44.7% and 5.0% for pea, but increased by 113.5% and 191.0% for soybean at the two N application rates, indicating different %N _dfa quantifications between the two methods. In experiment 2, %N _dfa of sole or intercropped faba bean was quantified by TNDM, INDM, and ¹⁵N natural abundance method (NA). The %N _dfa only by INDM correlated significantly with that from NA. Both interspecific root interactions and N loss affect %N _dfa estimation. Our results suggested that INDM could be more suitable than TNDM for quantifying %N _dfa of a N₂-fixing plant in intercropping systems.     

Estimation of nitrogen fixation by Trifoliumsubterraneum L. and Medicago truncatula gaertn. grown in pots using a non-destructive acetylene reduction assay

Non-destructive acetylene reduction assays were successfully performed using small (1.21) incubation chambers and a 1 h incubation at 20°C. The concentration of C₂H₂ substrate used in the assays reached saturation at a partial pressure of 10 kPa for nodulated subterranean clover and barrel medic grown in a sandy loam. The optimum rates of C₂H₂ reduction associated with both species occurred within the range of 25–50% of the soil moisture content at field capacity (33 kPa). The ratio of moles N₂ fixed to moles C₂H₂ reduced was calculated to be 1:2.9 for the subterranean clover—Rhizobium symbiosis and 1:3.3 for the barrel medic—Rhizobium symbiosis.     

Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil

Reduction of N₂O in moist soil was inhibited completely by 10^?2 atm C₂H₂ and partially by 10^?5 atm C₂H₂. The effect of C₂H₄ was 10⁴ times less than that of C₂H₂. Denitrification of NO^?₃ occurred in anaerobically or aerobically incubated waterlogged soil and in anaerobic but not in aerobic moist soil. In the absence of C₂H₂ there was transient accumulation of N₂O. In the presence of C₂H₂ there was stoichiometric conversion of NO^?₃ to N₂O. Some kinetics of the reduction of N₂O and of NO^?₃ to N₂O are presented. Denitrification of 1 μg added NO^?₃-N.g^? could be measured within 1 h. Stoichiometries of production of N₂O from NO^?₂ and NO^?₃, respectively, and production of CO₂ attributable to denitrification were consistent with reported energy yields. Reduction of C₂H₂ to C₂H₄ occurred immediately following complete denitrification of added NO^?₃. The incubation of soil in the presence and in the absence of C₂H₂ thus permits assay of both denitrification and N₂ fixation and provides information on the mole fraction of N₂O in the products of denitrification.     

Application of N2/Ar ratio method for N2 fixation studies in submerged soil

Abstract

Recently there has been developments in the measurement of N₂ fixation due mainly to the C₂H₂ reduction method (1). This method, however, has several disadvantages, especially for submerged soil, and the estimated amount of fixed N₂ on the basis of the C₂H₂ reduction activity is not very reliable. The tracer ¹⁵N₂ technique which gives a reliable estimation of the fixed N₂ is too expensive for common use. Development of an alternative method suitable for submerged soil would therefore be desirable. The present authors expected that the measurement of the ratio N₂/Ar in the soil solution might provide advantages for the estimation of the fixed N₂ in submerged soil.     

放线菌与非豆科植物共生固氮的生态研究

与放线菌结瘤共生固氮的已知200多种非豆科植物,他们同时得到菌根菌的共生,所以,不仅有类似于豆科植物的高固氮力,而且固氮时间长,生长旺盛、迅速,抗逆性强,是许多不良生态环境改造的先锋树种。其树种与菌种作为资源研究利用,均会对人类生产、生活带来显著效益。     

Nitrogen fixation by intact grass-soil cores using 15N2 and acetylene reduction

To determine N₂ fixation by intact grass-soil cores, samples were collected from 25 sites in central Texas during the summer. Three cores (32 cm² each) were extracted immediately adjacent to one another from single grass clumps or sods. Two of these cores were incubated under 10% C₂H₂ in air and the third core was incubated for 12 h in an atmosphere with 10% ¹⁵N₂ enrichment. Following incubation with ¹⁵N₂ the same core was assayed for rate of C₂H₂ reduction (AR). Rates of AR were generally low and quite variable (0–7.6 μmol C₂H₄ core^?1 day^?1). ¹⁵N₂ was incorporated into root and shoot tissues within 12–24 h. Extrapolated values of N₂ fixation based on ¹⁵N₂ incorporation ranged from 0 to 20 kg N ha^?1100 day^?1. The ratio of C₂H₂ reduced (μ mol C₂H₄ core^?1 day^?1) to N₂ fixed (μ mol N₂ fixed core^?1 day^?1) was highly variable ranging from 0 to 12. This study confirmed that N₂ is fixed in the rhizosphere of grasses grown in Texas through the use of ¹⁵N₂ and demonstrated that incorporation of fixed N into shoots was relatively rapid.     

Diurnal changes in acetylene reduction in field-grown cowpeas and soybeans

The effects of five environmental factors on variation in the rate of C₂H₂ reduction in two cowpea (Vigna unguiculata L. Walp.) and two soybean (Glycine max L. Merrill) cultivars were examined at two stages of growth in the field. Diurnal changes in C₂H₂ reduction, estimated as μmoles C₂H₄ produced·g^?1 nodules·h^?1, over a 30 h period were compared with changes in soil, canopy and air temperatures, global radiation, and vapour pressure deficit. Although the environmental factors showed one maximum and one minimum during a diurnal cycle, the C₂H₂ reduction rate in nodulated cowpea roots showed two peaks: one between 0600–1200 and another between 1800–2400 and two minima: one between 1200–1600 and another between 2400–0600. Variation in C₂H₂ reduction in soybean nodules did not show any definite pattern. Vapour pressure deficit appeared the most likely factor influencing the decline in C₂H₂ reduction between 1200–1600.The rate of C₂H₂ reduction in cowpeas was greater at the pre-flower than at the early pod-fill period; the rate in soybeans was not greatly different at early flower or early pod-fill.     

Limited inhibition of nitrous oxide reduction in soil in the presence of acetylene

Anaerobic flasks with two different soils contained microorganisms which effectively reduced NO₃⁻ to N₂ in the absence of C₂H₂ and in the presence or absence of CO₂. In the presence of C₂H₂, the microorganisms reduced NO₃⁻ to N₂O and the further reduction of N₂O to N₂ was temporarily inhibited. This was shown for two partial pressures of C₂H₂ (0.1 kPa and 1.0 kPa). However. after a maximum of 168 h, microorganisms were able to reduce N₂O to N₂ in the presence of C₂H₂. This was shown in the presence of CO₂ for both partial pressures of C₂H₂ and in the absence of CO₂ for 1.0 kPa C₂H₂. The absence of CO₂ delayed the complete reduction of N₂O. Microorganisms which had reduced N₂O in the presence of C₂H₂ retained this ability for at least 3 days after the original atmosphere containing C₂H₂ had been removed.     

Non-isotopic method for the quantification of biological nitrogen fixation and wheat production under field conditions

 A field experiment was conducted in the Inshas area (Sharkeia governorate) to study the potential of biofertilizers, when the quantities of commercial fertilizers were reduced, for optimal wheat production. The different treatments were arranged in a completely randomized block design with seven replicates. N fertilizer was applied in three treatments with one control, i.e. zero, full, half and one-fourth rates, in the presence or absence of inocula. Azospirillum brasilense strain Sp245 was used as a biofertilizer. Generally, inoculation increased the accumulation of shoot dry matter and grain yield by about 35%, relative to the control treatment. Similar trends were observed in the case of N and P uptake by shoots and grains, as well as the efficient use of both, where inoculation increased the acquisition of the two elements as compared with the uninoculated plants. The obtained data showed that N₂ fixed by shoots and grains ranged from 2 to 10 kg N ha^–1 and from 8 to 19 kg N ha^–1, respectively. With respect to fixed N₂, the best treatment was inoculation combined with the one-fourth dose of N, followed by inoculation combined with half of the recommended N dose. Most of the fixed N was utilized by grains and the results clearly reflected the negative effect of high N fertilizer rates on biological N fixation. It is obvious that inoculation, in general, enhanced the N fertilizer utilized by both shoots and grains of wheat plants. In conclusion, the application of biofertilization technology to a light-textured soil with low fertility had a positive effect on plant growth, N gained from the air and enhancement of fertilizer N uptake (apparent recovery fraction). Received: 22 April 1999