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.     

Nitrogenase activity associated with three tropical grasses growing in undisturbed soil cores

Nitrogenase activity associated with the root system of three tropical grasses Axonopus compressus, Digitaria decumbens var. pangolaand Paspalum notatum was measured by C₂H₂ reduction assay of soil-plant cores. The cores were incubated in perspex chambers in which 10% of the air was replaced with C₂H₂. Gas samples were taken at 7, 24, 31, 48, 55 and 72 h. No lag before onset of C₂H₄ production was evident and good agreement was obtained between replicates. Cumulative C₂H₄ production maintained a linear trend during the 72 h incubation.The largest increase in N₂-ase activity was detected in the A. compressus—gleyed podzolic system while D. decumbens (lateritic podzolic) and P. notatum (sandy yellow podzolic) had smaller activities. Variation between sampling sites in the second year of sampling of the experiment was associated with large variations in soil moisture.N₂ fixation estimated from N₂-ase activity in soil-plant cores was similar to the amount of N accumulated in the above-ground herbage in the field during 12 weeks.Response curves relating N₂-ase activity to soil moisture and soil temperature were established for all species. P. notatum and D. decumbens responded similarly to changes in both soil temperature and soil moisture while A. compressus contrasted sharply to the other two species in its reaction to both.     

Nitrogenase activity associated with pasture grasses in Northern New South Wales

Nitrogenase activity associated with roots of grasses was initially examined at 67 sites in New South Wales using an enriched malate medium. Twenty six of the 39 grass species examined produced at least 10 nmol C₂H₄cm^?1 root h^?1—a level accepted as positive presumptive evidence of N₂-fixation: 40 of the 288 samples exceeded 100nmol C₂H₄h^?1. The seasonal N₂ase activity of up to 4 grass species collected in soil cores at 6 sites was measured over 16 months.Activity at field moisture levels, but incubated at 30°C was greatest for cores collected in summer months. Activity was increased in 33.5% of samples by raising soil moisture to field capacity. No single species of grass consistently supported higher activity than any other.Nitrogenase activity was compared in cores of Kikuyu grass (Pennisetum clandestinum) watered to in excess of field capacity and allowed to drain for between 7 and 28 days before re-watering. Activity declined rapidly in the first 7 days and although recovery was also rapid, integration of N₂ase activity over time showed a loss of 20 and 61% for 7 and 28 days drainage respectively. N₂ase activity was greatest at 30°C.Maximum N₂ase activity in field samples was only 246 nmol C₂H₄ core^?1 h^?1 indicating that fixation of N₂ would not be of agronomic significance.     

Carbon monoxide as an inhibitor of N2ase activity(C2H2) in control measurements of endogenous formation of ethylene by forest soils

Two problems with the recently suggested method to measure endogenous formation of C₂H₄ in an atmosphere enriched with C₂H₂ and CO in studies of N₂ase activity (C₂H₂) in forest soils were analysed, namely the effect of consumption of CO during incubation and the effect of water-saturated conditions.After an initial addition of 100 ml C₂H₂ and 20 ml CO 1^?1 to soil incubation vessels, CO was gradually consumed and followed by a recovery of N₂ase activity when the concentration of CO was lower than about 10 ml 1^?1. The shortest period within which this concentration was achieved was 1 day when incubating fresh soil cores at 15°C, and it was concluded that longer incubations should be avoided.The inhibition of N₂ase activity by CO was strongly suppressed when all soil pores were filled with water. Dissolved inorganic N (0.1% of dry mass soil) was much more efficient in inhibiting N₂ase activity under such conditions.     

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.     

A gas-flow soil core method to measure field denitrification rates

A method was developed for rapid measurement of soil denitrification under conditions where natural soil structure and aeration status is maintained. Air was continuously recirculated by means of a membrane pump through a soil core and a sample loop of a gas chromatograph equipped with an electron capture detector. Addition of acetylene to the recirculating air permitted measurement of denitrification in the soil core. Because of the rapid distribution of C₂H₂ and removal of N₂O provided by the gas flow, denitrification rates could usually be determined in less than 2 h. By means of external 6-way and 8-way valves, four soil cores could be simultaneously analyzed on one gas Chromatograph equipped with dual detectors. Soil cores could also be stored at 4°C for later analysis without affecting the denitrification rate. The detection limit for denitrification rate measurements was 0.5 ngN g^?1 soil day^?1 or approximately 2.6 g N ha^?1 day^?1. Coefficients of variation for repeated measurements on the same soil core were usually less than 15%, but coefficients of variation for repacked or natural cores of the same soil were much higher (70–90%) Disruption of the natural soil structure by sieving increased the denitrification rate in an aggregated clay loam soil, but decreased the rate in a non-aggregated sandy soil. These results illustrate the importance of maintaining natural soil structure during denitrification measurements. The effect of pumping gas through soil was evaluated by comparing denitrification rates in soil cores where C₂H₂ was allowed to distribute into the soil by passive diffusion with rates obtained by pumping. Lower denitrification rates were observed in the static incubation presumably due to limited diffusion of C₂H₂ into or N₂O out of the denitrifying sites in the soil. This diffusion limitation could be overcome in the static incubations if C₂H₂ was initially distributed through the soil by pumping. This gas flow method is well suited to the study of soil denitrification rates under nearly natural conditions because the indigenous substrates and anaerobic microsites are preserved, the rapidity in which denitrification rates can be measured, and the high sensitivity and relatively low analytical variability of the method.     

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.     

Acetylene reduction by bacteria isolated from the rhizosphere of wheat

An asymbiotic bacillus possessing N₂-ase activity and capable of growth on low-N media was consistently isolated from the rhizosphere of a chromosome substitution line of spring wheat in which a pair of chromosomes 5D from the cultivar Rescue had been substituted for those of ‘Cadet’. None of the bacilli were isolated from either of the two parent cultivars, Rescue and Cadet, or from the corresponding substitution line involving a homoeologous chromosome 5B. Maximum C₂H₂ reduction to C₂H₄ by pure culture isolates was found to occur at a partial pressure of 0.2 atm C₂H₂. C₂H₂ reduction was diminished in the presence of O₂ and nearly ceased at a partial pressure of 0.08 atm O₂.The data presented suggest that altering the genetics of the wheat plant can change the root environment to favor the establishment of bacilli that exhibit N₂-ase activity in pure culture.     

Side Effects of Acetylene on the Conversion of Nitrate in Soil

Laboratory studies were conducted to determine if acetylene affects the rate of NO₃^? reduction to N₂O and N₂, if C₂H₂ ist anaerobically metabolized in the presence of nitrate, and if C₂H₂ affects soil carbon metabolism. These studies in water saturated soil, incubated under air or N₂ atmosphere, with or without acetone-free acetylene, show that C₂H₂ can accelerate NO₃^? reduction. Acetylene inhibited carbon mineralization when NO₃^? was limited but accelerated it when sufficient NO₃^? was available. After three days, only two per cent or less of the added C₂H₂ was directly oxidized to CO₂, however, up to 28 % of the added C₂H₂ carbon remained in the soil. The residual C₂H₂ carbon was oxidized aerobically and anaerobically when NO₃^? was added. These data suggest that when C₂H₂ is used in denitrification studies, the results must be carefully scrutinized. Once a soil is exposed to C₂H₂ it should not be used again soon to assess denitrification.     

Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method

¹⁴C-labelled glucose and ¹⁵N-labelled KNO₃ were added to soil and the microbial biomass during 42 days' incubation was estimated using the chloroform fumigation-incubation method (CFIM). By day 1, most of the glucose (1577 μgCg^?1 soil) was metabolized and 110 μg NO^?₃-Ng^?1 soil were immobilized. In situ values for the proportions of biomass C (k_C) and biomass N (k_N) mineralized during the 10 days after CHCl₃ fumigation were determined on the basis that the immobilized labelled C and N remaining in the soil at this time were present as living microbial cells and their associated metabolites. The tracer data indicated that biomass C could be calculated by applying a k_c value of 0.41 to the CO₂-C evolved from the fumigated sample without subtraction of an unfumigated “control”. Biomass N was estimated from the net NH₄^?-N accumulation during the fumigation-incubation. The problem of reimmobilization of NH⁺₄-N where organisms of wide C:N ratio occur was overcome by adjusting the value of k_N according to the ratio of CO₂-C evolved: net NH₄⁺-N accumulated during the fumigation-incubation (C_F:N_F).A C_F:N_F ratio of 6:1 resulted in a k_N of 0.30 whereas a ratio of 13:1 indicated a k_N of 0.20.     

Analysis of factors limiting associative N2-Fixation (C2H2 reduction) with two cultivars of Sorghum nutans

A 5–10 times larger nitrogenase activity per plant (max. 8 μmol C₂H₄ h⁻¹ plant⁻¹) was found in Sorghum nutans cultivar CSV 5 compared to the cultivar IS 5218 (max. 0.9 μmol C₂H₄ h⁻¹ plant⁻¹). This significant difference was reproduced for all water regimes from 8 to 21% soil moisture. The number of chemoorganotrophic bacteria (“total cell number”) on a medium with six carbon sources was very similar for both cultivars (3−4 × 10⁸ cells g⁻¹ rhizosphere soil). On the other hand, in the “high fixing” variety CSV 5 the number of N₂-fixing bacteria was greater than in IS 5218 by a factor of 3, the number of actinomycetes was reduced ten-fold and Arthrobacter strains to one-third or one-quarter. The number of aerobic N₂-fixing bacteria growing in an O₂ gradient system was increased 100-fold. Cultivar CSV 5 has a reduced photosynthetic area (20–30%), a reduced root weight (up to 50%, depending on the water regime) and a reduced transpiration rate (30–40%) compared to cv. IS 5218. Grain production however is 10–20% greater. At all soil moisture regimes the pH in the rhizosphere of cv. CSV 5 is 0.5–1.5 pH units below the values for cv. IS 5218.     

Critical evaluation of the acetylene reduction test for estimating the activity of nitrogen-fixing bacteria associated with the roots of wheat and barley

The reliability of the C₂H₂ reduction test for estimating the activity of N₂-fixing bacteria associated with the roots of cereals has been evaluated in Scottish soils. Six wheat cultivars, including two chromosome substitution lines, and five barley cultivars were grown in a glasshouse in nine soils from the North East of Scotland. All the soils exhibited C₂H₄ oxidase activity which was completely inhibited by 0.0001–0.1 atm C₂H₂. Over-estimation of C₂H₂ reduction, resulting from the accumulation of endogenous C₂H₄, could, therefore, occur in assays of undisturbed plants, with the real possibility of deducing the existence of N₂-fixation where none existed. However, radiolabelled C₂H₂ reduction tests on undisturbed plants producing 2.4–18.0 μmol C₂H₄ day^?1, showed that all the C₂H₄ had been derived from the C₂H₂. With less active plants, the source of the C₂H₄ could not be accurately determined by this tracer method. These low rates of C₂H₄ production (< 2.4 μmol C₂H₄ day^?1), referred to as apparent C₂H₂ reduction, should, therefore, not be considered proof of N₂-fixation.The highest C₂H₂ reduction activities were observed in soils at maximum water holding capacity (MWHC). Roots removed from these soils reduced C₂H₂ immediately, if the initial partial pressure of O₂ (pO₂) was < 0.1 atm. Roots washed free of soil did not oxidize C₂H₄ during the 8 h assay. The C₂H₂ reduction activities of these excised roots could not be related to the activity of plants in soil for three reasons. (1) Development of C₂H₂ reduction was dependent on protein synthesis (inhibited by chloramphenicol), indicating re-establishment of activity destroyed by exposure to atmospheric pO₂, rather than continuation of the activity of undisturbed roots. (2) A lag period, dependent on the volume of the incubation vessel, was observed, indicating the involvement of root respiration in the assay. (3) Growth of N₂-fixing bacteria on compounds released from the roots (reducing sugars, amino acids and α-keto acids) occurred during the assay.Even with the possibility of over-estimation of N₂-fixation, the C₂H₂ reduction activities measured were considered to be too low to contribute significantly to the nitrogen requirement of the cereals grown under field conditions in Scotland.Some guidelines for screening programmes of N₂-fixation associated with the roots of grasses are suggested.     

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.     

Denitrification potentials: Measurement of seasonal variation using a short-term anaerobic incubation technique

A short-term anaerobic incubation technique using the C₂H₂ inhibition of N₂O-reductase for comparing denitrification potentials of soils is described. Twenty grams of soil with added NO^?1₃ are incubated in the presence of He and 0.1 atm C₂H₂ at 25°C and 0 soil matric potential for 8 h. N₂O evolution is linear within 60 to 120 min. The denitrification potential of soils stored at 4°C decreased markedly over 21 days of storage in accordance with changes in the available C. Denitrification under an anaerobic atmosphere was observed at 4 C. Denitrification potentials were independent of NO^?3₃ concentrations above 25 μg NO^?₃-N g^?1 soil. Biphasic linear rates of N₂O evolution were observed in one soil. Incubation of this soil with chloramphenicol suggested the first linear phase is attributable to the in situ enzyme activity at the time of sampling. The second linear phase is indicative of the dentrification potential and is attributed to the full induction of denitrifying enzymes. The denitrification potential of a soil was maintained at or close to the maximum for 8 months of the year. During midsummer months the denitrification potential decreased markedly and the soil demonstrated a biphasic rate of denitrification suggesting an in situ denitrification activity less than the maximum potential. Results indicate that the maximum denitrification potential of this soil may often be limited not by NO^?₃ but by available C.     

Acetylene reduction (N2-fixation) by nodules of Acacia cyanophylla

The specific C₂H₂ reducing activity of excised nodules of Acacia cyanophylla was found to be 0.442 nmoles C₂H₄ produced min^?1 mg dry wt nodule^?1 and the apparent Km value for C₂H₂ between 8.5–11.0 × 10^?3atm. Subjection of plants of A. cyanophylla to drought, waterlogging, shading and defoliation led to drastic reduction or complete cessation of the ability of their nodules to reduce C₂H₂ to C₂H₄ When water-stressed and waterlogged plants were returned to a regular watering regime and the shaded plants to normal daylight the ability of their nodules to reduce C₂H₂ to C₂H₄ was partially or totally restored.     

Soil core incubation system for the field measurement of denitrification using acetylene-inhibition

Two systems for determining rates of denitrification, both based on the acetylene-inhibition technique, have been compared. One system involved in situ treatment of soil with C₂H₂ using an enclosure (500 × 150mm) placed over the soil surface followed by measurement of nitrous oxide (N₂O) emission. The other involved incubation, in the field, of soil cores with 5% (v/v) C₂H₂ in modified fruit preserving jars. Agreement between the two systems of measurement was close for well-drained soils over a wide range of rates of denitrification (0.005–1.27 kg N ha⁻¹ day⁻¹). Results obtained with poorly-drained soils having low air-filled porosities indicated that the incubation system with soil cores overcame the problems associated with applying the enclosure technique to soils of this type. Denitrification in the incubation system could be terminated by the addition of chloroform after an appropriate period (usually 24 h). The N₂O concentration in the air space of the jars then remained essentially constant for 14 days. This provided a second advantage in allowing the system to be used at sites remote from analytical facilities.     

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.     

Influence of the nitrogen content of straw amendments on nitrogenase activity in waterlogged soil

The effect of the nitrogen content of barley (Hordeum vulgare) and rape (Brassica campestris) straw on the nitrogenase activity in waterlogged straw-amended soil was investigated in laboratory incubation experiments. Nitrogenase activity was measured with the C₂H₂ reduction assay periodically throughout the incubation of soil-straw mixtures for 34 days at 25°C and 87 days at 16°C. Suppressed nitrogenase activity in soil amended with rape straw in preliminary experiments was attributed to the high N content of the straw. Removal of the water-soluble fraction of both barley and rape straw generally increased N₂(C₂H₂) fixation and the increase was highly significantly correlated (r = 0.80) with the water-soluble N content of the straw. Incorporation of NH₄⁺ and NO₃^? in amounts equivalent to those present in the original straw delayed the onset of nitrogenase activity but did not significantly affect the amount of N₂(C₂H₂) fixation in soil amended with the water-insoluble fraction of straw. However, when the water-soluble fraction was added with the water-insoluble fraction, N₂(C₂H₂) fixation was suppressed.     

Nitrogen fixation as influenced by moisture content,ammonium sulphate and organic sources in a paddy soil

The effect of moisture and (NH₄)₂SO₄ on N₂ fixation in a paddy soil was investigated employing C₂H₂ reduction assay and ¹⁵N-tracers. N₂ fixation was negligible under nonflooded conditions. Soil submergence accelerated N₂ fixation; with a further increase in N₂ fixation when the flooded soil was incubated under an Ar atmosphere. Rice straw additions to both moist and flooded soils enhanced N₂ fixation. N₂-ase activity in the soil decreased with increasing concentration of added N although complete suppression of the activity was not evident even at concentrations as high as 160–320 parts/10⁶ N. A similar trend of inhibition by N was also noticed in soils amended with glucose or cellulose in combination with N. However, the inhibitory effect of N decreased with increased incubation of soil except at 320 parts/10⁶ N.     

Persistence of glyphosate in a sandy loam

Glyphosale was added to samples of a sandy loam at rates of 0, 2, 5 and 10μg g^?1soil. After 120 days, soil was transferred to pots which were planted with subterranean clover. Plants were inoculated with Rhizohium trifolii and N₂ fixation (C₂H₂-reduction) was recorded after 9, 13, 15 and 19 weeks of growth. Nodule numbers and root weights were determined after the final C₂H₂-reduction assays had been performed. Decreased C₂H₂-reduction, nodule numbers and root weights associated with plants growing in glyphosate-treated soil indicated that this herbicide was not inactivated during the 120-days before planting.