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.     

Biological nitrogen fixation associated with roots of field-grown barley (Hordeum vulgare L.)

Nitrogenase (C₂H₂) activity was measured in microbial media inoculated with barley root segments or corresponding rhizosphere soil. Three different media were used, Döbereiner's malate medium, a modified Ashby medium, and an acid nitrogen-free medium. Only Döbereiner's medium gave consistently positive results, and cultures inoculated with roots showed higher activity than cultures inoculated with corresponding rhizosphere soil. Similar experiments with roots and rhizosphere soil from wheat gave only negligible nitrogenase activity, whereas the tropical grass, Cynodon dactylon, gave higher activity than barley. Measurements on intact soil cores containing barley root systems showed an initial lag phase followed by a rather stable activity level over a period from 12 h to 48 h, and then the activity again decreased. The activity during the stable period corresponded to fixation of about 100 to 200 g N₂ ha^?1 24 h^?1. Measurements on isolated, washed barley roots showed only negligible nitrogenase activity.     

Non-symbiotic nitrogen fixation in the rhizospheres of rice,maize and different tropical grasses

Nitrogenase activity estimated in the rhizospheres of rice, maize and different tropical grasses grown under controlled laboratory conditions was shown to depend upon plant species. High nitrogenase activity (2000–6000 nmoles C₂H₄ h^?1 g^?1 dry root) occurred in rice rhizosphere, this activity being only 10 times lower than that of symbiotic systems; in the rhizosphere of many other grasses grown in a similar way nitrogenase activity was as low as 10 nmoles C₂H₄ h^?1 g^?1 dry root. The influence of soil type on nitrogenase activity was impressive; but the exact nature of the factors implicated could not be established. A rather weak flush of nitrogenase activity in the rhizosphere occurred in the early stage of the plant growth; it was probably due to the exudation of compounds from the seed and lasted 2 or 4 days according to the size of the seed. When the plant entered into its intense photosynthetic phase, the nitrogenase activity gradually increased. When the shoots were severed, nitrogenase activity in the rhizosphere ceased. Nitrogenase activity in the rhizosphere responded greatly to light intensity. Extrapolation of these laboratory findings to the field is discussed.     

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.     

Acetylene reduction in soil and litter from pine and eucalypt forests in south-eastern Australia

Rates of C₂H₂-reduction in surface soil and litter from pine and eucalypt forests were measured for 1 yr. Rates of reduction increased significantly with moisture content, and mean rates (nmol kg^?1 h^?1) decreased in the order pine litter (339), eucalypt litter (220), eucalypt soil (54), pine soil (7). Asymbiotic N₂-fixation in litter and surface soil was estimated to be 108 mg m^?2 yr^?1 in eucalypt forest and 64 mg m^?2 yr^?1 in pine forest. About 80% of total fixation in eucalypt was in the soil, while 80% of the total in pine was in the litter. N₂ase was active in rotting wood but not in fresh foliage.     

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.     

Contribution of nitrogen fixed by Azospirillum to the n nutrition of spring wheat in Israel

Acetylene reduction activity (ARA) was measured in cores containing roots of various Israeli wild and cultivated wheat lines colonized by Azospirillum. The inoculated plants were grown under greenhouse or field conditions. Although, no measurable ARA was detected during earlier stages of wheat development, 50–600 nmol C₂H₄ g^?1 dry root h^?1 was measured during heading and flowering stages. By using N yield balance and ¹⁵N dilution techniques, it was found that Triticum aestivum cv. Miriam inoculated with Azospirillum accumulated 20% more N (¹⁴N and ¹⁵N) at the booting stage than did the uninoculated control. This difference in N content became less apparent in grains. No significant ¹⁵N dilution could be found and the contribution of atmospheric N₂ to the N content of grains of inoculated plants was negligible. It was concluded that the potential contribution of biological N₂ fixation to spring wheat cultivation in Israel is very low.     

Nitrogenase (C2H2) Activity in Roots of Non-Cultivated and Cereal Plants: Influence of Nitrogen Fertilizer on Populations and Activity of Nitrogen-Fixing Bacteria

Root samples of 11 non-cultivated monocotyledonous and 7 dicotyledonous species taken during a wet summer had low mean nitrogenase activities of 10.2 and 7.1 nmol C₂H₄·g^?1 DW·h^?1 after preincubation at pO₂ 0.02, respectively. Maxima of 139–169 nmol·g^?1·h^?1 were observed with Agrostis vulgaris and Agropyron repens on a sandy soil poor in C_org. Three of 6 early, but none of 4 late fodder maize cultivars had a very low activity up to 0.5 nmol·g^?1h^?1. Oat, rye and wheat roots from plots with organic or mineral N fertilizers had activities between 1.3 and 7.3 nmol·g^?1h^?1 at flowering, which were not correlated with their Azospirillum populations (10²-10⁷·g^?1 after preincubation). Winter wheat and barley roots given 0, 40, 80 and 120 kg. ha^?1 NH₄NO₃-N in 0–3 applications had mean activities of 0.08, 4.06, 0.09 and 0.08 nmol or 1.77, 2.67, 0.36 and 0.23 nmol C₂H₄g^?1·h^?1 after flowering, respectively. An appreciable part of this activity could be removed by root washing. In preincubated rhizosphere soil of wheat and barley populations of N₂-fixing, facultative anaerobic Klebsiella and Enterobacter spp. were 10–100 times higher than those of Azospirillum sp., both being higher in O N than in 80 kg N·ha^?1 trials.     

Nitrogenase activity associated with soil cores of grasses in Brazil

The N₂-ase activities of field-grown Brazilian grasses were measured with C₂H₂ reduction by soil cores containing the plants. C₂H₂ and C₂H₄ were observed to diffuse at similar rates through soil and equilibrated across the Brazilian soil in 3 h, but could take up to 30 h or more with some British soils. The diurnal fluctuation in the rates of N₂-ase activity by Brachiaria mutica and Sorghum vulgare were similar and the variation in rate was correlated with soil temperature. Estimates of N₂-fixation by measurement of C₂H₂ reduction by soil cores ranged from 14.7 to 51.4 g N ha^?1 day^?1 and were much lower than with “pre-incubated” excised roots from the cores or taken directly from the field. The merits of the soil core and the “pre-incubated” excised root assays are discussed. ft1|Present address: U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Northeastern Region, Room 309, Building 001, Beltsville, MD 20705, U.S.A.     

Nitrogenase activity on the roots of tropical forage grasses

Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method. Values varied considerably between sites but indicate the possible economic importance of several of the species studied. Maximal nitrogenase activity measured (nmoles C₂H₄g^?1 dry roots h^?1) was 754 for Pennisetum purpureum, 750 for Brachiaria mutica, 341 for Digitaria decumbens, 299 for Panicum maximum, 283 for Paspalum notatum, 269 for Cynodon dactylon, 41 for Melinis minutiflora and 29 for Hyparrhenia rufa. Nitrogenase activity varied considerably with season and was maximal during active vegetative growth of two of the grasses. Significant differences between Paspalum notatum ecotypes and cultivars. in Azotohacter paspali occurrence and nitrogen fixation, indicate the possibility of plant breeding to enhance nitrogen fixation in grass rhizospherc associations. Other research lines of agronomic importance are fertilizer effects. In intact soil plant cores with the Paspalum system 10 parts/10⁶ NH_4⁺J-N inhibited nitrogenase activity within 2 h and 10 parts/10⁶ NO^?₃-N within 4 h. but after 1 week these effects were negligible. In the field, nitrogenase activity on roots of P. purpureum and D. decumbens, assayed 2 weeks after top dressings of 20 kg N ha^?1 as NH₄NO₃. was not affected even after eight such dressings.     

Nitrogenase activity (Acetylene reduction) during the growth cycle of spring barley (Hordeum vulgare L.)

The nitrogenase activity (C₂H₂-reduction) was measured during the growth cycle of field grown spring barley in soil cores both with and without barley plants, and at two levels of nitrogen application, 30 and 120 kg N ha^?1 year^?1 respectively. The main purpose of the investigation was to study the effects of the growing barley plants on nitrogenase activity in the soil, and temperature and moisture contents were kept constant in all experiments. Therefore, the results cannot be used to calculate actual amounts of fixed nitrogen in the field, but should be considered rather as potential values. The nitrogenase activity was found to vary during the growth cycle, and seemed to be correlated to the photosynthetic activity of the plants. Relatively low nitrogenase activity was found in the early growth stages, and the activity increased up to a maximum in the late reproductive stage, followed by a rapid decrease during the grain filling stage. The mean values of nitrogenase activity in samples without barley plants and with barley plants were 40 and 78 nmoles C₂H₄ g soil dwt^?1 24 h^?1 respectively. The positive effect of barley plants on nitrogenase activity was stronger at 120 kg N than at 30 kg N fertilization. As a mean of the whole growth cycle the ratio between samples with and without barley plants was 1.7 with 30 kgN and 2.3 with 120 kg N fertilization. The inhibitory effect of nitrogen application on nitrogenase activity was measurable until 6–7 weeks after application, and it was strongest in cores without plants.     

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.     

Nitrogen-fixing homogenates of Myrica pensylvanica (bayberry) non-legume root nodules

A procedure was developed for preparing active N₂-fixing (C₂H₂-reducing) homogenates of root nodules from the non-legume Myrica pensylvanica L. Plants were grown in sand culture and inoculated with a crushed nodule suspension. Homogenates were prepared by freezing and crushing 4–6 month old nodules to a fine powder in liquid N₂. The homogenate was taken up in buffered sodium dithionite under Ar. Such homogenates reduced up to 2 μmol C₂H₂ h^?1 g^?1 fresh nodule for at least 3 h when assayed anaerobically in the presence of ATP, Mg²⁺, and sodium dithionite. All activity was particle bound. Acetylene reduction was prolonged by sequestering inhibitory phenolics with solid polyvinylpolypyrrolidone. Highest responses were obtained with 30 mm ATP and Mg²⁺ and 150 mm sodium dithionite. The high concentrations of ATP, Mg²⁺, and sodium dithionite required may indicate barriers to the uptake. The temperature optimum was between 25–28°C. Activity was retained after storage for 30 min at ?10, 5° and 10°C but was lost at 50°C and reduced by 60% at 27°C. The preparation of active C₂H₂-reducing homogenates from this symbiotic association opens the way to further physiological and biochemical studies on the bayberry nitrogenase.     

Nitrous oxide production by denitrification and nitrification in temperate forest, grassland and agricultural soils

Nitrous oxide is produced in soils by biological denitrification and nitrification. To improve the fundamental understanding of the processes leading to N₂O fluxes from soils, the production of N₂O from denitrification and nitrification in spruce forest, beech forest, riparian grassland, coastal grassland and an agricultural field were studied. Samples were taken at a high and a low position along a topographic gradient in each site in the spring and autumn when the largest N₂O fluxes were expected. They were incubated after being amended with N, and C₂H₂ was used as biological inhibitor to distinguish nitrification and denitrification. The N₂O production in the low landscape position varied between 32 and 121 ng N cm^?3 h^?1 in the riparian grassland. 9 and 26 ng N cm^?3 h^?1 in the coastal grassland, and 135 and 195 ng N cm^?3 h^?1 in the agricultural field which was 10–100 times more than in the high positions where rates ranged between 3 and 5 ng N cm^?3 h^?1, 0.3 and 0.4 ng N cm^?3 h^?1, and 7 and 10 ng N cm^?3 h^?1, respectively. These differences almost certainly arose because the soil in the low positions was wetter and contained more organic matter. In the two forests N₂O production was less than 1 ng N cm^?3 h^?1, strongly inhibited by O₂, and not influenced by landscape position. Nitrification contributed to more than 60% of total N₂O production in the riparian grassland. In the agricultural field nitrification produced 13–74% of the total N₂O in the low position, and 10–88% in the high position. Denitrification was the dominant source of N₂O in the coastal grassland except at the low position in the autumn where nitrification produced 60% of the total N₂O. In the two forests where the soil had small nitrification potentials denitrification was the only source of N₂O. In the other sites nitrification and denitrification potentials were large and of identical magnitude. The results emphasize the need to separate nitrification and denitrification at the process level and to recognize topography at the field scale when modelling N₂O effluxes from soil.     

Nitrogen fixing activity in warty lenticellate tree barks

Detached lenticellate warty bark of Inga laurina was first shown to develop acetylene reducing activity (ARA) (18 nmol C₂H₂red·g^-1 (F.W.)·hr^-1) after a few days' incubation with ambient air containing 0.10 atm C₂H₂. Similar N₂ase development was found to occur with warty barks of many other plant species including 12 leguminous and 9 non-leguminous trees growing in wet tropical and temperate forests. Among them higher activities (>7 nmol C₂H₂ red·g^-1 (F.W.)·hr^-1) were recorded in Inga laurina, Cynometra ramiflora, Cassia siamea, Robinia pseudo-acacia, Albizia julibrissin in the leguminous plants, and Ilex crenata, Ilex pedunculosa, Rhizophora mucronata, Bruguiera gymnorrhiza and Mallotus japonicus in the non-leguminous plants. The development of ARA was often accelerated under lower pO₂ (0.05 atm) and pC₂H₂ (0.05 atm). Complete replacement of O₂ with N₂ or Ar in the incubation atmosphere resulted in full suppression of the development of N₂ase activity.

When ARA measurement was made with intact barks of natural stands (Robinia pseudo-acacia, Ilex pedunculosa and Mallotus japonicus), it was found that all of these intact barks were reducing C₂H₂ linearly without any time lag. The activities recorded were 4.6–11.5 nmol C₂H₂ red·g^-1 (F.W.)·hr^-1 corresponding to 1.19–1.27 nmol C₂H₂ red·cm^-2hr^-1. These values roughly coincided with ARA of detached warty barks of the same plant species. The present results suggest that in situ N₂-fixation in tree barks of the forests would amount to several 10 kg·ha^-1·year^-1 in temperate and tropical wet forests     

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.     

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.     

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.     

Bacterial composition and N2-fixation of some Egyptian soils cultivated with wheat (Triticum aestivum L.)

The composition of the microflora, N₂-fixing bacteria particularly, in different soils cultivated with wheat in Egypt was investigated in some samples collected from the fields after applying the agricultural practices recommended for wheat cultivation and just before sowing. The influence of carbon sources, mineral nitrogen and water regimes on potential dinitrogen fixation (acetylene reduction assay) in soils was investigated. The bacterial population densities including-N₂-fixing organisms were related to a number of environmental factors such as organic matter content. Among diazotrophs, Azotobacter spp. and Azospirillum spp. were encountered in higher densities in comparison with clostridia. Unamended soils showed a lower acetylene-reducing activity (0.5–61.5 nmoles C₂H₄ g^?1 h^?1). Addition of glucose (1% w/w) greatly enhanced such activity being the highest (86.9–2846.5 nmoles C₂H₄ g^?1 h^?1) in the clay soil with the highest organic carbon content (1.42%). Glucose amendment had no significant influence on acetylene reduction in the saline soil. N₂-fixation in barley straw-amended (1%) soils was not much higher than in unamended soils. Concentrations of up to 70 ppm ammonium-nitrogen depressed N₂-fixation in soils that received barley straw. Acetylene reduction in submerged soil increased after addition of cellulose. Non-flooded conditions favoured N₂-fixation in the fertile clay soil amended with sucrose.