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.     

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.     

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.     

Gaseous nitrogen losses from fertilized soil during nitrification and denitrification using the acetylene blockage technique

Summary A sandy soil amended with different forms and amounts of fertilizer nitrogen (urea, ammonium sulphate and potassium nitrate) was investigated in model experiments for N₂O emission, which may be evolved during both oxidation of ammonia to nitrate and anaerobic respiration of nitrate. Since C₂H₂ inhibits both nitrification and the reduction of N₂O to N₂ during denitrification, the amount of N₂O evolved in the presence and absence of C₂H₂ represents the nitrogen released through nitrification and denitrification.Results show that amounts of N₂O-N lost from soils incubated anaerobically with 0.1% C₂H₂ and treated with potassium nitrate (23.1 µg N-NO ₃ ^– /g dry soil) exceeded those from soils incubated in the presence of 20% oxygen and treated with even larger amounts of nitrogen as urea and ammonium sulphate. This indicates that nitrogen losses by denitrification may potentially be higher than those occurring through nitrification.     

Acetylene and N-serve effects upon N2O emissions from NH4+ and NO3− treated soils under aerobic and anaerobic conditions

N₂O emissions from soils treated with NH₄⁺-N under aerobic conditions in the laboratory were 3- to 4-fold higher than those from controls (no extra N added) or when NO₃^?-N was added. Although the emission of N₂O-N in these field and laboratory experiments represented only 0.1–0.8% of the applied fertilizer NH₄⁺-N and are therefore not significant from an agronomic standpoint, these studies have conclusively demonstrated that the oxidation of applied ammoniacal fertilizers (nitrification) could contribute significantly to the stratospheric N₂O pool.Like N-serve, acetylene was shown to be a potent inhibitor of nitrification as it stopped the oxidation of NH₄⁺-N to (NO₃⁺-N + NO₂^?)-N and hence reduced the evolution of N₂O from nitrification within 60 min after its addition.Although high amounts of NO₃^?-N were present, the rate of denitrification was very low from soils with moisture up to 60% saturation. The further increase in the degree of saturation resulted in several-fold increase of denitrification which eventually became the predominant mechanism of gaseous N losses under anaerobic conditions.     

Nitrification in three soils amended with zinc sulfate

Zinc as ZnSO₄ was added to three soils at rates of 0, 10, 100 and 1000 μg Zn g^?1 soil. The soils were uniformly treated with 100 μg Ng^?1 as nh₄cl, incubated at 30°C and NH₄⁺-N and (NO₃^? + NO₂^?)-N determined weekly for 7 weeks. Nitrification in all three soils was totally inhibited by 1000 μg Zn g^?1. At the 100 μg Zn g^?1 rate, nitrification was significantly reduced in two of the three soils during some part of the incubation. This differential effect on nitrification at the 100 μg Zn g^?1 rate was related to differences in soil properties. These results imply that, with respect to nitrification, care should-be taken not to apply Zn-containing materials indiscriminately to soils.     

Effect of ammonium-, nitrite- and nitrate nitrogen on anaerobic nitrogenase activity in soil

Sandy loam soil, with added glucose, was incubated anaerobically under N₂ and subjected to repeated 1-h C₂H₂ reduction assays. In the presence of 1% glucose the addition of 50 μg NH₄⁺ ?N/g or of 20 μg NO^?₃ N/g (untreated soil contained 1.2 μg NH⁺₄?N and 7.10 μg NO^?₃-N/g) caused at least some suppression of nitrogenase activity. Activity developed when the KCl-extractable soil inorganic nitrogen concentration dropped below 35 μg/g. In the presence of 0.1 or 0.05% glucose the addition of 5 μg NH⁺₄?N/g caused some suppression of nitrogenase activity. However, activity developed when the soil NH₄⁺-N concentration dropped below about 4 μg/g. With 0.1% glucose and 5 μg added NO^?₂ N/g, activity did not develop until the soil NO^?₂ -N concentration dropped to zero. Added NO^?₃ N was rapidly reduced and denitrified to NO^?₂- N, N₂O-N and NH⁺₄ N and furthermore caused some inhibition of CO₂ evolution. The data from NH₄^?-addition experiments are consistent with a nitrogenase repression/ derepression threshold of 4 and 35μg NH⁺₄-N/g at 0.05 and 1% glucose concentrations, respectively. The data from NO^?₂- and NO^?₃-addition experiments suggest a combination of repression and toxicity effects in the presence of added NO^?₃ N.     

Effects of nitrogen dioxide on selected soil processes

Nitrogen dioxide gas was rapidly absorbed by soil. After a 15 min incubation at 25°C, soil at a moisture content of 16% absorbed 99% of the NO₂ introduced into the gas-phase volume of a closed system. The presence of microorganisms hatl no influence on the rate of absorption of the gas by soil. The absorption of NO₂ by sandy clay loam soil was not an oxygen- or temperature-dependent process nor did it depend upon the moisture content of the soil. These physical factors acquired significance only in determining the initial rate of absorption of the gas and the rate at which NO₂ diffused through the soil. Exposure of soil to NO₂ resulted in substantial increases in the levels of NO _inf2 ^sup? N in the soil. Chemical oxidation of the NO _inf2 ^sup? N resulted in an increase in NO _inf3 ^sup? N levels. During a 14-day incubation, NO _inf2 ^sup? N concentrations in sterile soil exposed to an atmosphere containing 100 μg ml^?1 of NO₂ decreased from 190 μg g^?1 of soil to 105 μg g^?1 with an accompanying increase in NO _inf3 ^sup? N from 2 μg g^? ₁ to 63 μg g^?1 of soil. Nitrogen dioxide severely inhibited the growth of both aerobic and anaerobic asymbiotic N₂-fixing bacteria in soil. After a 48 h incubation at 25°C, soil aggregates exposed to an atmosphere containing 100 μg ml^?1 of NO₂ contained 88% and 98% fewer aerobic and anaerobic N₂-fixing bacteria, respectively. C₂H₂-reduction measurements showed that nitrogenase synthesis and activity in artificial soil aggregates amended with 2% glucose were inhibited by 20% and 48%, respectively, when exposed to atmospheric concentrations of 35 and 3.5 μg ml^?1 of NO₂, respectively.     

A method of selective inhibition to distinguish between nitrification and denitrification as sources of nitrous oxide in soil

Summary Nitrapyrin and C₂H₂ were evaluated as nitrification inhibitors in soil to determine the relative contributions of denitrification and nitrification to total N₂O production. In laboratory experiments nitrapyrin, or its solvent xylene, stimulated denitrification directly or indirectly and was therefore considered unsuitable. Low partial pressures of C₂H₂ (2.5–5.0 Pa) inhibited nitrification and had only a small effect on denitrification, which made it possible to estimate the contribution of denitrification. The contribution of nitrification was estimated by subtracting the denitrification value from total N₂O production (samples without C₂H₂). The critical C₂H₂ concentrations needed to achieve inhibition of nitrification, without affecting the N₂O reductase in denitrifiers, must be individually determined for each set of experimental conditions.     

Effect of sodium chloride on CO2 evolution,ammonification, and nitrification in a Sassafras sandy loam

Sodium chloride, at rates up to 100 mg g^?1, was added to a Sassafras sandy loam amended with finely-ground alfalfa to determine the effect of NaCl on CO₂ evolution, ammonification, and nitrification in a 14-week study. A NaCl concentration of 0.25 mg g^?1 significantly reduced CO₂ evolution by 16% in unamended soil and 5% in alfalfa-amended soil. Increasing NaCl progressively reduced CO₂ evolution, with no CO₂ evolved from the soil receiving 100 mg NaCl g^?1. A 0.50 mg NaCl g^?1 rate was required before a significant reduction in decomposition of the alfalfa occurred. The NO^?₂-N + NO^?₃-N content of the soil was significantly reduced from 40 to 37 μg g^?1 at 0 and 0.25 mg NaCl g^?1, respectively in the unamended soil. In the alfalfa amended soil, nitrification was significantly reduced at 5 mg NaCl g^?1. At 10 mg NaCl g^?1, nitrification was completely inhibited, there being only 6 and 2 μg NO^?₂-N + NO^?₃-N g^?1 in the alfalfa amended and unamended soil, respectively. In the alfalfa amended soil NH⁺₄-N accumulated from 6 μg g^?1 at the 0 NaCl rate to a maximum of 54 μg g^?1 with 25 mg NaCl g^?1. These higher NH⁺₄-N values resulted in a 0.5 unit increase in the pHw over that of the 0 NaCl rate in the alfalfa amended soil. At NaCl concentrations above 25 mg g^?1 there was a reduction in NH⁺₄-N. The addition of alfalfa to the soil helped to alleviate the adverse affects of NaCl on CO₂ evolution and nitrification.     

Nitrification in soils incubated with pig slurry or ammonium sulphate

The effects of temperature, moisture content and the addition of pig slurry on nitrification in two soils were studed. There was no accumulation of NO₂^?-N under the incubation conditions investigated and the accumulation of NO₃^?-N was linear for additions of 50–250 μg NH₄⁺-N g^? soil, either as ammonium sulphate or as pig slurry. Nitrate formation was treated as a single step, zero order process to enable a rate constant to be calculated. Nitrification rate increased with increasing moisture content up to the highest level tested, soil water potential ?8.0 kPa, corresponding to approximately 60% of water holding capacity in both soils. Measurable nitrification was found in both soils at the lowest moisture content (soil water potential ?1.5 MPa) and temperature (5° C) tested. The nitrification rate constant in soils treated with 50 μg NH₄⁺-N g^? soil was not significantly affected (P = 0.05) by the form of ammonium added. Addition of 250 μg NH₄⁺-N as ammonium sulphate caused a marked inhibition of nitrification at all moisture contents and temperatures. Addition of 250 μg NH₄⁺-N as pig slurry caused a marked increase in nitrification rate, the increase being greater at the higher temperatures and moisture contents.     

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.     

The influence of glucose and nitrate concentrations upon denitrification rates in sandy soils

Dentrification rates in two soils were assessed separately as a function of NO₃^? concentration while providing a constant initial glucose concentration, and as a function of glucose concentration while providing a constant initial NO₃^?-N concentration. Of the soils used, a Hanford sandy loam and a Coachella fine sand, the bacteria in the former produced higher rates of denitrification with a maximum loss of 1500 μg NO₃^?-N/ml day^?1 as compared to a loss of 150 μg NO₃^?-N/ml day^?1 from the latter. Rates of loss closely approximated Michaelis Menten kinetics in the Coachella sand, and K_m values for glucose-C and NO₃^?-N were 500 μg/ml and 170 μg/ml, respectively. Rates of loss of NO₃^?-N from the Hanford soil did not approximate Michaelis-Menten kinetics, and this was attributed to failure to saturate enzyme systems in the denitrifying bacteria with glucose and nitrogen when each was held constant. C/N ratios around 2 appeared to provide the greatest rates of denitrification. High C/N ratios or high glucose concentrations (1.8 per cent) retarded denitrification, with fungal growth and a subsequent drop in pH occuring. A Pseudomonas was incubated aerobically for 24 h followed by a 72 h anaerobic incubation with nitrate as the sole nitrogen source at 0, 10, 50, 100, 250 and 500mg N/ml concentrations. Assimilatory nitrate reduction never exceeded 75 mg N/ml, and it was concluded that this mode of nitrate reduction is insignificant at higher nitrate concentrations by comparison to dissimilatory nitrate reduction, i.e. denitrification.     

区分土壤中硝化与反硝化对N2O产生贡献的方法

土壤是产生N₂O的最主要来源之一。硝化和反硝化反应是产生N₂O的主要机理，由于硝化和反硝化微生物同时存在于土壤中，因而硝化和反硝化作用能同时产生N₂O。N₂O的来源可通过使用选择性抑制剂，杀菌剂以及加入的标记底物确定。通过对生成N₂O反应的每一步分析，主要从抑制反应发生的催化酶和细菌着手，总结了测量区分硝化、反硝化和DNRA反应对N₂O产生的贡献方法。并对¹⁵N标记底物法，乙炔抑制法和环境因子抑制法作了详细介绍。     

Effects of pentachlorophenol on asymbiotic nitrogen fixation in soil

The effect of 50, 100, 150, and 400 μg sodium pentachlorophenate (Na-PCP) per gram soil was studied in nonsterile soil incubated under aerobic and anaerobic conditions, and in sterilized soil inoculated withAzotobacter sp. isolated from the soil. N₂ fixation was determined by acetylene reduction. Pentachlorophenate at a concentration of 50 μg g^?1 had an inhibitory effect in nonsterile soil incubated aerobically while strong inhibition of dinitrogen fixation in nonsterile soil occurred in the presence of 100 μg g^?1 and above. The EC₅₀ values for the inhibition of nitrogenase activity in nonsterile soil incubated aerobically and anaerobically and in sterilized soil inoculated withAzotobacter sp. suspensions were 49.8±1.4 μg Na-PCP g^?1, 186.8±2.8 μg Na-PCP g^?1, and 660.8±29.3 μg Na-PCP g^?1, respectively.     

Processes responsible for the nitrous oxide emission from a Costa Rican Andosol under a coffee agroforestry plantation

We used the inhibitor acetylene (C₂H₂) at partial pressures of 10 Pa and 10 kPa to inhibit autotrophic nitrification and the reduction of nitrous oxide (N₂O) to N₂, respectively. Soils (Andosol) from a Coffea arabica plantation shaded by Inga densiflora in Costa Rica were adjusted to 39, 58, 76 and 87% water-filled pore space (WFPS) and incubated for 6 days in the absence or presence of C₂H₂. Soil respiration, nitrification rates and N₂O emissions by both processes were measured in relation to soil moisture conditions. At all WFPS studied, rates of N₂O and N₂ productions were small (4.8; 14.7; 23 and 239.6 ng N–N₂O g⁻¹ d.w. d⁻¹ at 39, 58, 76 and 87% WFPS, respectively), and despite a low soil pH (4.7), N₂O was mainly produced by nitrification, which was responsible for 85, 91, 84 and 87% of the total N₂O emissions at 39, 58, 76 and 87% WFPS, respectively. At the three smaller values of WFPS, a linear relationship was established between WFPS, soil respiration, nitrification and N₂O released by nitrification; no N₂ was produced by denitrification. At more anaerobic conditions achieved by a WFPS of 87%, a large rate of N₂O production was measured during nitrification, and N₂ production accounted for 84% of the gaseous N fluxes caused by denitrification.     

Nitrous oxide production at different soil moisture contents in an arable soil in China

Abstract

Laboratory incubations were conducted to investigate nitrous oxide (N₂O) production from a subtropical arable soil (Typic Plinthodults) incubated at different soil moisture contents (SMC) and with different nitrogen sources using a 10% (v/v) acetylene (C₂H₂) inhibitory technique at 25°C. The production of N₂O and CO₂ was monitored during the incubations and changes in the contents of KCl-extractable NO^? ₃-N and NH⁺ ₄-N were determined. The production of N₂O increased slightly with an increase in SMC from 40% water-holding capacity (WHC) to 70% WHC, but increased dramatically at 100% WHC. After incubation the NO^? ₃-N content increased even at a SMC of 100% WHC. At a SMC of 100% WHC, the addition of NH⁺ ₄-N promoted the production of N₂O and CO₂, whereas the addition of NO^? ₃-N decreased N₂O production. Compared with the incubation without C₂H₂, the presence of C₂H₂ increased NH⁺ ₄-N content, but decreased NO^? ₃-N content, and there was no significant difference in N₂O production. These results indicate that heterotrophic nitrification contributes to N₂O production in the soil.     

Nitrous oxide production in grassland soils: assessing the contribution of nitrifier denitrification

Nitrifier denitrification is the reduction of NO₂⁻ to N₂ by nitrifiers. It leads to the production of the greenhouse gas nitrous oxide (N₂O) as an intermediate and possible end product. It is not known how important nitrifier denitrification is for the production of N₂O in soils. We explored N₂O production by nitrifier denitrification in relation to other N₂O producing processes such as nitrification and denitrification under different soil conditions. The influence of aeration of the soil, different N sources, and pH were tested in four experiments. To differentiate between sources of N₂O, an incubation method with inhibitors was used [Biol. Fertil. Soils 22 (1996) 331]. Sets of four incubations included controls without addition of inhibitors, incubations with addition of small concentrations of C₂H₂ (0.01-0.1 kPa), large concentrations of O₂ (100 kPa), or a combination of C₂H₂ and O₂. The results indicate that the availability of NO₂⁻ stimulated the apparent N₂O production by nitrifier denitrification. A decreasing O₂ content increased the total N₂O production, but decreased N₂O production by nitrifier denitrification. No significant effect of pH could be found. The study revealed problems concerning the use of the inhibitors C₂H₂ and O₂. Almost one-third of all incubations with inhibitors produced more N₂O than the controls. Possible reasons for the problems are discussed. The inhibitors C₂H₂ and O₂ need to be tested thoroughly for their effects on different N₂O producing processes before further application.     

Heterotrophic N2-fixation in arid soil crusts

The cryptogamic soil crusts of the Great Basin Artemisia, Ceratoides, and Atriplex plant communities contain a significant heterotrophic N₂-fixing microbial population in addition to the predominating filamentous cyanobacteria. The bacterial association with the cyanobacteria exhibits a phycosphere-like effect. Heterotrophically fixed N gains reached 17.5 μg N· g^?1 of soil (23.1% increase above the initial soil N content) and 45.9 μg N·g^?1 of soil (57.4% increase) after 3 and 5 weeks, respectively. (NH₄)₂SO₄ and native plant material amendments to soil resulted in a 41–100% reduction in N₂-fixation. The potential input of N to soil crusts may be reduced in the presence of shrub-produced allelochemic agents and by concurrent denitrification.     

Effect of Inorganic N Composition of Fertilizers on Nitrous Oxide Emission Associated with Nitrification and Denitrification

We studied the effect of repeated application (once every 2 d) of a fertilizer solution with different ratios of NH₄ ⁺ - and NO₃ ^?-N on N₂O emission from soil. After the excess fertilizer solution was drained from soil, the water content of soil was adjusted to 50% of the maximum water-holding capacity by suction at 6 × 10³ Pa. Repeated application of NH₄ ⁺- rich fertilizer solution stimulated nitrification in soil more than NO₃ ^?-rich fertilizer. Although the evolution of N₂O through nitrifier denitrification tended to increase with the repeated addition of a fertilizer solution rich in NH₄ ⁺ rather than in NO₃ ^?, the contribution of nitrifier denitrification remained at levels of 20 to 36% of the total emission regardless of the inorganic N composition. The total emission of N₂O also tended to increase with the application of NH₄ ⁺- rather than NO₃ ^?-rich fertilizer. It was suggested that the coupled process of nitrification and denitrification at micro-aerobic sites became important when fertilizer rich in NH₄ ⁺ was applied to soil under relatively aerobic conditions.