Comparison of two different methods to measure nitric oxide turnover in soils

 The NO turnover in soils was measured in two different experimental set-ups, a flow-through system, which is very time-consuming and needs rather sophisticated equipment, and a closed system using serum bottles. We compared the NO turnover parameters (NO consumption rate constant, NO production rate, NO compensation concentration) that were measured with both systems in different soils, under different conditions and in the presence of 10 Pa acetylene to inhibit nitrification. The values of the NO turnover parameters that were measured with the two systems under oxic conditions were usually comparable. The addition of acetylene did not affect the NO consumption rate constants of the soils with the exception of soil G1. However, the NO production rates and the NO compensation concentrations decreased significantly in the presence of acetylene, indicating that nitrification was the main source of NO in these soils. Only one soil (Bol) showed no nitrifying activity. Increasing soil moisture content resulted in decreasing NO consumption rate constants and NO production rates. Even at a high soil moisture content of 80% water holding capacity, nitrification was the main source of NO. The values of the NO turnover parameters that were measured with the two systems were not comparable under anoxic conditions. The NO consumption rate constants and the NO production rates were much lower in the closed than in the flow-through system, indicating that the NO consumption activity became saturated by the high NO concentrations accumulating in the closed system. Under oxic conditions, however, closed serum bottles were a cheap, easy and reliable tool with which to determine NO turnover parameters and to distinguish between nitrification and denitrification as sources of NO. Received: 21 April 1998     

Influence of soil properties on the turnover of nitric oxide and nitrous oxide by nitrification and denitrification at constant temperature and moisture

 Soils are a major source of atmospheric NO and N₂O. Since the soil properties that regulate the production and consumption of NO and N₂O are still largely unknown, we studied N trace gas turnover by nitrification and denitrification in 20 soils as a function of various soil variables. Since fertilizer treatment, temperature and moisture are already known to affect N trace gas turnover, we avoided the masking effect of these soil variables by conducting the experiments in non-fertilized soils at constant temperature and moisture. In all soils nitrification was the dominant process of NO production, and in 50% of the soils nitrification was also the dominant process of N₂O production. Factor analysis extracted three factors which together explained 71% of the variance and identified three different soil groups. Group I contained acidic soils, which showed only low rates of microbial respiration and low contents of total and inorganic nitrogen. Group II mainly contained acidic forest soils, which showed relatively high respiration rates and high contents of total N and NH₄ ⁺. Group III mainly contained neutral agricultural soils with high potential rates of nitrification. The soils of group I produced the lowest amounts of NO and N₂O. The results of linear multiple regression conducted separately for each soil group explained between 44–100% of the variance. The soil variables that regulated consumption of NO, total production of NO and N₂O, and production of NO and N₂O by either nitrification or denitrification differed among the different soil groups. The soil pH, the contents of NH₄ ⁺, NO₂ ^– and NO₃ ^–, the texture, and the rates of microbial respiration and nitrification were among the important variables. Received: 28 October 1999     

土壤N2O和NO产生机制研究进展

N2O和NO是大气中两种重要的活性氮气体,强烈影响着全球变化和生态环境。土壤是N2O和NO的重要排放源,生物和非生物途径均可产生N2O和NO。本文详细论述了自养硝化、异养硝化、生物反硝化、化学反硝化、硝化细菌反硝化和硝态氮异化还原成铵作用产生N2O和（或）NO的机制,并对研究中存在的一些问题进行了探讨。     

Contribution of denitrification and autotrophic and heterotrophic nitrification to nitrous oxide production in andosols

To quantify the contribution of denitrification and autotrophic and heterotrophic nitrification to N₂O production in Andosols with a relatively high organic matter content, we first examined the effect of C₂H₂ concentrations on N₂O production and on changes in mineral N contents. The optimum C₂H₂ concentration for inhibiting autotrophic nitrification was 10 Pa. Secondly, and Andosol taken from an arable field was incubated for 32 days at 30°C at 60, 80, and 100% water-holding capacity with or without the addition of NH ₄ ⁺ or NO _inf3 ^sup- (200 mg N kg^-1), and subsamples collected every 4–8 days were further incubated for 24 h with or without C₂H₂ (10 Pa). At 60 and 80% water-holding capacity with NH ₄ ⁺ added, 87–92% of N₂O produced (200–250 g N₂O–N kg^-1) was derived from autotrophic nitrification. In contrast, at 100% water-holding capacity with or without added NO _inf3 ^sup- , enormous amounts of N₂O (29–90 mg N₂O–N kg^-1) were produced rapidly, mostly by denitrification (96–98% of total production). Thirdly, to examine N₂O production by heterotrophic nitrification, the Andosol was amended with peptone or NH ₄ ⁺ (both 1000 mg N kg^-1)+citric acid (20 g C kg^-1) and with or without dicyandiamide (200 mg N kg^-1). Treatment with citric acid alone or with citric acid+dicyandiamide suppressed N₂O production. In contrast, peptone increased N₂O production (5.66 mg N₂O–N kg^-1) mainly by denitrification (80% of total production). However, dicyandiamide reduced N₂O production to 1.1 mg N₂O–N kg^-1. These results indicate that autotrophic nitrification was the main process for N₂O production except at 100% water-holding capacity where denitrification became dominant and that heterotrophic nitrification had a lesser importance in the soils examine.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday     

Relationships between soil moisture content and nitrous oxide production during nitrification and denitrification

Summary The effect of soil water content [60%–100% water-holding capacity (WHC)] on N₂O production during autotrophic nitrification and denitrification in a loam soil was studied in a laboratory experiment by selectively inhibiting nitrification with a low C₂H₂ concentration (2.1 Pa). Nitrifiers usually produced more N₂O than denitrifiers. During an initial experimental period of 0–6 days the nitrifiers produced more N₂O than the denitrifiers by a factor ranging from 1.4 to 16.5, depending on the water content and length of incubation. The highest N₂O production rate by nitrifiers was observed at 90% WHC, when the soil had become partly anaerobic, as indicated by the high denitrification rate. At 100% WHC there were large gaseous losses from denitrification, while nitrification losses were smaller except for the first period of measurement, when there was still some O₂ remaining in the soil. The use of 10 kPa C₂H₂ to inhibit reduction of N₂O to N₂ stimulated the denitrification process during prolonged incubation over several days; thus the method is unsuitable for long-term studies.     

Temperature dependence of nitrification,denitrification, and turnover of nitric oxide in different soils

Summary The temperature dependence of the NO production rate and the NO consumption rate constant was measured in an Egyptian soil, a soil from the Bavarian Forest, and a soil from the Donau valley, together with the temperature dependence of the potential rates of ammonium oxidation, nitrite oxidation, and denitrification, and the temperature dependence of the growth of NH _inf4 ^sup+ -oxidizing, NO _inf2 ^sup- -oxidizing, and NO _inf3 ^sup- -reducing bacteria in most probable number assays. In the acidic Bavarian Forest soil, NO production was only stimulated by the addition of NO _inf3 ^sup- but not NH _inf4 ^sup+ . However, NO production showed no temperature optimum, indicating that it was due to chemical processes. Most probable numbers and potential activities of nitrifiers were very low. NO consumption, in contrast, showed a temperature optimum at 25°C, demonstrating that consumption and production of NO were regulated individually by the soil temperature. In the neutral, subtropical Egyptian soil, NO production was stimulated only by the addition of NH _inf4 ^sup+ but not NO _inf3 ^sup- . All activities and most probable numbers showed a temperature optimum at 25° or 30°C and exhibited apparent activation energies between 61 and 202 kJ mol^-1. However, a few nitrifiers and denitrifiers were also able to grow at 8° or 50°C. Similar temperature characteristics were observed in the Donau valley soil, although it originated from a temperate region. In this soil NO production was stimulated by the addition of NH _inf4 ^sup+ or of NO _inf3 ^sup- . Both NO production and consumption were stimulated by drying and rewetting.     

Controls on nitric oxide emissions from tropical pasture and rain forest soils

In field studies, forest soils in the Atlantic Lowlands of Costa Rica emitted greater amounts of nitric oxide (NO) than soils from pastures that had been actively grazed for over 20 years following their conversion from forest. We measured NO production from intact soil cores from these land uses. Laboratory tests using ammonium(NH ₄ ⁺ ), nitrate (NO ₃ ^– ), nitrite (NO ₂ ^– ), water, and acetylene (C₂H₂) additions demonstrate a response consistent with field studies.Forest soil cores produced more NO than pasture cores regardless of treatment. In forest soil the response toNH ₄ ⁺ solution was significantly greater than response to water or an ambient moisture control. Addition of 10 kPa C₂H₂ caused a marked decrease in NO production in forest soil cores. These responses suggest a nitrification-linked control over NO production. Large and rapid responses toNO ₂ ^– additions suggest that chemical decomposition of this ion may contribute to NO production. Pasture soil cores did not show a significant response to any of the treatments including NO ₂ ^– . Low porosity in the pasture soils may restrict emission of NO produced therein.     

Mechanistic model for nitrous oxide emission via nitrification and denitrification

Seasonal and annual N₂O fluxes from urine-affected pasture were approximated with a mechanistic model based on Michaelis-Menten kinetics. The model combined the effects of soil nitrate-N, soil ammonium-N, soil temperature and soil moisture (all from the top 5cm) to calculate N₂O emissions from nitrification (F _nit ) and denitrification (F _den ), with total N₂O emission being the sum of the two (F _tot =F _nit +F _den ). Best results were obtained when different kinetic parameters were used for periods of constant soil moisture conditions and after heavy rainfalls when a rapid change of the soil moisture status occurred. Modelled N₂O emissions over a year were within the range of uncertainties of measured N₂O emissions. Results indicate that the spatial variability of N₂O emissions at times when all the model inupt variables were constant may be related to microorganism growth dynamics or enzyme production rates. Received: 2 October 1995     

Field method to determine N2O emission from nitrification and denitrification

 Nitrous oxide (N₂O) emissions via the nitrification (I _nit) and denitrification (I _den) pathways were successfully measured with in-field incubation of soil cores in preserving jars at 0 Pa and 5–10 Pa acetylene. From the incubations, fractions of nitrification – N₂O over total N₂O (I _nit / I _tot) – and denitrification – N₂O over total N₂O (I _den / I _tot) – were obtained. Actual field emissions of N₂O via nitrification (F _nit) and denitrification (F _den) were calculated by multiplying the fractions from the incubation technique with the daily N₂O emission (F _day) determined with a direct soil cover method. The approach presented here was successful for a whole range of soil moisture conditions in intensive grassland. F _nit and F _den followed the trends of soil ammonium and soil nitrate. Received: 31 October 1997     

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。     

Responses of nitrous oxide, dinitrogen and carbon dioxide production and oxygen consumption to temperature in forest and agricultural light-textured soils determined by model experiment

 The experiment, carried out on a forest and arable light-textured soil, was designed to study the temperature response of autotrophic and heterotrophic N₂O production and investigate how the N₂O flux relates to soil respiration and O₂ consumption. Although N₂O production seemed to be stimulated by a temperature increase in both soils, the relationship between production rate and temperature was different in the two soils. This seemed to depend on the different contribution of nitrification and denitrification to the overall N₂O flux. In the forest soil, almost all N₂O was derived from nitrification, and its production rate rose linearly from 2  °C to 40  °C. A stronger effect of temperature on N₂O production was observed in the arable soil, apparently as a result of an incremental contribution of denitrification to the overall N₂O flux with rising temperature. The soil respiration rate increased exponentially with temperature and was significantly correlated with N₂O production. O₂ consumption stimulated denitrification in both soils. In the arable soil, N₂O and N₂ production increased exponentially with decreasing O₂ concentration, though N₂O was the main gas produced at any temperature. In the forest soil, only the N₂ flux was related exponentially to O₂ consumption and it outweighed the rate of N₂O production only at >34  °C. Thus, it appears that in the forest soil, where nitrification was the main source of N₂O, temperature affected the N₂O flux less dramatically than in the arable soil, where a temperature increase strongly stimulated N₂O production by enhancing favourable conditions for denitrification. Received: 26 August 1998     

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。     

Influence of thiosulfate on nitrification,denitrification, and production of nitric oxide and nitrous oxide in soil

Thiosulfate and CS₂ inhibit nitrification. The effect of the addition of thiosulfate on the turnover of inorganic N compounds was tested in an Egyptian and a German arable soil under nitrifying and denitrifying conditions. For nitrification, the soils were amended with NH _inf4 ^sup+ and incubated under aerobic conditions. For denitrification, the soils were amended with NO _inf3 ^sup- and incubated under anaerobic conditions. In both cases, the thiosulfate decreased with time while tetrathionate accumulated to an intermediate extent. Both compounds disappeared completely after <25 days. Production of CS₂ was not observed. Carbonyl sulfide was produced only in the Egyptian soil, but production decreased with increasing amounts of added thiosulfate. Under nitrifying conditions, the addition of increasing amounts of thiosulfate (25, 50, and 100 g S g^-1 dry weight) resulted in decreasing rates of NH _inf4 ^sup+ oxidation to NO _inf3 ^sup- ; it also resulted in an increasing intermediate accumulation of NO _inf2 ^sup- and NO, and in an increasing production of N₂O. Under denitrifying conditions, the addition of increasing amounts of thiosulfate did not significantly affect the rate of NO _inf3 ^sup- reduction, and resulted in an increasing intermediate accumulation of NO _inf2 ^sup- and of NO only in the German soil in which the production of N₂O was slightly inhibited by thiosulfate. These results demonstrate that the nitrification of NH _inf4 ^sup+ and NO _inf2 ^sup- was inhibited by increasing concentrations of thiosulfate and/or tetrathionate without involving the formation of volatile S compounds as potential nitrification inhibitors. Denitrification was not affected by the addition of thiosulfate.     

Comparison of two versions of the acetylene inhibition/soil core method for measuring denitrification loss from an irrigated wheat field

 Two versions of the acetylene inhibition (AI)/soil core method were compared for the measurement of denitrification loss from an irrigated wheat field receiving urea-N at a rate of 100 kg ha^–1. With AI/soil core method A, the denitrification rate was measured by analysing the headspace N₂O, followed by estimation of N₂O dissolved in the solution phase using Bunsen absorption coefficients. With AI/soil core method B, N₂O entrapped in the soil was measured in addition to that released from soil cores into the headspace of incubation vessels. In addition, the two methods were also compared for measurement of the soil respiration rate. Of the total N₂O produced, 6–77% (average 40%) remained entrapped in the soil, whereas for CO₂, the corresponding figures ranged from 12–65% (average 44%). The amount of the entrapped N₂O was significantly correlated with the water-filled pore space (WFPS) and with the N₂O concentration in the headspace, whereas CO₂ entrapment was dependent on the headspace CO₂ concentration but not on the WFPS. Due to the entrapment of N₂O and CO₂ in soil, the denitrification rate on several (18 of the 41) sampling dates, and soil respiration rate on almost all (27 of the 30) sampling dates were significantly higher with method B compared to method A. Averaged across sampling dates, the denitrification rate measured with method B (0.30 kg N ha^–1 day^–1) was twice the rate measured with method A, whereas the soil respiration rate measured with method B (34.9 kg C ha^–1 day^–1) was 1.6 times the rate measured with method A. Results of this study suggest that the N₂O and CO₂ entrapped in soil should also be measured to ensure the recovery of the gaseous products of denitrification by the soil core method. Received: 12 May 1998     

Automated sampling system for long-term monitoring of nitrous oxide and methane fluxes from soils

We describe an automated gas sampling system for monitoring trace gas fluxes from soils. The sampling system allows automated collection of gas samples in glass vials using a syringe pump connected to an automated static chamber installed in the field. The gas samples are transferred to a laboratory and then analyzed using a gas chromatography system. Comparisons between manual and automated sampling of standard gases showed good agreement ( r ² = 0.99996 for N₂O, r ² = 0.999 for CH₄ and r ² = 0.998 for CO₂). In a field test, replicated flux measurements using two chambers generally showed good agreement. The sampling system allows frequent and long-term monitoring of fluxes under a wide range of weather conditions (tested temperatures ranged from –6.5 to 40°C; 127 mm day⁻¹ max precipitation). The major advantages of the system are its increased portability, ease of operation and cost effectiveness compared with on-line automated sampling/analytical systems.     

Fluxes and production pathways of nitrous oxide in different types of tropical forest soils in Thailand

Nitrous oxide (N₂O) emissions from the soil surface of five different forest types in Thailand were measured using the closed chamber method. Soil samples were also taken to study the N₂O production pathways. The monthly average emissions (±SD, n?=?12) of N₂O from dry evergreen forest (DEF), hill evergreen forest (HEF), moist evergreen forest (MEF), mixed deciduous forest (MDF) and acacia reforestation (ARF) were 13.0?±?8.2, 5.7?±?7.1, 1.2?±?12.1, 7.3?±?8.5 and 16.7?±?9.2?µg N m^?2 h^?1, respectively. Large seasonal variations in fluxes were observed. Emission was relatively higher during the wet season than during the dry season, indicating that soil moisture and denitrification were probably the main controlling factors. Net N₂O uptake was also observed occasionally. Laboratory studies were conducted to further investigate the influence of moisture and the N₂O production pathways. Production rates at 30% water holding capacity (WHC) were 3.9?±?0.2, 0.5?±?0.06 and 0.87?±?0.01?ng N₂O-nitrogen (N) g-dw^?1day^?1 in DEF, HEF and MEF respectively. At 60% WHC, N₂O production rates in DEF, HEF and MEF soils increased by factors of 68, 9 and 502, respectively. Denitrification was found to be the main N₂O production pathway in these soils except in MEF.     

Effects of various nitrogen fertilizers on emission of nitrous oxide from soils

Summary Field studies of the effects of different N fertilizers on emission of nitrous oxide (N20) from three Iowa soils showed that the N₂O emissions induced by application of 180 kg ha^–1 fertilizer N as anhydrous ammonia greatly exceeded those induced by application of the same amount of fertilizer N as aqueous ammonia or urea. On average, the emission of N₂O-N induced by anhydrous ammonia was more than 13 times that induced by aqueous ammonia or urea and represented 1.2% of the anhydrous ammonia N applied. Experiments with one soil showed that the N₂O emission induced by anhydrous ammonia was more than 17 times that induced by the same amount of N as calcium nitrate. These findings confirm indications from previous work that anhydrous ammonia has a much greater effect on emission of N₂O from soils than do other commonly used N fertilizers and merits special attention in research relating to the potential adverse climatic effect of N fertilization of soils.Laboratory studies of the effect of different amounts of NH₄OH on emission of N₂O from Webster soil showed that the emission of N₂O-N induced by addition of 100 g NH₄OH-N g^–1 soil represented only 0.18% of the N applied, whereas the emissions induced by additions of 500 and 1 000 g NH4OH-N g^–1 soil represented 1.15% and 1.19%, respectively, of the N applied. This suggests that the exceptionally large emissions of N₂O induced by anhydrous ammonia fertilization are due, at least in part, to the fact that the customary method of applying this fertilizer by injection into soil produces highly alkaline soil zones of high ammonium-N concentration that do not occur when urea or aqueous ammonia fertilizers are broadcast and incorporated into soil.     

Effect of increasing oxygen concentration on total denitrification and nitrous oxide release from soil by different bacteria

Summary The effect of increasing oxygen concentrations (0, 5, 10 and 20 Vol% O₂) on total denitrification and N₂0 release was studied in model experiments using a neutral pH loamy soil relatively rich in easily decomposable organic matter and supplied with nitrate (300 g nitrate N/g dry soil). The sterilized soil was inoculated with three different denitrifying bacteria (Bacillus licheniformis,Aeromonas denitrificans andAzospirillum lipoferum) and incubated (80% WHC, 30°C). The gas volume was analysed for O₂, CO₂, N₂O, NO and N₂ by gas chromatography and the soil investigated for changes in ammonium, nitrite, nitrate, pH, total N and C as well as water-extractable C. WithB. licheniformis andAeromonas denitrificans total denitrification increased remarkably with increasing pO₂ as the result of intensified mineralization.Azospirillum lipoferum, however, showed the highest activity at 5 vol% O₂. WithB. licheniformis N₂O was released only in anaerobic conditions and at 5 Vol% O₂ (maximum) or 10 Vol% 02, but not at 20 Vol%, whereasAeromonas denitrificans produced N₂O only in the presence of He gas (maximum) or at 5 Vol% O₂. In contrast to these bacteria, N₂O production withAzospirillum lipoferum was restricted to 10 Vol% O₂ (maximum) and to 20 Vol% 02, with some traces at 5 vol% O₂. With a certain set of conditions, total denitrification and N₂O formation seem to be governed by the mineralization rate of the organisms in question. The increased demand for electron acceptors by a high turnover rate rather than the presence of anaerobic conditions seems to have determined the rate of denitrification.     

Rapid shift from denitrification to nitrification in soil after biogas residue application as indicated by nitrous oxide isotopomers

Nitrous oxide (N₂O) is one of the major greenhouse gases emitted from soils, where it is mainly produced by nitrification and denitrification. It is well known that rates of N₂O release from soils are mainly determined by the availability of substrates and oxygen, but N₂O source apportioning, highly needed to advance N₂O mitigation strategies, still remains challenging. In this study, using an automated soil incubation system, the N₂O site preference, i.e. the intramolecular ¹⁵N distribution, was analyzed to evaluate the progression in N₂O source processes following organic soil amendment. Biogas fermentation residue (BGR; originating from food waste fermentation) was applied to repacked grassland soil cores and compared to ammonium sulfate (AS) application, both at rates equivalent to 160 kg NH₄⁺-N ha⁻¹, and to unamended soil (control). The soil cores were incubated in a helium-oxygen atmosphere with 20 kPa O₂ for 43 days at 80% water-filled pore space. 43-day cumulative N₂O emissions were highest with BGR treated soil accounting for about 1.68 kg N₂O-N ha⁻¹ while application of AS caused much lower fluxes of c. 0.23 kg N₂O-N ha⁻¹. Also, after BGR application, carbon dioxide (CO₂) fluxes showed a pronounced initial peak with steep decline until day 21 whereas with ammonium addition they remained at the background level. N₂O dual isotope and isotopomer analysis of gas samples collected from BGR treated soil indicated bacterial denitrification to be the main N₂O generating process during the first three weeks when high CO₂ fluxes signified high carbon availability. In contrast, in the second half after all added labile carbon substrates had been consumed, nitrification, i.e. the generation of N₂O via oxidation of hydroxylamine, gained in importance reaching roughly the same N₂O production rate compared to bacterial denitrification as indicated by N₂O SP. Overall in this study, bacterial denitrification seemed to be the main N₂O forming process after application of biogas residues and fluxes were mainly driven by available organic carbon.