N2O emissions and product ratios of nitrification and denitrification as affected by freezing and thawing

Agricultural soils contribute significantly to atmospheric nitrous oxide (N₂O). A considerable part of the annual N₂O emission may occur during the cold season, possibly supported by high product ratios in denitrification (N₂O/(N₂+N₂O)) and nitrification (N₂O-N/(NO₃⁻-N+NO₂⁻-N)) at low temperatures and/or in response to freeze-thaw perturbation. Water-soluble organic materials released from frost-sensitive catch crops and green manure may further increase winter emissions. We conducted short-term laboratory incubations under standardized moisture and oxygen (O₂) conditions, using nitrogen (N) tracers (¹⁵N) to determine process rates and sources of emitted N₂O after freeze-thaw treatment of soil or after addition of freeze-thaw extract from clover. Soil respiration and N₂O production was stimulated by freeze-thaw or addition of plant extract. The N₂O emission response was inversely related to O₂ concentration, indicating denitrification as the quantitatively prevailing process. Denitrification product ratios in the two studied soils (pH 4.5 and 7.0) remained largely unaltered by freeze-thaw or freeze-thaw-released plant material, refuting the hypothesis that high winter emissions are due to frost damage of N₂O reductase activity. Nitrification rates estimated by nitrate (NO₃⁻) pool enrichment were 1.5-1.8 μg NO₃-N g⁻¹ dw soil d⁻¹ in freeze-thaw-treated soil when incubated at O₂ concentrations above 2.3 vol% and one order of magnitude lower at 0.8 vol% O₂. Thus, the experiments captured a situation with severely O₂-limited nitrification. As expected, the O₂ stress at 0.8 vol% resulted in a high nitrification product ratio (0.3 g g⁻¹). Despite this high product ratio, only 4.4% of the measured N₂O accumulation originated from nitrification, reaffirming that denitrification was the main N₂O source at the various tested O₂ concentrations in freeze-thaw-affected soil. N₂O emission response to both freeze-thaw and plant extract addition appeared strongly linked to stimulation of carbon (C) respiration, suggesting that freeze-thaw-induced release of decomposable organic C was the major driving force for N₂O emissions in our soils, both by fuelling denitrifiers and by depleting O₂. The soluble C (applied as plant extract) necessary to induce a CO₂ and N₂O production rate comparable with that of freeze-thaw was 20-30 μg C g⁻¹ soil dw. This is in the range of estimates for over-winter soluble C loss from catch crops and green manure plots reported in the literature. Thus, freeze-thaw-released organic C from plants may play a significant role in freeze-thaw-related N₂O emissions.     

Nitrous oxide production by nitrification and denitrification in soil aggregates as affected by O2 concentration

Nitrous oxide emitted by soils can be produced either by denitrification in anoxic conditions or by nitrification in presence of O₂. The relative importance of the two processes, particularly under varied partial pressures of O₂, is not always known. This paper focuses on the influence of O₂ concentration on N₂O production by nitrification and denitrification in an arable Orthic Luvisol. Soil aggregates (2-3 mm size), water unsaturated, received 116 mg N kg⁻¹ as ammonium sulphate labelled with ¹⁵N and were incubated during 14 days at different O₂ partial pressures: 0, 0.35, 0.76, 1.5, 4.3 and 20.4 kPa. A ¹⁵N tracing technique was used to quantify nitrification and denitrification rates. ¹⁵N₂O and ¹⁵N₂ were measured. Oxygen pressure appeared to strongly influence both nitrification and denitrification rates and also N₂O emissions. Nitrification rates were reduced by a factor of 6-9 when O₂ decreased from 20.4 to 0.35 kPa. They were highly correlated with O₂ consumption rates. Denitrification mainly occurred in complete anoxic conditions. The proportion of N₂O emitted by denitrification was estimated by two independent methods: one based on ¹⁵N tracing using isotope composition of NH₄, NO₃ and N₂O, the other based on the measurement of the ¹⁵N₂O:¹⁵N₂ ratio. The two methods gave close results. The highest N₂O emissions were obtained under complete anoxic conditions and were due to denitrification. However, N₂O emissions almost as important were obtained at day 14 with 1.5 kPa O₂ pressure, and they were due to nitrification. Nitrification was the main source of N₂O at O₂ concentrations greater than 0.35 kPa. The amounts of N₂O-N emitted by nitrification were linearly related to the amounts of N nitrified, but the slope of the regression was highly dependent on O₂ concentration: it varied from 0.16 to 1.48% when O₂ concentration was reduced from 20.4 to 0.76 kPa. Emissions of N₂O by nitrification may then be quite significant if nitrification occurs at a reduced O₂ concentration.     

Effect of wax-coated calcium carbide and nitrapyrin on nitrogen loss and methane emission from dry-seeded flooded rice

Summary The effectiveness of wax-coated calcium carbide (as a slow-release source of acetylene) and nitrapyrin in inhibiting nitrification and emission of the greenhouse gases N₂O and CH₄ was evaluated in a microplot study with dry-seeded flooded rice grown on a grey clay near Griffith, NSW, Australia. The treatments consisted of factorial combinations of N levels with nitrification inhibitors (control, wax-coated calcium carbide, and nitrapyrin). The rate of nitrification was slowed considerably by the addition of wax-coated calcium carbide, but it was inhibited only slightly by the addition of nitrapyrin. As a result, the emission of N₂O was markedly reduced by the application of wax-coated calcium carbide, whereas there was no significant difference in rates of N₂O emission between the control and nitrapyrin treatments. Both nitrification inhibitors significantly reduced CH₄ emission, but the lowest emission rates were observed in the wax-coated calcium carbide treatment. At the end of the experiment 84% of the applied N was recovered from the wax-coated calcium carbide treatment compared with 43% for the nitrapyrin and control treatments.     

Fractionation of N2O isotopomers during production by denitrifier

Isotopomer ratios of N₂O, which include intramolecular ¹⁵N-site preference in addition to conventional isotope ratios for N and O in NNO (we designate N^α and N^β for the center and end N atom, respectively, in the asymmetric molecule), reflect production and consumption processes of this greenhouse gas. Therefore, they are useful parameters for deducing global N₂O budget. This paper reports the first precise measurement of ¹⁵N-site preference in N₂O produced by two species of denitrifying bacteria, Pseudomonas fluorescens (ATCC 13525) and Paracoccus denitrificans (ATCC 17741).Cultures were incubated in a batch mode with a liquid medium that contains KNO₃ as unique nitrogen supply under acetylene/helium (10% v/v) atmosphere at 27 °C. Enrichment factors for ¹⁵N in bulk nitrogen in N₂O (average for N^α and N^β) fluctuated in a few tens permil showing a slight difference between the species. In contrast, ¹⁵N-site preference (difference in isotope ratios between N^α and N^β) showed nearly constant and distinct value for the two species (23.3±4.2 and −5.1±1.8‰ for P. fluorescens and P. denitrificans, respectively). The site preference was also measured for N₂O produced by inorganic reactions (nitrite reduction and hydroxylamine oxidation); a unique value (about 30‰ for the both reactions) was obtained. These results and those recently reported for nitrifying bacteria suggest that ¹⁵N-site preference in N₂O can be used to identify the production processes of N₂O on the level of bacterial species or enzymes involved.     

Nitrifier dominance of Arctic soil nitrous oxide emissions arises due to fungal competition with denitrifiers for nitrate

Arctic soils emit nitrous oxide, which is a potent greenhouse gas and also represents an important loss of nitrogen to oligotrophic Arctic ecosystems. However, little is known about the temperature sensitivity of nitrous oxide release in Arctic soils or the organisms mainly responsible for it. We investigated controls on nitrous oxide emissions in an Arctic soil across a typical temperature range (between 4 and 13 °C) on Truelove Lowland, Devon Island, Canada (75°40′N 84°35′W) at two different moisture contents. When fertilized with ammonia or nitrate, nitrous oxide emissions and temperature dependence of nitrous oxide emissions were insensitive to soil moisture content but linked to nitrification rates. Stable isotope analysis revealed that nitrous oxide was predominantly released by nitrifiers. However, nitrous oxide emissions were not linked to nitrifier prevalence with an insignificant (P < 0.219) increase in amoA genes and a (P < 0.01) decrease in archaeal nitrifiers. In contrast, denitrifier nosZ prevalence was 10,000 times greater than that of nitrifiers and was related to nitrous oxide emission potential when soils were fertilized with nitrate. Manipulating water-filled pore space should have changed the pattern of N₂O emissions. We used selective inhibitors to further explore why denitrification did not occur under field conditions when we manipulated water-filled pore space or when we used ¹⁵N analysis. When fungi were inhibited in the soil, nitrous oxide emissions from denitrifiers increased with no change in nitrous oxide released by nitrifiers. When fungi were active in the soil, there was little available nitrate but when fungi were inhibited, available soil nitrate increased over the incubation period. The dominance of nitrifiers in nitrous oxide emissions from Arctic soils under field conditions is linked to the competition for nitrate between fungi and denitrifiers.     

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.     

盐碱土壤N2O排放与amoA和narG功能基因丰度的响应规律

为揭示盐碱土壤中参与氨氧化过程和硝酸盐还原过程的amoA和narG基因丰度与N_2O排放的响应规律,本研究选取内蒙古河套灌区3种不同盐碱程度土壤(轻度盐土SA、强度盐土SB和盐土SC),通过控制室内温度和土壤质量含水量进行室内培养试验,并运用荧光定量PCR(real-time PCR)技术研究了盐碱土壤中N_2O排放速率、氨氧化细菌和narG(膜结合型硝酸还原酶)型反硝化细菌丰度与土壤环境因子之间的偶联关系。结果表明:SA、SB和SC3种盐碱土壤中,N_2O平均排放速率随着土壤盐碱程度的升高而升高,值分别为16.9、30.8、69.6μg/(kg·d);氨氧化细菌和narG型反硝化细菌丰度分别为0.415×10~4、6.91×10~4、9.44×10~4 copies和2.61×10~4、5.36×10~4、13.5×10~4 copies,表明在一定盐分条件下,土壤中的盐分能够促进氨氧化细菌和narG型反硝化细菌丰度。RDA分析结果显示,N2O平均排放速率与氨氧化细菌和narG型反硝化细菌丰度具有显著的正相关(r=0.863、0.975,P0.01);土壤pH、EC、速效钾和有机碳是盐碱土壤中影响N2O排放速率的主要环境因子,其中,土壤pH、EC、速效钾和N_2O排放速率存在显著正相关(r=0.968、0.983、0.987,P0.01),土壤有机碳和N_2O排放速率存在负相关(r=–0.800,P0.05),土壤有效磷和总氮与N_2O排放速率的相关性未达到显著水平(P0.05)。     

Dinitrogen and N2O emissions in arable soils: Effect of tillage, N source and soil moisture

A laboratory investigation was performed to compare the fluxes of dinitrogen (N₂), N₂O and carbon dioxide (CO₂) from no-till (NT) and conventional till (CT) soils under the same water, mineral nitrogen and temperature status. Intact soil cores (0-10 cm) were incubated for 2 weeks at 25 °C at either 75% or 60% water-filled pore space (WFPS) with ¹⁵N-labeled fertilizers (100 mg N kg⁻¹ soil). Gas and soil samples were collected at 1-4 day intervals during the incubation period. The N₂O and CO₂ fluxes were measured by a gas chromatography (GC) system while total N₂ and N₂O losses and their ¹⁵N mole fractions in the soil mineral N pool were determined by a mass spectrometer. The daily accumulative fluxes of N₂ and N₂O were significantly affected by tillage, N source and soil moisture. We observed higher (P<0.05) fluxes of N₂+N₂O, N₂O and CO₂ from the NT soils than from the CT soils. Compared with the addition of nitrate (NO₃⁻), the addition of ammonium (NH₄⁺) enhanced the emissions of these N and C gases in the CT and NT soils, but the effect of NH₄⁺ on the N₂ and/or N₂O fluxes was evident only at 60% WFPS, indicating that nitrification and subsequent denitrification contributed largely to the gaseous N losses and N₂O emission under the lower moisture condition. Total and fertilizer-induced emissions of N₂ and/or N₂O were higher (P<0.05) at 75% WFPS than with 60% WFPS, while CO₂ fluxes were not influenced by the two moisture levels. These laboratory results indicate that there is greater potential for N₂O loss from NT soils than CT soils. Avoiding wet soil conditions (>60% WFPS) and applying a NO₃⁻ form of N fertilizer would reduce potential N₂O emissions from arable soils.     

Phytotron studies to compare nitrogen losses from corn-planted soil by the 15-N balance or direct dinitrogen and nitrous oxide measurements

Summary Containers filled with soil mixed with potassium nitrate highly enriched in ¹⁵N were planted with corn (Zea mays L.) and kept in a phytotron under controlled conditions for 79 days. Soil water content was normally maintained at exactly 60% water-holding capacity (–33 kPa), but it was increased several times to 85% (–5 kPa) for short periods to favour denitrification. The soil headspace was sealed from the phytotron atmosphere and aerated by a continuous stream of air. Nitrous oxide emission was measured by estimating the N₂O concentration differences in the air entering and leaving the containers. Emission of N₂ was estimated by mass spectroscopy from changes in the N₂ composition in the temporarily enclosed soil headspace. Both methods were carefully checked for accuracy by different tests. At specific times during the experiment the distribution of ¹⁵N between plants and soil was determined and a ¹⁵N balance established. Emission of N gases peaked at times of increased water content and reached maxima of 149 and 142 g N pot^–1 day^–1 for N₂O and N₂, respectively. While N losses of 5% ± 2% were indicated by the ¹⁵N balance, only 1.1% ± 0.3% loss from 2.7 g applied N was estimated from the N₂O and N₂ measurements after 79 days. Possible reasons for these differences are discussed.     

Assessing the potential of ammonia oxidizing bacteria to produce nitrous oxide in soils of a high arctic lowland ecosystem on Devon Island, Canada

The contribution of nitrifiers (ammonia-oxidizing bacteria (AOB)) and denitrifiers to nitrous oxide (N₂O) emission from arctic soils remains inconclusive. Based on preliminary experiments, we hypothesized that AOB are the primary producers of N₂O in a high arctic lowland ecosystem on Devon Island, Nunavut, Canada. In part 1 of the study, flux chambers were installed in a catena to determine in situ fluxes of gases (N₂O and carbon dioxide (CO₂)) from 16 June to 13 July 2004. Although fluxes were low, N₂O production occurred in the wettest area of the landscape when ammonium levels were high. As ammonium, but not nitrate, levels declined in the wet sedge meadow, N₂O emissions correspondingly decreased. In part 2, the contribution of nitrification and denitrification to N₂O production was assessed by Acetylene Inhibition Assay and ¹⁵N isotopically enriched incubations. Ammonium fertilization stimulated N₂O emissions to a greater extent than nitrate, and acetylene had a greater impact on N₂O emissions in ammonium-fertilized soils than in nitrate-amended soils. Stable isotope analysis indicated that at 50-55% water filled pore space, nitrification was the dominant (>80%) N₂O emitting process. In part 3, molecular analyses of the two N₂O producing groups indicated the both nitrifiers and denitrifiers did not differ between landforms. Our results suggest nitrifier denitrification is the dominant process occurring in these arctic soils and that the role of denitrifiers in N₂O release from arctic soils needs to be re-evaluated.     

三氯生和三氯卡班对水稻土好氧氮转化及N2O排放的影响

三氯生(Triclosan, TCS)和三氯卡班(Triclocarban, TCC)是典型的药品与个人护理用品,在土壤生态系统中被广泛检出,且存在增加土壤微生物抗药性及抑制土壤呼吸的潜在风险,但目前有关TCS和TCC对土壤氮转化过程及氧化亚氮(N_2O)排放的影响尚不清楚。基于此,采用室内培养实验和15N稀释-富集法,结合氮转化数值模型,研究了不同浓度梯度下TCS(2和5mg·kg~(-1))和TCC(1和2 mg·kg~(-1))的单独及联合存在对水稻土氮初级转化速率以及N_2O排放的影响。结果表明,1mg·kg~(-1)TCC及5mg·kg~(-1)TCS+2mg·kg~(-1)TCC处理对水稻土氮素的矿化-同化无显著影响,其余TCS和TCC处理均显著促进了氮的矿化-同化循环。此外,TCS和TCC处理显著降低了自养硝化速率、硝态氮的微生物固定速率以及硝酸盐异化还原成铵(Dissimilatory nitrate reduction to ammonium, DNRA)速率(2 mg·kg~(-1)TCS处理及5mg·kg~(-1)TCS+2mg·kg~(-1)TCC对DNRA速率无显著影响)。值得关注的是,TCS和TCC单一和联合处理均显著增加了N_2O的累积排放量,其累积排放量为对照的1.13倍～1.44倍。本研究表明,TCS和TCC改变了水稻土好氧氮转化过程,可能对稻田生态系统氮循环产生不利影响;TCC和TCS对水稻土N_2O排放的促进作用也增加了稻田生态系统对温室效应和臭氧层破坏的潜在贡献,因此,未来评价TCS和TCC土壤生态风险时,应考虑其对氮转化过程和N_2O排放的潜在影响。     

Nitrification activity in submerged soils and its relation to denitrification loss

Summary Nitrification activity (formation of NO ₂ ^– + NO ₃ ^– per unit soil weight) was measured in the surface layer of 15 presubmerged soils incubated in petri dishes under flooded but aerobic conditions. soils with pH above 5 nitrified quickly, whereas soils with pH below this level did not nitrify or nitrified slowly. The pH values between 7 and 8.5 were optimal for nitrification. Organic-matter levels in the 15 soils of our study did not influence their nitrification activities. In a follow-up greenhouse pot study, after a period of 3 weeks, ¹⁵N-balance measurements showed that the loss of N through apparent denitrification did not follow the nitrification patterns of the soils observed in the petri dishes. Apparent denitrification accounted for 16.8% and 18.9% loss of ¹⁵N from a soil with insignificant nitrification activity and a soil with high nitrification activity, respectively. These results, thus, indicate a lack of correspondence between the nitrification activities of soil and the denitrification loss of N when the former was measured in the dark and the latter was estimated in the light. Soils that nitrified in the darkness of the incubator did not nitrify in the daylight in the greenhouse.     

丛枝菌根真菌调控土壤氧化亚氮排放的机制

氮素是陆地生态系统初级生产力的主要限制因子,自Haber-Bosch反应以来,氮肥的生产和施用极大地提高了粮食产量.然而过量施用氮肥导致氮肥利用率低,并造成了严重的环境污染,包括氮沉降、硝态氮淋洗以及N2O排放等.微生物直接参与土壤氮素循环,固氮微生物、氨氧化和反硝化微生物分别在土壤固氮、铵态氮转化和硝态氮转化过程中起...     

亚硝酸盐添加对土壤硝化和反硝化基因转录活性及N2O排放的影响

设施菜田土壤氧化亚氮（N2O）脉冲式排放期间通常伴随着亚硝酸盐（NO2-）的大量积累,为揭示NO2-对设施菜田土壤N2O排放的影响机制,以两种典型蔬菜种植区土壤（碱性土壤/酸性土壤）为研究对象,通过室内培养试验,对比厌氧和好氧培养条件下添加NO2-后两种土壤无机氮转化与N2O、氮气（N2）和二氧化碳（CO2）等气体排放,以及氨氧化单加氧酶α亚基调控基因（amoA）、亚硝酸盐还原酶调控基因（nirK和 nirS,统称nir）和N2O还原酶调控基因（nosZ）的丰度和转录情况。结果显示：受pH等环境因素影响,土壤中NO2-含量并不一定与N2O排放之间存在相关性,但添加NO2-的处理显著增加了两种土壤的N2O排放量和N2O/(N2O+N2)指数（IN2O）（P<0.05）。碱性土壤中,60 mg?kg-1外源NO2-对土壤CO2排放无明显抑制作用,厌氧培养条件下nirK基因、好氧培养条件下amoA和nirS基因均出现了添加NO2-后转录拷贝数显著高于空白处理的现象,而nosZ基因无此现象。酸性土壤中,amoA转录活性整体较低,好氧空白处理时nirS基因转录拷贝数随培养时间的延长而增加（P<0.05）;60 mg?kg-1外源NO2-明显降低了酸性土壤的CO2排放量、相关基因的丰度及转录拷贝数。上述结果显示,土壤中积累的NO2-会通过诱导nir基因转录与N2O还原酶竞争电子和抑制N2O还原酶活性等途径,增加土壤的IN2O,影响有氧条件下N2O的排放途径,研究结果将为探索设施菜田土壤氮素高效利用和N2O减排提供科学依据。