铜绿假单胞菌norB基因的克隆及表达

为使norB基因得到有效表达,利用分子生物学技术将铜绿假单胞菌B136-33的norB基因克隆至表达载体pET-28a上。首先,根据GenBank公布的norB基因序列及表达载体pET-28a上的多克隆位点进行引物(含EcoRⅠ和HindⅢ酶切位点)设计;然后以B136-33基因组为模板,扩增目的片段norB;产物经双酶切克隆至pET-28a后,化学转化至克隆菌DH5α,得到含有重组质粒的转化子DH5α-pET-28a-norB;再经双酶切和测序鉴定正确后,将重组质粒pET-28a-norB转化至表达菌BL21;最后,用SDS-PAGE鉴定norB基因表达产物的分子量。结果表明,norB能在BL21中得到正确有效的表达。     

Effect of chemical fertilizer form on N2O, NO and NO2 fluxes from Andisol field

N₂O, NO and NO₂ fluxes from an Andosol soil in Japan after fertilization were measured 6 times per day for 10 months from June 1997 to April 1998 with a fully automated flux monitoring system in lysimeters. Three nitrogen chemical fertilizers were applied to the soil–calcium nitrate (NI), controlled-release urea (CU), and controlled-release calcium nitrate (CN), and also no nitrogen fertilizer (NN). The total amount of nitrogen applied was 15 g N m^–2 in the first and the second cultivation period of Chinese vegetable. In the first measuremnt period of 89 days, the total N₂O emissions from NI, CN, CU, and NN were 18.4, 16.3, 48.7, and 9.60 mgN m^–2, respectively. The total NO emissions from NI, CN, CU, and NN were 48.4, 33.7, 149, and 13.7 mgN m^–2, respectively. In the second measurement period of 53 days, the total N₂O emissions from NI, CN, and CU were 9.66, 7.23, and 20.6 mgN m^–2, respectively. The total NO emissions from NI, CN, and CU were 24.7, 2.60 and 34.2 mgN m^–2, respectively. The total N₂O emission from CU was significantly higher than CN. In the third cultivation period, all plots were applied with 10 g N m^–2 of ammonium phosphate (AP) and winter barley was cultivated. In the third measurement period of 155 days, the total N₂O and NO emissions were 9.02 mgN m^–2 and 10.2 mgN m^–2, respectively. N₂O and NO peaks were observed just after the fertilization for 30 days and 15 days, respectively. N₂O, NO and NO₂ fluxes for the year were estimated to be 38.6 81.5, 48.2 181, and –24.8 to –39.3 mgN m^–2, respectively. NO₂ was absorbed in all the plots, and a negative correlation was found between NO₂ flux and the NO₂ concentration just after the chamber closed. NO was absorbed in the winter period, and a negative correlation was found between NO flux and the NO concentration just after the chamber closed. A diurnal pattern was observed in N₂O and NO fluxes in the summer, similar to air and soil temperature. We could find a negative relationship between flux ratio of NO-N to N₂O-N and water-filled pore space (WFPS), and a positive relationship between NO-N and N₂O-N fluxes and temperature. Q₁₀ values were 3.1 for N₂O and 8.7 for NO between 530 °C.     

Nitrite in soils: accumulation and role in the formation of gaseous N compounds

Nitrite is an intermediary compound formed during nitrification as well as denitrifiication. It occasionally accumulates in soils and drainage water. The nitrite can then undergo transformations to gaseous nitrogen compounds such as NO and NO₂. Soil pH controls the abiotic nitrite decomposition to a large extent. Under acidic conditions(pH <5.5), nitrous acid spontaneously decomposes preferentially to NO and NO₂. Nitrite also undergoes reactions with metallic cations (especially ferrous iron) and with organic matter. As a result of these reactions gaseous compounds such as NO, NO₂, N₂O and CH₃ONO can be formed. Through reaction of nitrite with phenolic compounds nitroand nitrosocompounds can be formed, building up organic N. With normal agricultural practices on slightly acidic soils, the nitrite instability usually does not lead to economically important N losses from soils. However, the compounds formed through its degradation or interaction with other soil constituents are linked to environmental problems such as tropospheric ozone formation, acid rain, the greenhouse effect and the destruction of the stratospheric ozone.     

Subsoils: chemo-and biological denitrification, N2O and N2 emissions

Agricultural practices, soil characteristics and meteorological conditions are responsible for eventual nitrate accumulation in the subsoil. There is a lot of evidence that denitrification occurs in the subsoil and rates up to 60–70 kg ha^-1 yr^-1 might be possible. It has also been shown that in the presence of Fe²⁺ (formed through weathering of minerals) and an alkaline pH, nitrate can be chemically reduced. Another possible pathway of disappearance is through the formation of nitrite, which is unstable in acid conditions. With regard to the emission of N₂O and N₂, it can be stated that all conditions whereby the denitrification process becomes marginal are favourable for N₂O formation rather than for N₂. Because of its high solubility, however, an important amount of N₂O might be transported with drainage water.     

Nitrous oxide production in riparian zones and groundwater

This paper addresses the question of whether riparian zones and groundwater are hotspots of nitrous oxide (N₂O) flux in the landscape. First, we describe how riparian zones and groundwater function as transformers of N, with a particular emphasis on mechanisms of N₂O production in these ecosystems. We then present specific data on N₂O flux in these ecosystems and attempt to reconcile these data with existing regional scale estimates of N flux for Norway and with estimates of N₂O flux for Norway produced using the OECD/IPCC/IEA Phase II methodology for calculation of regional and global N₂O budgets. While the OECD/IPCC/IEA approach produces estimates of riparian and groundwater N₂O flux that are reasonable, given what we know about regional scale N balances and actual data on N₂O flux, it does not allow us to determine if riparian zones and groundwater are hotspots of N₂O production in the landscape. The approach fails to answer this question because it is unable to account for spatially explicit phenomena such as riparian and groundwater processing of excess agricultural N. Research needs that would allow us to address this question are discussed.     

Quantification of N-losses as NH3, NO, and N2O and N2 from fertilized maize fields in southwestern France

Emissions of nitrogen compounds (NO, NH₃, N₂O and N₂) from heavily fertilized (280 kg(N) ha^-1) and irrigated maize fields were studied over an annual cultivation cycle in southwestern France. NO and N₂O emissions were measured by chamber techniques throughout the year. During fertilization and maize growth periods, chamber measurements were intensified and complemented by flux-gradient micrometeorological measurements of NO_x and NH₃. The two methods used, Bowen ratio and a simplified aerodynamical techniques, agree quite well and quantify NO_x and NH₃ flux variations during the period of intense emission which followed fertilizer application. Over a yearly cycle, nitrogen loss in the form of NH₃, NO and N₂O were calculated using micrometeorological flux measurements and emission algorithms calibrated with field data (chambers). The soil denitrification potential represented by the ratio N₂O/(N₂O+N₂) was measured in the laboratory to calculate potential total gaseous nitrogen loss. Taking into account all uncertainties, the total N loss into the atmosphere represents 30 to 110 kg(N) ha^-1 with about less than 1% as NH₃, 40% as NO, 14% as N₂O and 46% as N₂. This is in agreement with the agronomic nitrogen budget based on the N fertilizer input and soil furniture and, on the N-output by crops and crop residues, which displays a net imbalance of 50 to 100 kg(N) ha^-1.     

Formation of N2O and (NO)2 During NO Adsorption on Au 3D Crystals

The adsorption of NO on Au 3D hemispherical crystals (field emitter tips) has been studied by means of pulsed field desorption mass spectrometry (PFDMS) under dynamic gas flow conditions and at 300 K. Local chemical probing of ~200 Au sites in the stepped surface region between the central (111) pole and the peripheral (001) plane leads to the detection of NO⁺, N₂O⁺ and (NO) species. Obviously, molecular NO adsorption on stepped Au surfaces can lead to dimerization. Nitrous oxide formation probably occurs via the dimer, (NO)₂.     

Influence of soil physical properties, fertiliser type and moisture tension on N2O and NO emissions from nearly saturated Japanese upland soils

In Japan, upland soils are an important source of nitrous oxide (N₂O) and nitric oxide (NO) gas emissions. This paper reports on an investigation of the effect of soil moisture near saturation on N₂O and NO emission rates from four upland soils in Japan of contrasting texture. The aim was to relate these effects to soil physical properties. Intact cores of each soil type were incubated in the laboratory at different moisture tensions after fertilisation with NH₄-N, NO₃-N or zero N. Emissions of N₂O and NO were measured regularly over a 16–20 day period. At the end of the incubation, soil cores were analysed for physical properties. Moisture and N fertiliser significantly affected rates of emissions of both N₂O and NO with large differences between the soil types. Nitrous oxide emissions were greatest in the finer-textured soils, whereas NO emissions were greater in the coarser-textured soils. Emissions of N₂O increased at higher moisture contents in all soils, but the magnitude of increase was much greater in finer-textured soils. Nitric oxide emissions were only significant in soils fertilised with NH₄-N and were negatively correlated with soil moisture. Analysis of soil properties showed that there was a strong relationship between the magnitude of emissions and soil physical properties. The importance of soil wetness to gas emissions was mainly through its influence on soil air-filled porosity, which itself was related to gas diffusivity. From the results of this research, we can now estimate likely effects of soil texture on emissions through the influence of soil type on soil aeration and soil drainage. This is of particular value in modelling N₂O and NO emissions from soil moisture status and land use inputs.     

Simultaneous catalytic reduction of NO and N2O using Fe-MFI prepared by solid-state ion exchange

Solid-state ion exchange in the presence of air has been used to prepare Fe-MFI catalysts that exhibit activities comparable to preparations obtained under anaerobic conditions. Preliminary results obtained during the simultaneous catalytic reduction of NO and N₂O with propane in the temperature range from 473 to 773 K show that Fe-MFI might be of interest for the treatment of exhaust gases containing NO_x and nitrous oxide. Undesirable CO formed in a side-reaction can be oxidized to CO₂ by promotion of Fe-MFI with small amounts of Pt. This revised version was published online in July 2006 with corrections to the Cover Date.     

N2O4-N2O5- HNO3体系液液平衡数据测定

采用浊点法测定了N2O4-N2O5-HNO3体系在258.2～281.2 K的液液平衡和固液平衡数据,得到了固液平衡临界点及N2O5在N2O4和HNO3中的溶解度、N2O4在HNO3中的溶解度和HNO3在N2O4中的溶解度数据,确定了液液平衡区域和近似固液平衡区域.结果表明,随着温度的升高,两相区逐渐减小;N2O5在N2O4和HNO3中的溶解度、N2O4在HNO3中的溶解度和HNO3在N2O4中的溶解度均增大.     

Effects of deep application of urea on NO and N2O emissions from an Andisol

A modeling study revealed that the depth of nitric oxide (NO) production in soil is crucial for its flux, while that of nitrous oxide (N₂O) is not. To verify this result, laboratory experiments with soil columns classified as Andisol (Hydric Hapludand) were conducted, with changing the depth of urea application, at 0–0.1 or 0.1–0.2 m. All the NO concentration profiles in soil exhibited a sharp peak at each fertilized layer within 5 days of fertilizer application. NO concentration in soil decreased abruptly as the distance from the fertilized layer increased. These findings imply that NO is produced mainly within the fertilized layer, but does not diffuse widely in the soil columns, because of rapid NO uptake within the soil. As a result, the NO flux from soil columns fertilized at 0.1–0.2 m depth over the 48-day study period was reduced to almost the same rate as that of the unfertilized one. The total NO emissions from soil columns unfertilized and fertilized at 0–0.1 and 0.1–0.2 m depth were 0.02, 1.39 (± 0.05) and 0.05 (± 0.03) kg N ha^–1, respectively, suggesting that NO emission derived from N fertilizer could be reduced to 2% by shifting the depth of fertilizer application by 0.1 m. On the other hand, soil N₂O concentration profiles exhibited a gentler peak, because of the lower uptake by soil. N₂O fluxes were affected more by the soil conditions, e.g. soil water content, than the distance between fertilized depth and soil surface. The total N₂O emissions from soil columns unfertilized and fertilized at 0–0.1 and 0.1–0.2 m were 0.02, 0.16 (± 0.03) and 0.25 (± 0.04) kg N ha^–1, respectively.     

N2O and NO emissions from a field of Chinese cabbage as influenced by band application of urea or controlled-release urea fertilizers

A field experiment was conducted in an Andosol in Tsukuba, Japan to study the effect of banded fertilizer applications or reduced rate of fertilizer N (20% less) on emissions of nitrous oxide (N₂O) and nitric oxide (NO), and also crop yields of Chinese cabbage during the growing season in 2000. Six treatments were applied by randomized design with three replications, which were; no N fertilizer (CK); broadcast application of urea (BC); band application of urea (B); band application of urea at a rate 20% lower than B (BL); band application of controlled-release urea (CB) and band application of controlled-release urea at a rate 20% lower than CB (CBL). The results showed that reduced application rates, applied in bands, of both urea (BL) and controlled-release urea fertilizer (CBL) produced yields that were not significantly lower than yields from the full rate of broadcast urea (BC). The emissions of N₂O and NO from the reduced fertilizer treatments (BL, CBL) were lower than that of normal fertilizer rates (B, CB). N₂O and NO emissions from controlled-release urea applied in band mode (CB, CBL) were less than those from urea applied in band mode (B, BL). The total emissions of N₂O and NO indicated that applying fertilizers in band mode mitigated NO emission from soils, but N₂O emissions from banded urea (B) were no lower than from broadcast urea (BC).     

生物脱氮中N2O产生过程的微生物作用

N2O是一种重要的温室气体.微生物的生物硝化反硝化过程是N2O产生的主要来源.从微生物学的角度阐述了脱氮过程中N2O的产生过程,并分析了不同脱氮过程中各菌种对N2O产生过程的作用.硝化过程中N2O主要产生于氨氧化细菌的氨氧化过程,亚硝酸盐氧化细菌的存在可以减少N2O的产量;反硝化过程中亚硝酸盐的积累,低氧和碳源不足都会导致N2O产生量的增加;另外,其他一些参与氮循环的微生物也会产生N2O.文章最后给出了污水脱氮过程中N2O减量化的策略以及今后研究的方向.     

硫磺/石灰石自养反硝化系统脱氮性能及N_2O排放规律研究

为了考察硫磺/石灰石自养反硝化系统的脱氮性能,并探究系统N_2O的产生和排放规律,采用均匀填充的上流式硫磺/石灰石生物滤池反应器,研究了2组HRT下,不同进水NO_3~--N浓度对系统脱氮效果的影响及N_2O的排放规律。结果表明,进水NO_3~--N浓度为(54.46±1.15)mg/L、HRT为2.5 h时,反应器容积负荷最大且对NO_3~--N去除率最高,可达99.93%,系统无NO_2~--N累积,出水N_2O低于0.86 mg/L;另外,研究发现NO_3~--N浓度随反应器高度增加而逐渐降低,N_2O浓度随着反应器下部NO_2~--N的富集逐渐增加,并随上部NO_2~--N的还原而逐渐减小;进水NO_3~--N浓度增大,N_2O累积量峰值点沿反应器高度逐渐上移,因此该系统仅能处理较低浓度NO_3~--N废水。     

Effects of the depth of NO and N2O productions in soil on their emission rates to the atmosphere: analysis by a simulation model

Many factors are concerned in the changing forms of nitrogen compounds in soil, so it is not easy to make precise models to simulate the concentration profiles of soil nitric oxide (NO) and nitrous oxide (N₂O) and their emission rates under various soil conditions. We prepared a simple mathematical simulation model based on soil concentration profiles of NO and N₂O. The profiles were measured at lysimeters filled with Andosol soil and fertilized with ammonium sulfate at rate of 200 kgNha^-1, incorporating to plow layer (Hirose & Tsuruta, 1996). In this model, diffusion of gases in soil followed Fick's law and the diffusion coefficient was adopted from Sallam et al. (1984). The gas production rate was set up at constant value in the site of gas production, and the gaseous consumption followed Michaelis-Menten kinetics. By changing only the depth of NO and N₂O production in soil in this model, we obtained the following results.(1) When the depth of gas production was set at near the soil surface (NO: 0–10 cm, N₂O: 0-8 cm), the emission rates of both gases corresponded with the results of the lysimeter-measurement.(2) When the depth of gas production was shifted down 10 cm deeper (NO: 10–20 cm, N₂O: 10-18 cm), the gas emission rate of NO decreased to 1.3% of (1), while that of N₂O was almost the same as (1).(3) In the case that the total intensity of produced gases was not changed from (1) or (2), but that the extent of gas productions expanded 3 times wider (NO: 0–30 cm, N₂O: 0–24 cm) than (1) or (2), the emission rates of NO and N₂O became 26% and 95% of (1), respectively.The above results suggest the possibility of mitigating NO emission by setting the site of gaseous production in deeper soil, e.g. by means of deep application of fertilizer.