不同根系分泌物对土壤N2O排放及同位素特征值的影响

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庄姗¹, 林伟¹, 丁军军¹, 郑欠¹, 寇馨月¹, 李巧珍¹, 李玉中^,¹^,²1 中国农业科学院农业环境与可持续发展研究所/农业农村部旱地节水农业重点实验室,北京 100081;
2 中国农业科学院环境稳定同位素实验室,北京 100081

Effects of Different Root Exudates on Soil N₂O Emissions and Isotopic Signature

ZHUANG Shan¹, LIN Wei¹, DING JunJun¹, ZHENG Qian¹, KOU XinYue¹, LI QiaoZhen¹, LI YuZhong^,¹^,²

1 Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences/Key Laboratory of Dryland Farming Agriculture, Ministry of Agriculture and Rural Affairs, Beijing 100081;

2 Environmental Stable Isotope Laboratory, Chinese Academy of Agricultural Sciences, Beijing 100081

通讯作者: 李玉中,Tel：18810871629;E-mail：liyuzhong@caas.cn

责任编辑: 李云霞
收稿日期:2019-08-2接受日期:2019-11-6网络出版日期:2020-05-16

基金资助:

国家自然科学基金.41473004
国家自然科学基金.41701308
国家重点研发计划项目.2017YFD0201702

Received:2019-08-2Accepted:2019-11-6Online:2020-05-16
作者简介 About authors
庄姗,Tel：18519190629;E-mail：489300802@qq.com。

摘要
【目的】探究植物根系分泌的主要组分（有机酸、氨基酸、糖类）对土壤N₂O排放及其微生物过程的影响,为选择适宜的植物进而控制土壤N₂O排放提供支撑。【方法】通过室内试验分别添加草酸、丝氨酸、葡萄糖于土壤中模拟根系的3种主要分泌物,每种分泌物设置两个浓度水平：低浓度（150 μg C·d ^-1）和高浓度（300 μg C·d ^-1）,另设置添加蒸馏水的对照组,共7个处理。将土壤置于120 mL玻璃瓶中进行培养,24 h内采集气体样品7次,每次培养2 h,获取N₂O排放速率、日累积排放量和同位素特征值（δ¹⁵N ^bulk、δ¹⁸O和SP（site preference,SP=δ¹⁵N ^α-δ¹⁵N ^β））。【结果】添加3种根系分泌物组分后,土壤N₂O排放速率均逐渐升高,且均高于对照。高浓度处理组N₂O累积排放量为：葡萄糖（（3.2±1.3）mg·kg ^-1·d ^-1）处理>丝氨酸（（2.6±0.5）mg·kg ^-1·d ^-1）处理>草酸（（1.4±0.2）mg·kg ^-1·d ^-1）处理,低浓度处理组为：草酸（（2.7±1.3）mg·kg ^-1·d ^-1）处理>丝氨酸（（1.8±0.4）mg·kg ^-1·d ^-1）处理>葡萄糖（（1.6±0.8）mg·kg ^-1·d ^-1）处理;添加根系分泌物的不同处理间土壤N₂O的δ¹⁸O值无明显差异,并稳定在24.1‰—25.6‰,且均显著高于对照（（20.1±1.5）‰）;土壤N₂O的δ¹⁵N ^bulk值与添加根系分泌物的种类有关,其中草酸处理组为（-20.06±2.22）‰、丝氨酸处理组为（-22.33±1.10）‰、葡萄糖处理组为（-13.86±1.11）‰、对照组为（-23.14±3.72）‰。各处理土壤N₂O的SP值的变化范围为13.13‰—15.03‰,根系分泌物浓度越高,SP值越低。综合分析不同处理4个指标（N₂O排放速率、N₂O的δ¹⁵N ^bulk、δ¹⁸O和SP值）的不同时刻的检测值与日均值的校正系数,添加根系分泌物后第16小时各处理4个指标的校正系数最接近于1。【结论】在NH+ 4-300 mg N·kg ^-1的土壤环境下根系分泌物促进N₂O的排放,且在培养期间（24 h）土壤N₂O排放速率逐渐升高。高浓度处理组葡萄糖对土壤N₂O排放速率促进效果最强,低浓度处理组草酸对土壤N₂O排放速率促进效果最强。与对照组相比,根系分泌物的添加使N₂O的δ¹⁸O值显著升高;与对照组相比,葡萄糖的添加使δ¹⁵N ^bulk值显著升高。根系分泌物浓度越高,反硝化作用对N₂O的贡献越大。
关键词： 根系分泌物;硝化作用;反硝化作用;N₂O;同位素特征值

Abstract

【Objective】The aim of this study was to investigate the effects of the main plant root exudates (organic acids, amino acids, sugars) on N₂O emission and its microbial processes, so as to provide a support for selecting suitable plants to control soil N₂O emissions. 【Method】Three main components of root exudates, including oxalic acid, serine and glucose, were added in the soil, and two levels of concentration were set for each component: low concentration (150 μg C·d ^-1) and high concentration (300 μg C·d ^-1). There were totally 7 treatments with a control treatment treated with distilled water, and all treatments were incubated in 120 mL glass bottles in incubator. The gas samples were sampled 7 times within 24 hours after 2-hour incubation each time. After sampling, the N₂O emission rate, daily cumulative emission and isotope signatures (δ¹⁵N ^bulk, δ¹⁸O and SP (intramolecular ¹⁵N site preference, δ¹⁵N ^α-δ¹⁵N ^β)) were measured, then the optimal sampling time was determined according to their daily variation rules. 【Result】The N₂O emission rate of soils increased gradually after adding three components of root exudates, which were higher than the control treatment. The cumulative emission of N₂O in the high concentration treatment was: Glucose treatments ((3.2±1.3) mg·kg ^-1·d ^-1)>Serine treatments ((2.6±0.5) mg·kg ^-1·d ^-1)>Oxalic acid treatments((1.4±0.2) mg·kg ^-1·d ^-1), low concentration treatment: Oxalic acid ((2.7±1.3) mg·kg ^-1·d ^-1)>Serine ((1.8±0.4) mg·kg ^-1·d ^-1)>Glucose ((1.6±0.8) mg·kg ^-1·d ^-1); the values of δ¹⁸O of N₂O were not different among different root exudate treatments and were stable at 24.1‰-25.6‰, but significantly higher than the control treatment ((20.1±1.5) ‰); the δ¹⁵N ^bulk value of N₂O was related to the component of root exudates added, which was (-20.06±2.22) ‰ of oxalic acid treatment, (-22.33±1.10) ‰ of serine treatment, (-13.86±1.11) ‰ treated of glucose treatment, and (-23.14±3.72) ‰ of the control treatment. The SP value of N₂O of each treatment ranged from 13.13‰ to 15.03‰, and the higher the root exudate concentration, the lower the SP value; after a comprehensive analysis of the correction coefficients of four indexes (N₂O emission rate, the value of δ¹⁵N ^bulk, δ¹⁸O and SP) at each sampling time and their daily mean values of 7 treatments, the correction coefficients of all treatments were closest to 1 at the 16th hour after the addition of root exudates. 【Conclusion】In the soil environment with NH+ 4- 300 mg N·kg ^-1, the root exudates promoted N₂O emission and the N₂O emission rate increased gradually during the culture time (24 hours). The promotion effect of glucose in high concentration group was the strongest, while that of oxalic acid in low concentration group was the strongest. Compared with the control treatment, the addition of root exudates significantly increased the δ¹⁸O value of N₂O; the addition of glucose significantly increased the δ¹⁵N ^bulk value of N₂O. The higher the concentration of root exudates, the stronger the contribution of denitrification to N₂O was detected.

Keywords：root exudates;nitrification;denitrification;N₂O;isotopic signature

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本文引用格式
庄姗, 林伟, 丁军军, 郑欠, 寇馨月, 李巧珍, 李玉中. 不同根系分泌物对土壤N₂O排放及同位素特征值的影响[J]. 中国农业科学, 2020, 53(9): 1860-1873 doi:10.3864/j.issn.0578-1752.2020.09.013
ZHUANG Shan, LIN Wei, DING JunJun, ZHENG Qian, KOU XinYue, LI QiaoZhen, LI YuZhong. Effects of Different Root Exudates on Soil N₂O Emissions and Isotopic Signature[J]. Scientia Acricultura Sinica, 2020, 53(9): 1860-1873 doi:10.3864/j.issn.0578-1752.2020.09.013

0 引言

【研究意义】N₂O是一种重要的温室气体,能够破坏平流层中的臭氧层且参与大气中光化学反应,从而进一步加剧温室效应^[1]。土壤是N₂O的主要排放源^[1,2],约占全球N₂O排放总量的65%^[3]。自20世纪初合成氨技术发明以来,氮肥被大量的投入到农业生产中,在增加作物产量的同时也使得N₂O排放量增加了19%^[4,5,6]。而菜地具有高水、高肥、肥料利用率低等特点^[7],造成了N₂O的大量排放。因此研究菜地N₂O排放对减缓全球变暖具有重要的理论和现实意义。【前人研究进展】硝化作用和反硝化作用是N₂O产生的主要过程^[8]。传统的研究方法主要有乙炔抑制法和同位素标记法等,这些方法在实际操作过程中都有一些局限性,如抑制剂（乙炔）和标记物在系统中扩散不均,易受系统干扰等^[9,10]。而自然丰度条件下的N₂O同位素位嗜值（SP值）不受N₂O前体同位素的影响,进而克服土壤与N作用的环境阻碍而直接评价N₂O产生的微生物过程,是估测N₂O来源及贡献的一种有效方法^[11]。尽管N₂O的δ¹⁵N^bulk和δ¹⁸O值易受底物同位素影响,但也能够在一定程度上反应微生物过程,其与SP值相结合进行分析有助于更加全面了解N₂O的排放行为。N₂O排放的影响因素很多,其中对土壤孔隙含水量（WFPS）^[12,13]、pH^[14]、O₂浓度^[15]、温度^[16,17,18]等研究较多。然而,植物通过根系分泌物对N₂O的产生也具有重要的影响。植物净光合固定的碳5%—25%通过根系分泌物流失到土壤中^[19,20,21],并以有机酸,氨基酸,糖类和其他类型分子的形式释放,是土壤重要的碳源,影响土壤硝化和反硝化作用^[22,23];根系分泌物还可以作为电子供体还原NO- 3和NO- 2^[24],改变N₂O产生的电子传递途径;还可通过改变根际周围微生物的群落结构影响N₂O的产生^[25]。草酸、丝氨酸、葡萄糖分别是根系分泌物中普遍存在并且含量较多的一种低分子有机酸、氨基酸、糖类^[26],它们在N₂O产生过程中的作用机理存在一定差异。研究发现草酸显著促进土壤中有机态氮和有机无机复合态氮的矿化速度,促进氮的释放^[27]。也有研究表明草酸促进N₂O还原基因的复制,对N₂O的还原产生影响^[28]。葡萄糖也能够促进氮的分解,但这种促进作用明显小于草酸,此外,优先利用葡萄糖的微生物可能导致氮分解率下降^[28]。丝氨酸不仅是细胞蛋白质的重要组成部分,也是微生物的能量来源,在土壤中可以矿化为NH+ 4,改变土壤的C/N和pH^[29]。【本研究切入点】不同植物根系分泌物的组成和含量不同,大量研究表明N₂O的排放与根系分泌物的组成和含量有关^{[24-25,29-30]},但鲜有系统研究不同组分单独作用对N₂O产生过程的影响。又由于根系分泌物组成和土壤环境的复杂,在自然条件下很难做到定量提取研究^[31],所以通过模拟根系分泌物组分和含量制备根系分泌物溶液添加到土壤中是研究根系分泌物作用下土壤N₂O排放特征的重要方法。尽管实验室环境能够控制土壤温度、湿度等环境因子以减少环境差异对N₂O排放的影响,但添加根系分泌物后微生物对各根系分泌物的相互作用和响应时间是影响N₂O排放量的关键,N₂O排放的时空变异性对定量分析不同根系分泌物组分对N₂O产生作用的影响仍无法排除。因此研究不同根系分泌物组分对土壤N₂O排放的影响,同时确定合理的气体取样时间,对于提高试验效率,深入研究根系分泌物对N₂O产生在时间和空间上的作用至关重要。【拟解决的关键问题】通过对根系分泌物中3种主要组分（有机酸、丝氨酸、糖类）的主要成分（草酸、丝氨酸、葡萄糖）作用下土壤N₂O排放和同位素特征的综合分析,揭示不同根系分泌物对土壤N₂O产生过程的影响及其作用机制,为选择适宜的植物进而减缓土壤N₂O排放提供支撑。

1 材料与方法

1.1 土壤采集

土壤采集于中国农业科学院农业环境与可持续发展研究所顺义实验基地,东经40°05′2″,北纬116°55′19″。基地为典型的暖温带半湿润大陆性季风气候,年平均日照2 684 h,年平均气温12.5℃,年平均降水量623.5 mm,无霜期195 d左右。土壤类型为潮褐土,土壤密度1.40 g·cm^-3,有机质16.48 g·kg^-1,全氮1.10 g·kg^-1, 全磷0.68 g·kg^-1,全钾20.31 g·kg^-1,pH 8.7。

土壤采集于2018年8月（雨后2 d）,取0—20 cm表层土壤于阴凉处自然风干。除去砂石、植物根系等杂质,过2 mm筛,室内保存备用。

1.2 试验方法

选择3种代表性有机物,即草酸代表有机酸,丝氨酸代表氨基酸,葡萄糖代表糖类,以此为研究对象。依据白菜根系分泌速率^[26],设置2个添加水平：150 μg C·d^-1（低浓度）和300 μg C·d^-1（高浓度）,另添加蒸馏水为空白对照,共7个处理,每次添加溶液体积为0.85 mL,每种处理设3次重复。用120 mL密封性良好的血清瓶作培养瓶,称取40 g风干土放入120 mL血清瓶中,共21个。用蒸馏水调节土壤孔隙含水量（WFPS：water-filled-pore-space）至75%左右（称重法）,放入25℃恒温培养箱黑暗预培养。根据培养前重量每4天补充一次水分（日蒸发量约为0.85 mL）,共补充3次。第12天补充水分的同时施入(NH 4)₂SO₄溶液（300 mgN·kg^-1干土,足量氮使微生物对根系分泌物敏感,且使土壤产生的N₂O同位素丰度在仪器测结果最准确的范围内）。在第14天用2.5 mL一次性注射器依次吸取草酸、丝氨酸、葡萄糖溶液和蒸馏水,插入土壤缓慢推动注射器使溶液缓慢送出,作为正式培养第1天,之后每隔24 h添加一次。尽管丝氨酸能够提供N素,但是培养期间添加丝氨酸N素的总量不足土壤总氮的3%,对N₂O排放量的影响较小。

1.3 主要药品及规格

草酸（H₂C₂O₄·2H₂O）：分析纯,北京化工厂;D-无水葡萄糖（C₆H₁₂O₆）：>99.9%,MYM Biological Technology;丝氨酸（C₃H₇NO₃）：99%,北京百灵威科技有限公司;硫酸铵（(NH 4)₂SO₄）：分析纯,天津市福晨化学试剂厂。

1.4 气体样品采集

在正式培养的第3天,添加根系分泌物溶液后的第2、4、8、12、16、20和24小时取样,共采集7次。采样前将培养瓶盖上橡胶塞和铝盖,并用密封器压紧密封,培养2 h。其他时间保持培养瓶敞口。用50 mL针筒抽取30 mL氦气注入培养瓶中,来回抽动针筒重复3次,将气体混匀,再将针筒抽到30 mL刻度处,待针筒不回弹后拔出并迅速插入已抽成真空的20 mL血清瓶,做好标记。取样的同时抽取实验室内空气作为N₂O的环境背景值（3次重复）。

1.5 样品测定与方法：

1.5.1 N₂O同位素特征值测定与校正稳定同位素质谱仪（Delta V Plus‐Precon, Thermo Fisher Scientific, Bremen, Germany）用来测定N₂O同位素特征值：δ¹⁵N^α、δ¹⁵N^bulk、δ¹⁸O。计算公式如下^[32]：

(1)$\delta^{15}N^i=\left( \frac{^{\text{15}}R_{\text{sa}}^{i}}{^{\text{15}}{{R}_{\text{st}}}}-\text{1} \right)\times \text{1000 (}i=\text{ }\!\!\alpha\!\!\text{ , }\!\!\beta\!\!\text{ ,bulk)}$

(2)$\delta^{18}O(‰)=\left( \frac{^{\text{18}}R_{\text{sa}}^{{}}}{^{\text{18}}{{R}_{\text{st}}}}-\text{1} \right)\times 1000$

(3)${{\delta }^{\text{15}}}{{\text{N}}^{\text{bulk}}}=\frac{{{\delta }^{\text{15}}}{{\text{N}}^{\text{ }\!\!\alpha\!\!\text{ }}}+{{\delta }^{\text{15}}}{{\text{N}}^{\text{ }\!\!\beta\!\!\text{ }}}}{\text{2}}$

式中,R=¹⁵N/¹⁴N,R=¹⁸O/¹⁶O。sa表示样品,st表示标准物。¹⁵N和¹⁸O的标准分别是大气中的N₂和维也纳标准平均海洋水（Vienna Standard Mean Ocean Water）,校准用标准参考气体（美国液化空气特种气体有限责任公司（Air Liquide America, Specialty Gases LLC））。

由于测定的N₂O为空气中和土壤产生的N₂O的混合气,受空气同位素值的影响,各检测结果（δ¹⁵N^α、δ¹⁵N^bulk、δ¹⁸O）需按照如下公式校正^[33]：

(4)${{\delta }_{\text{soil}-\text{emitted}}}=\frac{{{\delta }_{\text{mix}}}\times {{c}_{\text{mix}}}-{{\delta }_{\text{air}}}\times {{c}_{\text{air}}}}{{{c}_{\text{mix}}}-{{c}_{\text{air}}}}$

式中,$\delta_{soil-emitted}$代表土壤排放N₂O的同位素值;δ_mix、c_mix分别表示土壤与空气混合气体的同位素值和N₂O浓度;δ_air、c_air分别表示空气背景的同位素值和N₂O浓度（0.63 mg·m^-3。c_mix-c_air为土壤排放的N₂O浓度。

由于土壤N₂O排放具有时间变异性,N₂O的日平均同位素特征值用通量的加权平均值来表示^[34]：

(5)${{\delta }_{\text{wa}}}=\frac{\sum\limits_{i=\text{1}}^{n}{\text{(}{{\text{e}}_{i}}\times {{\delta }_{\iota }}\text{)}}}{\sum\limits_{i=\text{1}}^{\text{n}}{{{\text{e}}_{i}}}}$

式中,δ_wa表示同位素的日通量加权平均值。e_i表示第i-1取样到i次取样时间内土壤排放N₂O的量（i时刻的排放速率×时间间隔（2 h 或4 h））。δ_i表示i次取样检测的同位素特征值。

1.5.2 N₂O来源确定的理论基础 N₂O为直线型分子：N^β-N^α=O。根据两个氮原子与氧原子的相对位置不同,则中间的氮原子命名为α氮原子,末端的氮原子为β氮原子^[32]。自然丰度条件下N₂O分子中¹⁵N原子出现在α位和β位的差异,称为同位素位嗜值（site preference）,即SP值,计算公式如下^[35,36]：

(6)$SP=\delta^{15} N^{a}-\delta^{15}N^{\beta}$

在不同微生物群落或酶参与下的不同反应过程产生的N₂O的SP值不同,这是SP值用于土壤N₂O溯源研究的重要理论基础。纯细菌培养下硝化作用和反硝化作用的SP值是已知的,且其值不受底物氮氧同位素的影响。但自然状况下这两种过程往往同时存在^[32],为测定气体样品中N₂O的同位素值,通常利用同位素二源混合模型计算硝化作用和反硝化作用对N₂O的贡献^[37]。公式如下：

(7)${{f}_{\text{D}}}=\frac{S{{P}_{\text{sa}}}-S{{P}_{\text{N}}}}{S{{P}_{\text{D}}}-S{{P}_{\text{N}}}}$

(8)$f_N=1-f_D$

式中,f_N和f_D分别表示N₂O来自土壤中硝化和反硝化作用的贡献比例,SP_N表示纯细菌培养条件下硝化作用的SP值,为33‰^[38,39];SP_D表示纯细菌培养条件下反硝化作用的SP值,为0‰^[40,41];SP_sa表示测得样品的SP值,样品SP值大于33‰或小于0‰均按33‰或0‰计算。

1.5.3 N₂O排放速率的计算采集的气体用稳定同位素质谱仪（Delta V Plus-Preco,Thermo Fisher Scientific,Bremen,Germany）结合痕量气体浓缩系统测定N₂O气体峰面积,根据N₂O在样品中峰面积与环境背景中峰面积的比值,计算样品N₂O浓度,根据培养前后瓶内N₂O的浓度变化计算单位时间内土壤N₂O的排放速率（培养前以及环境背景气体N₂O的体积分数为320 nmol·mol^-1）。公式如下^[42]：

(9)$F=\frac{\text{(}{{c}_{\text{sa}}}-{{c}_{\text{air}}}\text{)}\times V\times M\frac{\text{273}}{\text{273}+T}}{\text{1000}\times m\times \text{ }\!\!\Delta\!\!\text{ }t\times \text{22}\text{.4}}$

式中,F表示N₂O排放速率（μg·kg·h^-1）,22.4为标准大气压下的摩尔体积（L·mol^-1）。M为标准状态下N₂O气体的摩尔质量（44 g·mol^-1）。1000为单位换算。V表示培养瓶的体积,0.09 L。c_sa、c_air分别表示培养后瓶内气体和空气中N₂O浓度（nmol·mol^-1）。Δt为培养时间,2 h。T为培养温度,25℃。

1.5.4 N₂O通量和同位素值的校正系数各采样时刻N₂O排放速率和日平均排放速率的校正,各采样时刻测得的同位素值和同位素的日均值之间的校正公式如下^[43,44]：

(10)${{C}_{i}}=\frac{{{F}_{\text{ave}}}}{{{F}_{i}}}$

式中,F_i（i=2、4、8、12、16、20、24）表示各时间的平均通量。C_i为校正系数,F_ave为N₂O日平均排放速率、δ¹⁵N^bulk、δ¹⁸O和SP的日平均值,其中δ¹⁵N^bulk、δ¹⁸O和SP的日平均值为各时刻排放速率的加权平均值（根据公式（5）计算）。F_i为实测的第i次实测通量,或第i次取样检测的同位素值。校正系数越接近1,则说明该时刻测量的N₂O排放速率和同位素值越接近日平均值,测量结果越具有代表性,即可采用该时刻作为最佳的取样时间^[45]。

1.6 数据处理与分析

采用Microsoft Excel·2016对试验数据进行处理和做图。用SPSS·25.0（SPSS Inc., Chicago, IL, USA）进行统计分析。各处理间N₂O累计排放量,δ¹⁸O,δ¹⁵N^bulk和SP用单因素方差分析（One-Way ANOVA）,LSD法进行多重比较。双因素方差分析（Two-Way ANOVA）比较根系分泌物组分和浓度及其交互作用对N₂O排放量和同位素特征值的影响。用一元线性回归分析最佳采样时间的检测值和日均值间的关系。

2 结果

2.1 N₂O排放速率的日变化

如图1所示,在取样期间所有处理的N₂O排放速率均呈上升趋势,添加根系分泌物处理组均高于对照。在培养时间内添加低浓度、高浓度草酸处理土壤N₂O排放速率迅速升高,分别从13.21 μg·kg^-1·h^-1增加到226.28 μg·kg^-1·h^-1和从17.84 μg·kg^-1·h^-1增加到97.21 μg·kg^-1·h^-1,添加草酸后土壤N₂O排放速率,低浓度处理组高于高浓度处理组;葡萄糖处理中低浓度组低于高浓度组,低浓度丝氨酸、高浓度丝氨酸、低浓度葡萄糖和高浓度葡萄糖处理的土壤N₂O排放速率分别从70.72 μg·kg^-1·h^-1增加到125.79 μg·kg^-1·h^-1、50.82 μg·kg^-1·h^-1增加到171.71 μg·kg^-1·h^-1、20.34 μg·kg^-1·h^-1增加126.69 μg·kg^-1·h^-1和20.46 μg·kg^-1·h^-1增加到185.21 μg·kg^-1·h^-1。

图1

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图1添加不同根系分泌物后不同时刻N₂O排放速率

误差线表示标准误差（n=3）。下同
Fig. 1N₂O emission rates at different observation time after adding root exudates

Error bars represent standard error (n=3). The same as below

双因素方差分析显示N₂O累计排放量受根系分泌物和浓度共同作用的影响（P<0.05）（表1）。N₂O累计排放量低浓度处理组：草酸处理>丝氨酸处理>葡萄糖处理,而在高浓度处理组：葡萄糖处理>丝氨酸处理>草酸处理。单因素方差分析显示,低浓度草酸、高浓度丝氨酸和高浓度葡萄糖处理土壤N₂O累计排放速率显著高于对照（P<0.05）。增加根系分泌物浓度时,仅高浓度葡萄糖处理N₂O累计排放量显著高于低浓度葡萄糖处理（P<0.05）。

Table 1
Table 1Cumulative emissions of N₂O and daily weighted average of isotope signature

处理 Treatment		N₂O累计排放量 (mg·kg^-1·d^-1)	δ¹⁸O (‰)	δ¹⁵N^bulk(‰)	SP (‰)
草酸 Oxalic acid	CL	2.68±1.34ab	24.16±1.55a	-19.82±7.41abc	17.03±0.40a
草酸 Oxalic acid	CH	1.38±0.24bc	24.08±1.22a	-20.31±5.82abc	13.13±2.70b
丝氨酸 Serine	AL	1.81±0.45abc	25.33±0.39a	-24.44±1.95c	15.71±0.78ab
丝氨酸 Serine	AH	2.56±0.45ab	24.68±1.11a	-20.21±2.11abc	14.35±1.47ab
葡萄糖 Glucose	PL	1.61±0.79bc	25.59±0.25a	-13.12±1.13a	16.16±1.40a
葡萄糖 Glucose	PH	3.23±1.33a	24.09±2.30a	-14.6±4.38ab	15.18±1.04ab
对照 Control treatment		0.90±0.09c	20.06±1.52b	-23.14±3.72c	13.29±1.40b
添加根系分泌物 Adding root exudates		<0.001**	0.196	0.089	0.032*
组分 Component		0.718	0.495	0.017*	0.718
浓度 Concentration		0.38	0.261	0.726	0.012*
组分×浓度 Component×Concentration		0.0278*	0.611	0.51	0.222

CL: 150 μg C·d^-1 Oxalic acid treatment; CH: 300 μg·C·d^-1 Oxalic acid treatment; AL:150 μg C·d^-1 serine treatment; AH:300 μg C·d^-1 serine Treatment; PL:150 μg C·d^-1 glucose treatment; PH: 300 μg C·d^-1 glucose treatment; Control: Distilled water treatment. The same as below
The weighted average of δ¹⁸O, δ¹⁵N^bulk and SP were calculated by equation (5). The same as below
The values followed by different little letters in the same column indicate the significant differences between treatments at P<0.05 level using One-Way ANOVA. * indicate the significant differences between treatments at P<0.05 level using Two-Way ANOVA
CL：150 μg C·d^-1草酸处理;CH：300 μg C·d^-1草酸处理;AL：150 μg C·d^-1丝氨酸处理;AH：300 μg·C·d^-1丝氨酸处理;PL：150 μg C·d^-1葡萄糖处理;PH：300 μg·C·d^-1葡萄糖处理;Control：蒸馏水处理（对照）。下同
δ¹⁸O,δ¹⁵N^bulk和SP的值为各处理不同采样时间同位素值的加权平均值,由公式（5）计算。下同
同列中小写字母表示单因素方差分析（One-Way ANOVA）显著性P<0.05。*表示双因素方差分析（Two-Way ANOVA）显著性P<0.05

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2.2 N₂O同位素值的日变化

不同处理土壤N₂O同位素特征值不同（图2）。添加根系分必物处理的δ¹⁸O变化基本一致,稳定在23.43‰—25.90‰;而对照则在20.69‰—22.13‰。3种不同根系分泌物处理N₂O的δ¹⁵N^bulk值不同,葡萄糖处理组δ¹⁵N^bulk值最高,变化范围分别是：-15.22‰— 13.55 ‰（低浓度）和-14.82‰—-10.70‰（高浓度）。低浓度丝氨酸处理δ¹⁵N^bulk值的范围是-26.56‰— 22.07‰,低于高浓度丝氨酸处理（-22.21‰— -16.66‰）。两浓度草酸处理δ¹⁵N^bulk值差异较小,低、高浓度的变化范围分别是-21.89‰—-15.27‰和-23.97‰—-16.75‰。3种根系分泌物浓度越高,SP值越低,低浓度草酸处理SP值从16.27‰增加至17.65‰,高浓度草酸处理SP值从16.69‰降至12.67‰;两浓度的丝氨酸处理SP值逐渐升高,低浓度丝氨酸和高浓度丝氨酸处理变化范围是：11.64‰—18.50‰和11.45‰—14.83‰;两浓度葡萄糖处理SP值先升高后降低,极值分别出现在添加葡萄糖后的第8小时（18.79‰）和第12小时（17.26‰）。

图2

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图2添加不同根系分泌物后N₂O同位素特征值(δ¹⁸O, δ¹⁵N^bulk, SP)随时间的变化

Fig. 2Isotope signature (δ¹⁸O, δ¹⁵N^bulk, SP) of N₂O at different observation time after adding root exudates

双因素方差分析显示,N₂O的δ¹⁵N^bulk受添加根系分泌物成分的影响（P<0.05）,其中葡萄糖处理δ¹⁵N^bulk值最高（（-13.86±1.11）‰）。SP值受添加根系分泌物浓度的影响（P<0.05）,浓度越高,SP值越低。单因素方差分析显示添加根系分泌物处理N₂O的δ¹⁸O值显著高于对照（P<0.05）,但不同组分处理间无差异;添加葡萄糖和丝氨酸的不同浓度间N₂O的SP值均无明显差异;添加草酸后土壤N₂O的SP值,低浓度处理组显著高于高浓度处理组（P<0.05）。

2.3 不同处理反硝化作用对N₂O排放的贡献

通过二元混合模型（公式（6）,（7））计算各处理反硝化作用和硝化作用对土壤N₂O排放的相对贡献率如图3所示。空白对照处理、低浓度草酸、高浓度草酸、低浓度丝氨酸、高浓度丝氨酸、低浓度葡萄糖、高浓度葡萄糖处理反硝化作用的贡献分别是：60%、48%、60%、52%、57%、51%、54%。添加草酸、丝氨酸、葡萄糖的浓度越高,反硝化作用越强。

图3

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图3不同处理反硝化作用-硝化作用对N₂O排放的贡献

Fig. 3Contribution of denitrification-nitrification on N₂O

2.4 各时刻N₂O排放速率和同位素特征值的校正系数

N₂O排放速率和同位素特征值随时间有一定的变化,添加根系分泌物后各时刻的检测值和日平均值的校正系数如图4所示。不同指标的校正系数的波动范围不同,N₂O排放速率的校正系数波动较大（0.49—8.46）,同位素值的校正系数波动较小（0.79—1.87）,其中各指标前8 h校正系数的波动较大,后逐渐减小。N₂O排放速率的校正系数为1时的点在添加根系分泌物后的第16 h;N₂O的δ¹⁸O值24 h内比较稳定,各处理校正系数稳定在1附近;N₂O的δ¹⁵N^bulk值的校正系数呈先增高后降低的趋势,校正系数为1的点出现在添加根系分泌物后的第2—8小时和第16—20小时;SP值的校正系数为1的点出现在添加根系分泌物后的第16—20小时。

图4

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图4添加根系分泌物后各时刻N₂O排放速率,稳定同位素值（δ¹⁸O, δ¹⁵N^bulk,SP）的校正系数

Fig. 4The correction coefficient of emission rate and isotope values (δ¹⁸O, δ¹⁵N^bulk, SP) at different observation time after adding root exudates

3 讨论

3.1 根系分泌物组分和浓度对N₂O排放量及其同位素特征值的影响

本研究发现添加根系分泌物处理土壤N₂O排放量高于对照,说明根系分泌物的添加会促进土壤N₂O的排放,这和以往研究结果一致^[46,47]。植物根系分泌物使植物通过光合作用固定的碳外流^[48],并以有机碳的形式释放到土壤中。微生物从根系分泌物中获取能量,使土壤酶数量增大,活性增强,导致N₂O排放量的增加^[49]。植物根系分泌物也可作为碳源改变微生物细胞内N₂O产生过程的电子传递途径^[50],并促进N₂O的产生。微生物对不同根系分泌物的响应机制不同,从而导致N₂O产生路径也会有差异,进而造成N₂O排放量的差异。

本研究发现添加3种根系分泌物处理间土壤N₂O的δ¹⁸O值无明显差异,且均显著高于对照（P<0.05）。$NO_{3}^{-}$还原为N₂O的反硝化过程（$NO_{3}^{-}$→$NO_{2}^{-}$→NO→ N₂O）可看作O逐渐去除的过程,¹⁶O比¹⁸O更容易断裂使得产物N₂O中富集¹⁸O从而产生同位素分馏^[38];同时,N₂O还原（N₂O→N₂）过程也会导致剩余N₂O的δ¹⁸O值升高^[51,52]。而$NO_{3}^{-}$在还原过程中产生的中间产物可能会与环境中的H₂O发生O交换,从而减弱$NO_{3}^{-}$在还原过程中发生的分馏效应^[38]。以上反硝化过程中的每一个还原步骤均是在不同酶的催化下发生,且导致不同的O同位素分馏^[53]。因此根系分泌物处理组土壤N₂O的δ¹⁸O值高于对照组可能是由于根系分泌物的添加促进了反硝化作用的进行,使反硝化还原酶活性改变所致;且3种根系分泌物可能共同作用了某一种对O同位素分馏起关键作用的酶而导致处理间N₂O的δ¹⁸O值无差异。另外,添加3种根系分泌物可能造成了不同程度的N₂O向N₂的还原。

各根系分泌物处理N₂O的δ¹⁵N^bulk值不同,δ¹⁵N^bulk值受根系分泌物组分的影响。在肥料$NH_4^+$充足的土壤条件下,N₂O的δ¹⁵N^bulk值的升高和降低是由多种原因造成的,同位素的分馏可能是重要的原因。硝化作用的产物$NO_3^-$可作为反硝化作用的底物,根据同位素分馏原理,微生物优先利用轻同位素而导致产物发生重同位素贫化^[54]使由反硝化过程产生的N₂O的δ¹⁵N^bulk值降低;其次,N₂O还原可能对δ¹⁵N^bulk值产生重要影响,N₂O还原过程中¹⁴N-O键比¹⁵N-O键更容易断裂,导致剩余N₂O的δ¹⁵N^bulk值升高;所以添加根系分泌物通过促进反硝化过程和N₂O还原可能会导致N₂O的δ¹⁵N^bulk值升高^[30]。又因为土壤中的不同微生物过程导致的N同位素分馏不同^[55],这使得不同根系分泌物处理的土壤N₂O的δ¹⁵N^bulk值有差异。由于葡萄糖会促进N₂O还原过程,该理论已在本课题组其他研究中得到验证^[56]。此外,SPEIR等^[57]通过¹⁵N标记肥料也发现,葡萄糖为易有效碳,而易有效碳的添加会促进反硝化过程进行完全,促进N₂O向N₂的还原过程,这可能是葡萄糖处理组N₂O的δ¹⁵N^bulk值显著高于对照的原因（P<0.05）。

SP值表征的是直线型分子N₂O（N-N-O）的分子内¹⁵N的位置嗜好性差异,该值和微生物过程有关,是一个被国际认可的N₂O溯源研究工具^[35]。本研究发现同一根系分泌物组分作用下,SP值随根系分泌物浓度的升高而降低。由于纯细菌培养下硝化作用下产生的N₂O的SP值为33‰^[38,39],反硝化作用下产生的N₂O的SP值为0‰^[40,41]。本研究所有处理SP值介于两者之间则说明产生的N₂O来源于硝化作用和反硝化作用的共同作用。根据硝化和反硝化的二元混合模型可知SP值的升高和降低分别代表土壤微生物过程趋于硝化作用方向和反硝化作用进行,SP值越低,则反硝化作用越强。根系分泌物的添加为土壤提供碳源,从而促进了异养微生物作用下的反硝化过程。在适宜范围内,反硝化作用随着土壤碳源浓度的升高而增强,则产生的N₂O的SP值也会因此而降低。

不同浓度处理组各根系分泌物累计排放量的顺序不同,我们发现在低浓度处理组：草酸处理>丝氨酸处理>葡萄糖处理,而在高浓度处理组：葡萄糖处理>丝氨酸处理>草酸处理。通过对土壤N₂O排放速率及δ¹⁸O、δ¹⁵N^bulk、SP值等指标的综合分析,有利于进一步明确不同根系分泌物组分对N₂O产生过程的影响。高浓度葡萄糖处理组土壤N₂O排放量是低浓度处理组的2倍,但这两种处理土壤产生N₂O的δ¹⁸O、δ¹⁵N^bulk值均无明显差异,在葡萄糖低、高两种浓度处理下,反硝化作用对土壤N₂O排放的贡献率分别为51%和54%。说明不同葡萄糖浓度下,硝化作用和反硝化作用对土壤N₂O排放的贡献均无显著差异（P>0.05）,本研究的这两种葡萄糖浓度并没有导致土壤产生N₂O的微生物结构的改变,这两种葡萄糖处理下土壤N₂O的N、O同位素分馏也因此无明显变化^[55],两种浓度葡萄糖处理下土壤产生的N₂O的δ¹⁸O、δ¹⁵N^bulk值也无明显差异。这和以往研究结果一致^[58],即葡萄糖对土壤微生物群落结构的影响较小。葡萄糖的添加对土壤产生N₂O的作用机制是增加微生物数量和提高酶活性^[58],葡萄糖的浓度越高,越有利于N₂O的产生。但也有研究发现,当葡萄糖达到一定浓度后,土壤N₂O排放量不会再继续增加反而出现下降,这是由于碳源的添加使土壤中的其他异养微生物大量增殖,并竞争N₂O的产生底物,从而使N₂O排放量降低^[59];同时,碳源会促进无机氮固定为有机氮,抑制有机氮的矿化,微生物死亡后与金属离子结合到超分子聚合体中^[60],也是导致N₂O排放量降低的因素。由于不同土壤类型^[61],微生物对添加的葡萄糖的响应机制不同,从而导致的N₂O排放量不同^[47]。此外,微生物对葡萄糖的响应还和底物氮源类型有关^[62,63,64]。本研究添加较高浓度的铵态氮肥后,导致土壤C/N比值降低,葡萄糖的添加为微生物提供了其限制性的碳源,这是本实验研究结果和其他研究不同的主要原因。丝氨酸在土壤中不仅能够被微生物以碳源的形式直接利用^[29],其分子内的N素还能转化为NH+ 4作为硝化作用的底物^[65]。但由于丝氨酸提供的N源浓度较低,对N₂O排放量的促进作用不明显。土壤排放N₂O的SP值随丝氨酸浓度升高而降低,说明添加的丝氨酸主要发挥了为土壤微生物提供碳源的作用,从而促进了反硝化作用。本研究发现草酸和葡萄糖浓度越高,土壤排放的N₂O的δ¹⁵N^bulk值越低,这主要是高浓度组分的根系分泌物促进反硝化作用,由于同位素分馏而导致N₂O的δ¹⁵N^bulk值降低。但土壤中由于底物NH+ 4充足,硝化作用为先导反应,这是两种浓度根系分泌物作用下N₂O的δ¹⁵N^bulk值虽然降低但未达到显著性水平的原因。而丝氨酸未表现出此规律,这可能是丝氨酸N原子参与N₂O产生过程的结果。本研究发现低浓度草酸处理组N₂O的排放量最高。说明低浓度下草酸对N₂O的促进效果最强。草酸会加速有机无机复合体的分解^[66,67],还可以增加一些特殊的微生物种类,如,K-战略类型的微生物（在一定环境中具有竞争能力的微生物）,并产生大量与氮转化有关的酶,增加矿物态氮的释放^[68],促进N₂O的排放。本研究利用的土壤为碱性（pH= 8.7）,而草酸为酸性,添加后会使土壤微环境的碱性减弱,当草酸达到一定浓度后对土壤碱性环境的削弱程度更强,有利于N₂O还原过程的进行,使更多的N₂O转化为N₂,从而促进N₂O向N₂的还原过程^[69]。这正好验证了本研究高浓度草酸处理组土壤N₂O排放量显著低于高浓度处理组的结果。与低浓度草酸处理组（48%）相比,高浓度草酸处理组（60%）反硝化作用对土壤N₂O排放的相对贡献率增加了12%,两浓度草酸处理土壤硝化和反硝化过程差异均显著（P<0.05）。这说明草酸处理改变了土壤微生物的结构,且促进了N₂O还原基因的复制而导致N₂O还原为N₂,其他研究中也有类似结果^[30],这不同于葡萄糖不改变微生物的群落结构而通过促进反硝化过程的完全进行而使N₂O还原为N₂,N₂O还原与N₂O产生同比例增加。高浓度处理组反硝化作用的增强,使通过硝化作用得到的可作为反硝化底物的$NO_3^-$的相对比例增加,进而使高浓度处理组由反硝化过程产生的N₂O的相对比例增加,根据同位素分馏效应,产物相对于底物会发生¹⁵N贫化^[54]。因此,添加高浓度草酸处理组土壤N₂O的δ¹⁵N^bulk值也会降低,高浓度草酸处理N₂O还原导致的δ¹⁵N^bulk值升高抵消了反硝化过程产物¹⁵N逐渐贫化的效果,使得两浓度处理下δ¹⁵N^bulk差异不显著。与此有关的证据还需进一步研究确定。

3.2 基于N₂O排放速率及同位素特征值确定最佳取样时间

在采取手动N₂O气体观测中,通常采用1次的检测结果而作为1日甚至多日的平均情况,所以采样时间的选择尤为重要。根据研究需要,基于不同指标的取样时间不同,N₂O排放速率、N₂O的δ¹⁵N^bulk及SP值的最佳取样时间分别为添加根系分泌物后的第12—16 h、4—8 h或16—20 h和16 h。由于N₂O的δ¹⁸O值随着培养时间无明显变化,因此,取样时间对N₂O的δ¹⁸O值的影响不大。综合N₂O的排放速率和各稳定同位素值的取样时间,选择添加根系分泌物后的第16 h为最佳取样时间,从而可以对各个指标进行综合分析,更有利于量化不同微生物过程对土壤N₂O排放的贡献率。对各处理最佳取样时间下得到的各个指标的结果分别与日平均值做相关分析显示（图5）。N₂O排放速率：R²=0.93（P<0.001）,N₂O的δ¹⁸O值：R²=0.71（P<0.001）,N₂O的δ¹⁵N^bulk值：R²=0.91（P<0.05）,SP值：R²=0.91（P<0.001）。因此,确定不同处理组的最佳取样时间是添加根系分泌物后的第16小时。此外,该时刻同位素值相对稳定,说明土壤内微生物过程稳定,利于分析N₂O产生的微生物过程,因此结果最具代表性。

图5

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图5最佳取样时间N₂O排放速率、稳定同位素值（δ¹⁸O, δ¹⁵N^bulk, SP）和日平均值的比较

Fig. 5Comparison of emission rate, isotope signature (δ¹⁸O, δ¹⁵N^bulk, SP) of N₂O at optimal sampling time with daily average

3.3 研究展望

鉴于稳定同位素技术在区分N₂O产生的微生物过程中存在优势,未来将继续利用同位素技术围绕不同根系分泌物组分及其组合作用下对土壤N₂O的贡献规律及其产生的多种微生物途径进行研究。由于N₂O还原对同位素值的影响较大,且促进N₂O还原为N₂是减少农业土壤N₂O排放的努力方向,所以未来研究将N₂O还原纳入考虑,同时引入国际稳定同位素先进技术和方法,如δ¹⁵N^bulk-δ¹⁸O模型和δ¹⁵N^bulk-SP模型,通过对反应底物浓度、底物同位素和前体同位素图谱的综合分析,进一步阐明植物根系分泌物对土壤N₂O产生的作用机制。

4 结论

通过外源添加根系分泌物3种主要组分,研究其对N₂O排放高峰期的排放速率和稳定同位素特征值的影响。在施加NH+ 4的土壤中添加草酸、丝氨酸、葡萄糖促进土壤N₂O的排放,高浓度组处理葡萄糖的促进效果最强,而低浓度组处理草酸的促进效果最强;根系分泌物作用下土壤排放N₂O的δ¹⁸O的值升高;根系分泌物不同组分作用下N₂O的δ¹⁵N^bulk值不同,葡萄糖的添加使δ¹⁵N^bulk值显著升高;各处理N₂O的SP值的范围为13.13‰—15.03‰,根系分泌物浓度越高SP值越低;反硝化作用对N₂O的贡献越强。通过对所测指标（N₂O排放速率、δ¹⁵N^bulk、δ¹⁸O和SP值）的校正系数和回归分析显示添加根系分泌物后的最佳取样时间为添加根系分泌物后的第16小时。

参考文献原文顺序
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Applied and Environmental Microbiology, 2006 72(1):638-644.

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The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

[11]

BAGGS

, MAIR

, MAHMOOD

. A review of stable isotope techniques for N₂O source partitioning in soils: recent progress, remaining challenges and future considerations
Rapid Communications in Mass Spectrometry, 2007,22(11):1664-1672.

DOI:10.1002/rcm.3456 URLPMID:18435506 [本文引用: 1]

Nitrous oxide is produced in soil during several processes, which may occur simultaneously within different micro-sites of the same soil. Stable isotope techniques have a crucial role to play in the attribution of N(2)O emissions to different microbial processes, through estimation (natural abundance, site preference) or quantification (enrichment) of processes based on the (15)N and (18)O signatures of N(2)O determined by isotope ratio mass spectrometry. These approaches have the potential to become even more powerful when linked with recent developments in secondary isotope mass spectrometry, with microbial ecology, and with modelling approaches, enabling sources of N(2)O to be considered at a wide range of scales and related to the underlying microbiology. Such source partitioning of N(2)O is inherently challenging, but is vital to close the N(2)O budget and to better understand controls on the different processes, with a view to developing appropriate management practices for mitigation of N(2)O. In this respect, it is essential that as many of the contributing processes as possible are considered in any study aimed at source attribution, as mitigation strategies for one process may not be appropriate for another. To aid such an approach, here the current state of the art is critically examined, remaining challenges are highlighted, and recommendations are made for future direction.

[12]

丁军军, 张薇, 李玉中, 林伟, 徐春英, 李巧珍 . 不同灌溉量对华北平原菜地N₂O排放及其来源的影响
应用生态学报, 2017,28(7):2269-2276.

DOI:10.13287/j.1001-9332.201707.026 URLPMID:29741059 [本文引用: 1]

To understand the mechanisms of agricultural N₂O emission, we investigated the N₂O emission dynamics, the N₂O isotope signatures, and the site preference value under different soil water conditions in the vegetable farmland of North China, by using the stable isotope technique and the acetylene inhibition method. The results demonstrated that N₂O emission was significantly affec-ted by the water condition, and N₂O emissions from soil with water-filled pore space (WFPS) of 70% were significantly higher than that with 50% WFPS. N₂O emission occurred mostly in the early stage of fertilization, and decreased rapidly in the later stage of fertilization. At 50% WFPS, nitrification was the major process generating N₂O during the early fertilization stage, accounting for approximately 90% of the N₂O emission. However, the contribution of nitrification decreased sharply, whereas denitrification became the dominant process, accounting for 80% of the N₂O emission 7 days after the fertilization. On the other hand, at 70% WFPS, denitrification was the main process releasing N₂O during the early fertilization stage, decreasing from 70% to 40% and then gradually increasing to 80% 10 days after the fertilization. Overall, N₂O emission was mainly dominated by the denitrification. The effect of different water treatments on soil nitrification and denitrification took place mainly in the early stage of fertilization, and N₂O emission was gradually dominated by the denitrification at the later stage. These results suggested we could reduce N₂O emission by approp-riately reducing the amount of irrigation in the vegetable farmland of North China.

DING J

, ZHANG

, LI Y

, LIN

, XU C

, LI Q

. Effects of soil water condition on N₂O emission and its sources in vegetable farmland of North China Plain
Chinese Journal of Applied Ecology, 2017,28(7):2269-2276. (in Chinese)

DOI:10.13287/j.1001-9332.201707.026 URLPMID:29741059 [本文引用: 1]

郑欠, 丁军军, 李玉中, 林伟, 徐春英, 李巧珍, 毛丽丽 . 土壤含水量对硝化和反硝化过程N₂O排放及同位素特征值的影响
中国农业科学, 2017,50(24):4747-4758.

[本文引用: 1]

ZHENG

, DING J

, LI Y

, LIN

, XU C

, LI Q

, MAO L

. The effects of soil water content on N₂O emissions and isotopic signature of nitrification and denitrification
Scientia Agricultura Sinica, 2017,50(24):4747-4758. (in Chinese)

[本文引用: 1]

[14]

?UHEL

, ?IMEK

, LAΜGHLIN

R J

, BRU

, CHENEBY

, WATSON

, PHILIPPOT

. Insights into the effect of soil pH on N₂O and N₂ emissions and denitrifier community size and activity
Applied & Environmental Microbiology, 2010,76(6):1870-1878.

DOI:10.1128/AEM.02484-09 URLPMID:20118356 [本文引用: 1]

The objective of this study was to investigate how changes in soil pH affect the N(2)O and N(2) emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H(2)SO(4) solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N(2)O and N(2) emissions using the (15)N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N(2)O/(N(2)O + N(2)) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N(2)O production were observed. Denitrification activity and N(2)O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N(2)O/(N(2)O + N(2)) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.

[15]

HANG-WEI

, DELI

, JI-ZHENG

. Microbial regulation of terrestrial nitrous oxide formation: understanding the biological pathways for prediction of emission rates
FEMS Microbiology Reviews, 2015,39(5):729-749.

DOI:10.1093/femsre/fuv021 URLPMID:25934121 [本文引用: 1]

The continuous increase of the greenhouse gas nitrous oxide (N2O) in the atmosphere due to increasing anthropogenic nitrogen input in agriculture has become a global concern. In recent years, identification of the microbial assemblages responsible for soil N2O production has substantially advanced with the development of molecular technologies and the discoveries of novel functional guilds and new types of metabolism. However, few practical tools are available to effectively reduce in situ soil N2O flux. Combating the negative impacts of increasing N2O fluxes poses considerable challenges and will be ineffective without successfully incorporating microbially regulated N2O processes into ecosystem modeling and mitigation strategies. Here, we synthesize the latest knowledge of (i) the key microbial pathways regulating N2O production and consumption processes in terrestrial ecosystems and the critical environmental factors influencing their occurrence, and (ii) the relative contributions of major biological pathways to soil N2O emissions by analyzing available natural isotopic signatures of N2O and by using stable isotope enrichment and inhibition techniques. We argue that it is urgently necessary to incorporate microbial traits into biogeochemical ecosystem modeling in order to increase the estimation reliability of N2O emissions. We further propose a molecular methodology oriented framework from gene to ecosystem scales for more robust prediction and mitigation of future N2O emissions.

[16]

SHUKRA RAJ

, OHKYUNG

, SAMIR KUMAR

, KARTIK

, SUNGPYO

, JAW WOO

. Effects of temperature on nitrous oxide (N2O) emission from intensive aquaculture system
Science of the Total Environment, 2015,518-519:16-23.

DOI:10.1016/j.scitotenv.2015.02.076 URLPMID:25747359 [本文引用: 1]

This study examines the effects of temperature on nitrous oxide (N2O) emissions in a bench-scale intensive aquaculture system rearing Koi fish. The water temperature varied from 15 to 24 °C at interval of 3 °C. Both volumetric and specific rate for nitrification and denitrification declined as the temperature decreased. The concentrations of ammonia and nitrite, however, were lower than the inhibitory level for Koi fish regardless of temperature. The effects of temperature on N2O emissions were significant, with the emission rate and emission factor increasing from 1.11 to 1.82 mg N2O-N/d and 0.49 to 0.94 mg N2O-N/kg fish as the temperature decreased from 24 to 15 °C. A global map of N2O emission from aquaculture was established by using the N2O emission factor depending on temperature. This study demonstrates that N2O emission from aquaculture is strongly dependent on regional water temperatures as well as on fish production.

[17]

LAI T

, FARQUHARSON

, DENTON M

. High soil temperatures alter the rates of nitrification, denitrification and associated N₂O emissions
Journal of Soils and Sediments. 2019,19(5):2176-2189.

[本文引用: 1]

[18]

BOUWMAN A

, BEUSEN

A H W

, GRIFFIOEN

, VAN GROENIGEN

J W

, HEFTING

M M

, OENEMA

, VAN PUIJENBROEK

P J T M

, SEITZINGE

R S

, SLOMP

C P

, STEHFEST

. Global trends and uncertainties in terrestrial denitrification and N₂O emissions
Philosophical Transactions of the Royal Society of London, 2013,368(1621):91-97.

DOI:10.1098/rstb.2013.0112 URLPMID:23713114 [本文引用: 1]

Soil nitrogen (N) budgets are used in a global, distributed flow-path model with 0.5° × 0.5° resolution, representing denitrification and N2O emissions from soils, groundwater and riparian zones for the period 1900-2000 and scenarios for the period 2000-2050 based on the Millennium Ecosystem Assessment. Total agricultural and natural N inputs from N fertilizers, animal manure, biological N2 fixation and atmospheric N deposition increased from 155 to 345 Tg N yr(-1) (Tg = teragram; 1 Tg = 10(12) g) between 1900 and 2000. Depending on the scenario, inputs are estimated to further increase to 408-510 Tg N yr(-1) by 2050. In the period 1900-2000, the soil N budget surplus (inputs minus withdrawal by plants) increased from 118 to 202 Tg yr(-1), and this may remain stable or further increase to 275 Tg yr(-1) by 2050, depending on the scenario. N2 production from denitrification increased from 52 to 96 Tg yr(-1) between 1900 and 2000, and N2O-N emissions from 10 to 12 Tg N yr(-1). The scenarios foresee a further increase to 142 Tg N2-N and 16 Tg N2O-N yr(-1) by 2050. Our results indicate that riparian buffer zones are an important source of N2O contributing an estimated 0.9 Tg N2O-N yr(-1) in 2000. Soils are key sites for denitrification and are much more important than groundwater and riparian zones in controlling the N flow to rivers and the oceans.

[19]

JONES D

, NGUYEN

, FINLAY R

. Carbon flow in the rhizosphere: Carbon trading at the soil-root interface
Plant & Soil. 2009,321(1/2):5-33.

[本文引用: 1]

[20]

BAIS H

, WEIR T

, PERRY L

, GILROY

, VIVANCO J

. The role of root exudates in rhizosphere interactions with plants and other organisms
Annual Review of Plant Biology, 2006,57(1):233-266.

[本文引用: 1]

[21]

HAICHAR F E

, SANTAELLA

, HEULIN

, ACHOUAK

. Root exudates mediated interactions belowground
Soil Biology & Biochemistry, 2014,77(7):69-80.

[本文引用: 1]

[22]

BATEMAN E

, BAGGS E

. Contributions of nitrification and denitrification to N₂O emissions from soils at different water-filled pore space
Biology & Fertility of Soils, 2005,41(6):379-388.

[本文引用: 1]

[23]

, WANG

, HE

, ZHANG

, LIANG

, SHEN

. Effects of root exudates on denitrifier gene abundance, community structure and activity in a micro-polluted constructed wetland
Science of the Total Environment, 2017,598:697-703.

DOI:10.1016/j.scitotenv.2017.04.150 URLPMID:28456121 [本文引用: 1]

In micro-polluted constructed wetland (CW), the low pollutant concentrations and the low COD/N ratios (chemical oxygen demand: total nitrogen in influent), make the biological treatment more difficult. It is expected that root exudates drive microbial-based transformations within plant rhizosphere. In this research, the roles of root exudates of three aquatic plants (Phragmites australis, Typha angustifolia and Cyperus alternifolius) in improving the growth of heterotrophic denitrifying bacteria were determined in a micro-polluted CW. In studied root rhizospheres, the total organic carbon (TOC) released from the plant roots varied significantly among plant species and seasons; the average TOC ranged from 0.1715 to 0.9221mgg^-1rootDMd^-1, which could fuel a denitrification rate of approximately 156-841kgNO₃^--Nha^-1year^-1 if all were used by the denitrifying bacteria; the abundances of nirK- and nirS-encoding bacteria were significantly influenced by the concentration of sucrose and glucose (0.869≤r≤0.933, p<0.05), and microbial community richness and diversity had response to root exudates. The results revealed that root exudates can act as endogenous carbon sources for heterotrophic denitrifying bacteria and ultimately determine the microbe distribution patterns in micro-polluted CW.

[24]

马舒坦, 颜晓元 . 甲酸盐和葡萄糖对两种土壤N₂O排放的刺激作用
农业环境科学学报, 2019,38(1):241-248.

[本文引用: 2]

MA S

, YAN X

. Effect of formate and glucose organic carbon on N₂O emission from two soils
Journal of Agro-Environment Science, 2019,38(1):241-248. (in Chinese)

[本文引用: 2]

[25]

LAPALANGARICA FUENTES

, MANRUBIA

, GILES M

, MITCHELL

, DANIELL T

. Effect of model root exudate on denitrifier community dynamics and activity at different water-filled pore space levels in a fertilised soil
Soil Biology & Biochemistry, 2018,120:70-79.

[本文引用: 2]

[26]

徐卫红, 刘吉振, 黄河, 熊治廷 . 高锌胁迫下不同大白菜品种生长、Zn吸收及根系分泌物的研究
中国农学通报, 2006,22(8):458.

[本文引用: 2]

XU W

, LIU J

, HUANG

, XIONG Z

. Study of Zn stress on plant growth, Zn uptake and root exudates in different cultivars of Chinese Cabbage
Chinese Agricultural Science Bulletin, 2006,22(8):458. (in Chinese)

[本文引用: 2]

[27]

YUAN Y

, ZHAO W

, ZHANG Z

, XIAO

, LI D

, LIU

, YIN H

. Impacts of oxalic acid and glucose additions on N transformation in microcosms via artificial roots
Soil Biology & Biochemistry, 2018,121:16-23.

[本文引用: 1]

[28]

BOL

, R?CKMANN

, BLACKWELL

, YAMULKI

. Influence of flooding on δ¹⁵N, δ18O, ¹δ¹⁵N and ²δ¹⁵N signatures of N₂O released from estuarine soils-A laboratory experiment using tidal flooding chambers
Rapid Communications in Mass Spectrometry, 2004,18(14):1561-1568.

DOI:10.1002/rcm.1519 URLPMID:15282780 [本文引用: 2]

The influence of flooding on N2O fluxes, denitrification rates, dual isotope (delta18O and delta15N) and isotopomer (1delta15N and 2delta15N) ratios of emitted N2O from estuarine intertidal zones was examined in a laboratory study using tidal flooding incubation chambers. Five replicate soil cores were collected from two differently managed intertidal zones in the estuary of the River Torridge (North Devon, UK): (1) a natural salt marsh fringing the estuary, and 2 a managed retreat site, previous agricultural land to which flooding was restored in summer 2001. Gas samples from the incubated soil cores were collected from the tidal chamber headspaces over a range of flooding conditions, and analysed for the delta18O, delta15N, 1delta15N and 2delta15N values of the emitted N2O. Isotope signals did not differ between the two sites, and nitrate addition to the flooding water did not change the isotopic content of emitted N2O. Under non-flooded conditions, the isotopic composition of the emitted N2O displayed a moderate variability in delta18O and 2delta15N delta values that was expected for microbial activity associated with denitrification. However, under flooded conditions, half of the samples showed strong and simultaneous depletions in 1delta15N and delta18O values, but not in 2delta15N. Such an isotope signal has not been reported in the literature, and it could point towards an unidentified N2O production pathway. Its signature differed from denitrification, which was generally the N2O production pathway in the salt marsh and the managed retreat site.

[29]

HU G

, HE H

, ZHANG

, ZHAO J

, CUI J

, LI

, ZHANG X

. The transformation and renewal of soil amino acids induced by the availability of extraneous C and N
Soil Biology & Biochemistry, 2016,96:86-96.

[本文引用: 3]

[30]

HENRY

, TEXIER

, HALLET

, BRU

, DAMBREVILLE

, CHèNEBY

, BIZOUARD

, GERMON

J C

, PHILIPPOT

. Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: Insight into the role of root exudates
Environmental Microbiology. 2010,10(11):3082-3092.

DOI:10.1111/j.1462-2920.2008.01599.x URLPMID:18393993 [本文引用: 3]

To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.

[31]

OBURGER

, JONES D

. Sampling root exudates - mission impossible?
Rhizosphere, 2018,6:116-133.

[本文引用: 1]

[32]

TOYODA

, YOSHIDA

. Determination of nitrogen isotopomers of nitrous oxide on a modified isotope ratio mass spectrometer
Analytical Chemistry, 1999,71(20):4711-4718.

[本文引用: 3]

[33]

OPDYKE M

, OSTROM N

, OSTROM P

. Evidence for the predominance of denitrification as a source of N₂O in temperate agricultural soils based on isotopologue measurements
Global Biogeochemical Cycles, 2009,23(4). DOI: 10.1029/2009GB003523.

[本文引用: 1]

[34]

房福力, 李玉中 . 基于同位素特征的华北平原菜地N₂O排放监测中取样时间的探讨
植物营养与肥料学报, 2016,22(4):978-987.

[本文引用: 1]

FANG F

, LI Y

. Preliminary research on N₂O sampling time based on isotopomer signature measurement of vegetable fields in the North China Plain
Journal of Plant Nutrition and Fertilizers, 2016,22(4):978-987. (in Chinese)

[本文引用: 1]

[35]

TOYODA

, MUTOBE

, YAMAGISHI

, YOSHIDA

, TANJI

. Fractionation of N₂O isotopomers during production by denitrifier
Soil Biology & Biochemistry, 2005,37(8):1535-1545.

[本文引用: 2]

[36]

DECOCK

, SIX

. How reliable is the intramolecular distribution of ¹⁵N in N₂O to source partition N₂O emitted from soil?
Soil Biology & Biochemistry, 2013,65:114-127.

[本文引用: 1]

[37]

ZHANG

, LI Y

, XU C

, LI Q

, LIN

. Isotope signatures of N₂O emitted from vegetable soil: Ammonia oxidation drives N₂O production in NH+ 4-fertilized soil of North China
Scientific Reports, 2016,6:29257.

DOI:10.1038/srep29257 URLPMID:27387280 [本文引用: 1]

Nitrous oxide (N2O) is a potent greenhouse gas. In North China, vegetable fields are amended with high levels of N fertilizer and irrigation water, which causes massive N2O flux. The aim of this study was to determine the contribution of microbial processes to N2O production and characterize isotopic signature effects on N2O source partitioning. We conducted a microcosm study that combined naturally abundant isotopologues and gas inhibitor techniques to analyze N2O flux and its isotopomer signatures [δ(15)N(bulk), δ(18)O, and SP (intramolecular (15)N site preference)] that emitted from vegetable soil after the addition of NH4(+) fertilizers. The results show that ammonia oxidation is the predominant process under high water content (70% water-filled pore space), and nitrifier denitrification contribution increases with increasing N content. δ(15)N(bulk) and δ(18)O of N2O may not provide information about microbial processes due to great shifts in precursor signatures and atom exchange, especially for soil treated with NH4(+) fertilizer. SP and associated two end-member mixing model are useful to distinguish N2O source and contribution. Further work is needed to explore isotopomer signature stability to improve N2O microbial process identification.

[38]

TOYODA

, MUTOBE

, YAMAGISHI

, YOSHIDA

, TANJI

. Fractionation of N₂O isotopomers during production by denitrifier
Soil Biology & Biochemistry. 2005,37(8):1535-1545.

[本文引用: 4]

[39]

JUNG M

, WELL

, MIN

, GIESEMANN

, RHEE S

. Isotopic signatures of N₂O produced by ammonia-oxidizing archaea from soils
Isme Journal, 2013,8(5):1115-1125.

DOI:10.1038/ismej.2013.205 URLPMID:24225887 [本文引用: 2]

YAMAZAKI

, HOZUKI

, ARAI

, TOYODA

, KOBA

, FUJIWARA

. Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria
Biogeosciences, 2014,11(10):2679-2689.

DOI:10.5194/bg-11-2679-2014 URL [本文引用: 2]

[41]

BRAKER

, LEWICKASZCZEBAK

. Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification a pure culture study
Rapid Communications in Mass Spectrometry, 2014,28(17):1893-1903.

DOI:10.1002/rcm.6975 URLPMID:25088133 [本文引用: 2]

The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria.

[42]

张仲新, 李玉娥, 华珞, 万运帆, 姜宁宁 . 不同施肥量对设施菜地N₂O排放通量的影响
农业工程学报, 2010,26(5):269-275.

URL [本文引用: 1]

To identify the characteristics of N2O emission from protected vegetable land in Beijing, and to seek a way that decreases N2O emission and increase or keep cucumber yield, with the method of static chamber-gas chromatograph technique, N2O emission was monitored in cucumber field from protected vegetable land in Beijing. The effects of different amounts of fertilization on N2O emission, vegetable yields and economic benefit were analyzed. The results showed that significant temporal variability of N2O flux from all treatments was observed in different growing stages of cucumber. Larger emission happened at the initial stage of the experiment. N2O emission decreased and remained stable with time. At the late stage, a peak emission happened and continued for a long time because of larger amount of top dressing. The order of total N2O emission was: T4 (conventional fertilization + chicken dug，in short “CF+CD”) >T3 (3/4CF+CD) > T1 (1/4CF+CD) > T2 (1/2CF+CD) > Tn (CD) > T0 (Control treatment), and there existed significant difference between treatments. By considering fertilizer rates, N2O emission and cucumber yield, it was concluded that the fertilization rate of T3 (3/4CF+CD) was very reasonable, which could provide basis for applying fertilizer rationally, reducing farm production costs, estimating greenhouse gas emissions from cropland and compiling national greenhouse gases emission inventory

ZHANG Z

, LI Y

, HUA

, WAN Y

, JIANG N

. Effects of different fertilizer levels on N₂O flux from protected vegetable land
Transactions of the Chinese Society of Agricultural Engineering, 2010,26(5):269-275. (in Chinese)

URL [本文引用: 1]

田慎重, 宁堂原, 迟淑筠, 王瑜, 王丙文, 韩惠芳, 李成庆, 李增嘉 . 不同耕作措施的温室气体排放日变化及最佳观测时间
生态学报, 2012,32(3):879-888.

[本文引用: 1]

TIAN S

, NING T

, CHI S

, WANG

, WANG B

, HAN H

, LI C

, LI Z

. Diurnal variations of the greenhouse gases emission and their optimal observation duration under different tillage systems
Acta Ecologica Sinica, 2012,32(3):879-888. (in Chinese)

[本文引用: 1]

[44]

徐钰, 刘兆辉, 石璟, 魏建林, 李国生, 王梅, 江丽华 . 北方设施菜地土壤N₂O排放通量日变化及最佳观测时间确定
中国农业气象, 2016,37(5):505-512.

[本文引用: 1]

, LIU Z

, SHI

, WEI J

, LI G

, WANG

, JIANG L

. Diurnal variation characteristic of nitrous oxide from greenhouse vegetable soil during emission peak and its optimal observation duration
Chinese Journal of Agrometeorology, 2016,37(5):505-512. (in Chinese)

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郑循华, 王明星, 王跃思 . 太湖地区农田NO排放不连续测量最佳时间
环境科学, 2000,21(1):1-6.

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ZHENG X

, WANG M

, WANG Y

. Diurnal variation of NO emission from an ecosystem and the optimum observation time for intermittent flux measurement
Environmental Science, 2000,21(1):1-6. (in Chinese)

[本文引用: 1]

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王琳, 周晓丽, 马银丽, 巨晓棠, 吉艳芝, 张丽娟 . 铵态氮源和碳源对土壤N₂O、CO₂释放的影响
农业资源与环境学报, 2016,33(1):23-28.

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WANG

, ZHOU X

, MA Y

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, JI Y

, ZHANG L

. Effect of ammonium nitrogen source and carbon source on the CO₂ and N₂O emissions of soil
Journal of Agricultural Resources and Environment, 2016,33(1):23-28. (in Chinese)

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李贵桐, 李保国, 黄元仿 . 碳源与底物对不同层次土壤产生N₂O能力的影响
土壤与环境, 2002,11(3):227-231.

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LI G

, LI B

, HUANG Y

. Effects of carbon and substrate on the N₂O productivity of different soil layers
Soil and Environmental Sciences, 2002,11(3):227-231. (in Chinese)

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[48]

NGUYEN

. Rhizodeposition of organic C by plants: Mechanisms and controls. Agronomie, 2003,23:375-396.

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HENRY

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, BIZOUARD

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J C

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. Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: Insight into the role of root exudates
Environmental Microbiology, 2010,10(11):3082-3092.

DOI:10.1111/j.1462-2920.2008.01599.x URLPMID:18393993 [本文引用: 1]

MOTHAPO

, CHEN

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, SHI

. Phylogenetic, taxonomic and functional diversity of fungal denitrifiers and associated N₂O production efficacy
Soil Biology & Biochemistry, 2015,83:160-175.

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KOOL D

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, OENEMA

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, VAN GROENIGEN

J W

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Rapid Communications in Mass Spectrometry, 2010,21(22):3569-3578.

DOI:10.1002/rcm.3249 URLPMID:17935120 [本文引用: 1]

Stable isotope analysis of oxygen (O) is increasingly used to determine the origin of nitrate (NO(3)-) and nitrous oxide (N(2)O) in the environment. The assumption underlying these studies is that the (18)O signature of NO(3)- and N(2)O provides information on the different O sources (O(2) and H(2)O) during the production of these compounds by various biochemical pathways. However, exchange of O atoms between H(2)O and intermediates of the (de)nitrification pathways may change the isotopic signal and thereby bias its interpretation for source determination. Chemical exchange of O between H(2)O and various nitrogenous oxides has been reported, but the probability and extent of its occurrence in terrestrial ecosystems remain unclear. Biochemical O exchange between H(2)O and nitrogenous oxides, NO(2)- in particular, has been reported for monocultures of many nitrifiers and denitrifiers that are abundant in nature, with exchange rates of up to 100%. Therefore, biochemical O exchange is likely to be important in most soil ecosystems, and should be taken into account in source determination studies. Failing to do so might lead to (i) an overestimation of nitrification as NO(3)- source, and (ii) an overestimation of nitrifier denitrification and nitrification-coupled denitrification as N(2)O production pathways. A method to quantify the rate and controls of biochemical O exchange in ecosystems is needed, and we argue this can only be done reliably with artificially enriched (18)O compounds. We conclude that in N source determination studies, the O isotopic signature of especially N(2)O should only be used with extreme caution.

[52]

CASCIOTTI K

, SIGMAN D

, HASTINGS M

, BOHLKE J

, HILKERT

. Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method
Analytical Chemistry, 2002,74(19):4905-4912.

DOI:10.1021/ac020113w URLPMID:12380811 [本文引用: 1]

We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate. (1) Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N20 analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O. Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N20 product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses. The analysis of appropriate isotopic reference materials can thus be used to correct the measured 18O/16O ratios of samples for both effects. This is the first method tested for 18O/16O analysis of nitrate in seawater. Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 microM NO3-), lack of interference by other solutes, and ease of sample preparation.

[53]

ROHE

, WELL

, LEWICKA SZCZEBAK

. Use of oxygen isotopes to differentiate between nitrous oxide produced by fungi or bacteria during denitrification
Rapid Communications in Mass Spectrometry, 2017,31(16):1297-1312.

DOI:10.1002/rcm.7909 URLPMID:28556299 [本文引用: 1]

Fungal denitrifiers can contribute substantially to N₂ O emissions from arable soil and show a distinct site preference for N₂ O (SP(N₂ O)). This study sought to identify another process-specific isotopic tool to improve precise identification of N₂ O of fungal origin by mass spectrometric analysis of the N₂ O produced.

[54]

PéREZ

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S C

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P A

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D W T

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Journal of Geophysical Research: Atmospheres, 2001,106(9):9869-9878.

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[55]

HYODO

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, MUSHINSKI R

, WEST J

. Biochar amendment suppresses N₂O emissions but has no impact on ¹⁵N site preference in an anaerobic soil
Rapid Communications in Mass Spectrometry, 2019,33(2):165-175.

DOI:10.1002/rcm.8305 URLPMID:30304571 [本文引用: 2]

Biochar amendments often decrease N₂ O gas production from soil, but the mechanisms and magnitudes are still not well characterized since N₂ O can be produced via several different microbial pathways. We evaluated the influence of biochar amendment on N₂ O emissions and N₂ O isotopic composition, including ¹⁵ N site preference (SP) under anaerobic conditions.

[56]

郑欠 . 土壤水分和pH对N₂O排放及同位素特征值影响的机理研究
[D]. 北京: 中国农业科学院, 2018.

[本文引用: 1]

ZHENG

. The study on mechanism of N₂O emissions and isotopic signature under different soil water content and pH
[D]. Beijing: Chinese Academy of Agricultural Sciences, 2018. (in Chinese)

[本文引用: 1]

[57]

SPEIR T

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, MORE R

. Aerobic emissions of N₂O and N₂ from soil cores: Factors influencing production from ¹³N-labelled NO₃ - and NH₄ +
Soil Biology & Biochemistry, 1995,27(10):1299-1306.

[本文引用: 1]

[58]

HENDERSON S

, DANDIE C

, GOYER

, PATTEN

, ZEBARTH B

, BURTON D

, TREVORS J

. Changes in denitrifier abundance, denitrification gene mRNA levels, nitrous oxide emissions, and denitrification in anoxic soil microcosms amended with glucose and plant residues
Applied and Environmental Microbiology, 2010,76:2155-2164.

DOI:10.1128/AEM.02993-09 URLPMID:20154105 [本文引用: 2]

In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N(2)O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirS(P)) increased only in glucose-amended soil: the nirS(P) guild abundance increased 5-fold over the 72-h incubation period (P &lt; 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P &lt; 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 x 10(7) gene copies and 6.9 x 10(7) transcripts g(-1) dry soil. Cumulative N(2)O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirS(P) and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios.

[59]

贾俊仙, 李忠佩, 车玉萍 . 添加葡萄糖对不同肥力红壤性水稻土氮素转化的影响
中国农业科学, 2010,43(8):1617-1624.

URL [本文引用: 1]

【Objective】 In order to understand the mechanisms of N transformation and manage N fertilization better, the effect of glucose addition on N transformation in paddy soils with a gradient of organic C content were determined. 【Method】 Changes of mineralization, nitrification and denitrification in paddy soils, as well as their response to glucose addition were measured by incubation experiment. 【Result】 Intensity of mineralization and denitrification were: soils with high fertility ＞ soils with middle fertility ＞ soils with low fertility. During the first week of incubation, net mineralization and denitrification rate in paddy soils with high fertility were 1.9 and 1.1 times of that in soil with middle fertility and 5.3 and 2.9 times of that in soils with low fertility, respectively. Addition of glucose decreased approx. 78.8%, 109.2%, and 177.4% of net mineralization in soils with high, middle, and low fertility, respectively. However, denitrification rate in soils with middle and low fertility increased by 14.4% and 166.2%. The highest nitrate content in all the tested soils was 0.62 mg•kg-1 and the highest nitrification ratio was 0.33%. Addition of glucose had no obvious effects on nitrate content and nitrification ratio. 【Conclusion】 It was suggested that the intensity of mineralization and denitrification were quite different in soils with different fertility. The intensity was increased with the increase of soil organic C content. Addition of glucose decreased mineralization, but increased denitrification. The effects of glucose addition were significantly different in different soils. The effect of glucose addition on soils with low organic C content were greater than that on soils with high organic C content. Neither addition of glucose nor inherent soil organic C had obvious effects on nitrification of test paddy soils.

JIA J

, LI Z

, CHE Y

. Effects of glucose addition on N transformations in paddy soils with a gradient of organic C content in subtropical China
Scientia Agricultura Sinica, 2010,43(8):1617-1624. (in Chinese)

URL [本文引用: 1]

崔敏, 冉炜, 沈其荣 . 水溶性有机质对土壤硝化作用过程的影响
生态与农村环境学报, 2006,22(3):45-50.

URL [本文引用: 1]

Effect of addition of dissolved organic matter （DOM） on nitrification in fluvo-aquic soil was studied.The soil used in the experiment was collected from Suqian,Jiangsu.The experiment had three treatments different in DOM addition rate,220,440 and 880 mg·L^-1,respectively.Results show that addition of DOM inhibited nitrification to a certain extent.After 16 days of incubation,nearly 100% of NH⁺₄-N in CK had changed into NO^-₃-N.Compared with CK,the three treatments,DOC220,DOC440 and DOC880,decreased by 7.83%,13.60% and 19.12%,respectively in nitrification,and hence the decrease in NH⁺₄-N concentration in the treatments was much slower than in CK.The addition of DOM,however,increased nitrite accumulation significantly.In CK,NO^-₂-N concentration peaked on D 12 in incubation,up to 67.83(mg·L^-1),whereas in Treatment DOC220,DOC440 and DOC880,it did on D 12,14 and 16,and 21.17%,33.91% and 59.90% higher,respectively.The addition of DOM also decreased the production rate of NO^-₃-N.The maximum concentration of NO^-₃-N,143.61 mg·L^-1,in CK was found on D 16,while only 41.97,78.09 and 91.30 mg·L^-1 in Treatment DOC220,DOC440 and DOC880 respectively,much lower than in CK.In order to eliminate the effect of extraneous nitrogen brought in with the addition of DOM,comparison was made of effects of DOM addition and application of（NH₄）₂SO₄ as the sole nitrogen source on nitrification in soils with same initial nitrogen concentration.Results also show increase in NO^-₂-N accumulation and decrease in NO^-₃-N production,which indicate that the organic carbon and low molecular organic compounds in DOM affected the process of nitrification.The findings of the experiment implied a high risk of NO^-₂-N accumulation existed in environments high in organic matter content,which may have some bad effects on the health of the terrestrial and aquatic systems.

CUI

, RAN

, SHEN Q

. Effects of dissolved organic matter on nitrification in soil
Journal of Ecology and Rural Environment, 2006,22(3):45-50. (in Chinese)

URL [本文引用: 1]

王风, 陈思, 杨厚花, 沈仕洲, 张克强, 王晓光 . 葡萄糖添加对室温和冻结过程土壤N₂O排放特征影响
生态科学, 2017,36(3):31-35.

[本文引用: 1]

WANG

, CHEN

, YANG H

, SHEN S

, ZHANG K

, WANG X

. Effect of glucose addition on N₂O emission from three types of cultivated soils under ambient and freezing temperature
Ecological Science, 2017,36(3):31-35. (in Chinese)

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姜宁宁, 李玉娥, 华珞, 万运帆, 石生伟 . 不同氮源及秸秆添加对菜地土壤N₂O排放影响
土壤通报, 2012,43(1):219-223.

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JANG N

, LI Y

, HUA

, WAN Y

, SHI S

. Effect of different nitrogen sources and straw adding on N₂O emission from vegetable soil
Chinese Journal of Soil Science, 2012,43(1):219-223. (in Chinese)

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林伟, 张薇, 李玉中, 徐春英, 徐春英, 李巧珍, 郑欠 . 有机肥与无机肥配施对菜地土壤N₂O排放及其来源的影响
农业工程学报, 2016,32(19):148-153.

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LIN

, ZHANG

, LI Y

, XU C

, LI Q

, ZHENG

. Effects of combined application of manure and inorganic fertilizer on N₂O emissions and sources in vegetable soils
Transactions of the Chinese Society of Agricultural Engineering, 2016,32(19):148-153. (in Chinese)

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林伟, 丁军军, 李玉中, 徐春英, 李巧珍, 郑欠, 庄姗 . 有机肥和无机肥对菜地土壤N₂O排放及其来源的影响
应用生态学报, 2018,29(5):100-108. (in Chinese)

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LIN

, DING J

, LI Y

, XU C

, LI Q

, ZHENG

, ZHUANG

. Effects of organic and inorganic fertilizers on emission and sources of N₂O in vegetable soils
Chinese Journal of Applied Ecology, 2018,29(5):100-108. (in Chinese)

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MCLAIN J E

, MARTENS D

. Nitrous oxide flux from soil amino acid mineralization
Soil Biology & Biochemistry, 2005,37(2):289-299.

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CLARHOLM

, SKYLLBERG

, ROSLING

. Organic acid induced release of nutrients from metal-stabilized soil organic matter- The unbutton model
Soil Biology & Biochemistry, 2015,84:168-176.

DOI:10.1016/j.soilbio.2015.02.019 URL [本文引用: 1]

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KEILUWEIT

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J J

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P S

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RIDGE J

, KLEBER

. Mineral protection of soil carbon counteracted by root exudates. Nature Climate Change, 2015,5(6):588-595.

DOI:10.1038/nclimate2580 URL [本文引用: 1]

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MALY

, KRáLOVEC

, HAMPEL

. Effects of long-term mineral fertilization on microbial biomass, microbial activity, and the presence of R and K -strategists in soil
Biology & Fertility of Soils, 2009,45(7):753-760.

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LIU

, BAKKEN L

, FROSTEGARD

. Denitrification gene pools, transcription and kinetics of NO, N₂O and N₂ production as affected by soil pH
Fems Microbiology Ecology, 2010,72(3):407-417.

DOI:10.1111/j.1574-6941.2010.00856.x URLPMID:20370831 [本文引用: 1]

The N(2)O : N(2) product ratio of denitrification is negatively correlated with soil pH, but the mechanisms involved are not clear. We compared soils from field experiments where the pH had been maintained at different levels (pH 4.0-8.0) by liming (&gt; or = 20 years), and quantified functional gene pools (nirS, nirK and nosZ), their transcription and gas kinetics (NO, N(2)O and N(2)) of denitrification as induced by anoxic incubation with and without a carbon substrate (glutamate). Denitrification in unamended soil appeared to be based largely on the activation of a pre-existing denitrification proteome, because constant rates of N(2) and N(2)O production were observed, and the transcription of functional genes was below the detection level. In contrast, glutamate-amended soils showed sharp peaks in the transcripts of nirS and nosZ, increasing the rates of denitrification and pH-dependent transient accumulation of N(2)O. The results indicate that the high N(2)O : N(2) product ratio at low pH is a post-transcriptional phenomenon, because the transcription rate of nosZ relative to that of nirS was higher at pH 6.1 than at pH 8.0. The most plausible explanation is that the translation/assembly of N(2)O reductase is more sensitive to low pH than that of the other reductases involved in denitrification.