土壤氮气排放研究进展*

张志君, 秦树平**, 袁海静, 张玉铭, 胡春胜



土壤氮气排放研究进展*

张志君1, 秦树平1**, 袁海静1, 张玉铭2, 胡春胜2

(1. 福建省土壤环境健康与调控重点实验室/福建农林大学资源与环境学院 福州 350002; 2. 中国科学院遗传与发育生物学研究所农业资源研究中心 石家庄 050022)

自20世纪初人类发明并掌握工业合成氨的技术以来, 氮肥施用量迅速增长。在一部分国家或地区, 氮肥的施入量已经超过作物对氮素的需求, 导致大量氮素损失到环境中, 造成氨挥发、氧化亚氮排放、地下水硝酸盐污染等环境问题。土壤在微生物的作用下可以通过反硝化、厌氧氨氧化等过程将活性氮素转化为惰性氮气, 达到清除过多活性氮的目的。由于大气中氮气背景浓度太高, 因此很难直接准确测定土壤的氮气排放速率, 导致土壤氮气排放通量、过程与调控机制研究远远落后于土壤氮循环的其他方面。本文综述了土壤氮气排放主要途径(反硝化、厌氧氨氧化与共反硝化)及其对土壤氮气排放的贡献; 测定土壤氮气排放速率的方法(乙炔抑制法、氮同位素示踪法、N2/Ar比率-膜进样质谱法、氦环境法与N2O同位素自然丰度法)及其优缺点; 调控土壤氮气排放通量的主要因素(氧气、可溶性有机碳、硝酸盐、微生物群落结构与功能基因表达等)及其相关作用机制。最后指出研发新的测定原位无扰动土壤氮气通量的方法是推进本领域相关研究的关键; 定量典型生态系统(如旱地农田、稻田、森林、草地与湿地)土壤氮气排放通量, 阐明其中的微生物学机制, 模拟并预测土壤氮气排放对全球变化的响应规律是本领域的研究热点与发展方向。

土壤; 氮气排放; 反硝化; 厌氧氨氧化; 氧化亚氮排放; 氮损失

土壤氮气排放是指活性氮(主要是指硝酸根、亚硝酸根、铵与氧化亚氮等)在微生物的作用下转化为惰性氮(氮气)的过程。这一过程在生态系统氮循环中具有重要作用, 它是氮循环的最后一步, 是闭合氮循环的关键过程, 影响生态系统活性氮归趋与氮素平衡。对于农业生态系统来说, 土壤氮气排放与氮肥损失及温室气体氧化亚氮的排放密切相关。大气中的氮气浓度很高(体积浓度约78%), 在如此高的氮气背景浓度下准确测定土壤氮气排放一直是国际土壤学领域的方法学难题。相对于土壤氮素循环的其他环节, 氮气排放方面的研究还十分薄弱。本文主要从土壤氮气产生与排放的主要途径、主要测定方法学与主要的环境及微生物调控机制3个方面进行综述。

1 土壤氮气产生与排放的主要途径

土壤产氮气的过程主要有反硝化(denitrification)、厌氧氨氧化(anammox)与共反硝化(co-denitrification) 3种途径[1]。土壤反硝化过程是指在微生物的作用下硝酸盐经由亚硝酸盐、一氧化亚氮、氧化亚氮等中间产物最终还原为氮气的过程[2-5], 一般认为土壤中反硝化过程主要由反硝化微生物驱动, 化学反硝化过程只在pH值很低的情况下发生[6-10]。厌氧氨氧化过程是指微生物在厌氧条件下利用铵盐为电子供体, 亚硝酸盐为电子受体, 最终产生氮气的过程[11-14]。除亚硝酸盐外, 近年来又发现一些微生物能利用其他电子受体, 如Fe(Ⅲ), 进行厌氧氨氧化产氮气, 称为铁厌氧氨氧化(feanammox)[15]。Ding等[16]将耕种年代不同序列的水稻土作为研究对象, 第一次在水稻土中验证了铁厌氧氨氧化现象的出现, 并提出氮气是该过程的主要产物。共反硝化是指微生物利用反硝化的中间产物亚硝酸盐为电子受体, 以铵、羟胺、叠氮化物等为电子供体, 以氮气为最终产物的过程[17]。在以上3种途径中, 反硝化过程被认为是土壤氮气产生的主要途径。厌氧氨氧化在高度厌氧的环境中, 如底泥与稻田土壤中对氮气排放具有一定贡献[18-19]。共反硝化在草地等真菌较多的生态系统中对氮气排放具有一定作用[17]。

2 土壤氮气排放的主要测定方法

受到大气中氮气高背景浓度的影响, 直接测定土壤氮气排放通量非常困难[20]。目前使用较多的主要有乙炔抑制法、氮同位素示踪法、N2/Ar比率-膜进样质谱法、氦环境法与N2O同位素自然丰度法[21]。

2.1 乙炔抑制法

乙炔抑制法的原理是利用高浓度乙炔(体积浓度>1%, 通常为10%)抑制土壤中氧化亚氮还原酶的活性, 进而阻止氧化亚氮还原为氮气, 最后通过测定氧化亚氮的累积量来间接表征土壤氮气排放通量[22-23]。该方法具有简单易操作等优点。因此该方法在过去的30年中被广泛用于土壤反硝化速率及氮气排放通量的测定, 积累了大量田间试验数据[24-25]。但是乙炔抑制法具有难以克服的缺点: 第一, 高浓度乙炔在抑制土壤氧化亚氮还原酶活性的同时会对土壤硝化过程产生抑制作用, 导致反硝化的底物——硝酸盐的供给减少[26-27]。第二, 一部分反硝化微生物对乙炔不敏感, 这些微生物在碳含量较高而硝酸盐浓度较低(<10 μmol∙L-1)的环境下能够促进氧化亚氮还原为氮气[28-29]。2010年, Zhong等[30]研究中国太湖梅梁湾表层沉积物潜在的反硝化速率的季节性变化, 发现沉积物反硝化速率在春季最高, 夏季和秋季最低, 主要是由于硝酸盐浓度和水温的季节差异,并且限制反硝化速率的关键因素是硝酸盐。夏季和秋季, 水体中的硝酸盐已被耗尽, 大大抑制了太湖反硝化作用对氮的有效去除。第三, 乙炔很难均匀地扩散到土壤中, 如果有一部分土壤空隙中没有乙炔, 则会导致该处氧化亚氮被还原为氮气[31]。2008年, Woodward等[32]通过研究河岸地带的土壤, 发现反硝化作用不是硝酸盐损失的主要途径, 仅占地下水中硝酸盐去除率的3%, 所以使用乙炔抑制法测定河岸地带的土壤反硝化速率会造成一定的低估, 主要因为河岸土壤较高的含水量阻碍了乙炔的扩散。第四, 一些微生物会将乙炔当作碳源而将其分解利用, 导致土壤中的乙炔浓度降低, 影响其对氧化亚氮还原的抑制作用[28]。最后, 在好氧条件下, 高浓度乙炔(>0.1 kPa)会促使反硝化的中间产物一氧化氮分解或者催化其氧化为二氧化氮, 导致氮气排放通量低估[33]。1997年, Bollmann等[34]采用乙炔抑制技术测定了29种土壤的反硝化速率、一氧化氮的生成量和氧化速率以及氧化亚氮的净释放速率。他们在10 Pa乙炔条件下测定了一氧化氮和氧化亚氮排放速率, 发现乙炔抑制了硝化作用而不是反硝化作用, 并且在乙炔浓度大于0.1 kPa时证明了乙炔能够增强对一氧化氮氧化的催化作用, 产生的二氧化氮被土壤吸收, 不能再进一步转换为氧化亚氮, 从而造成对反硝化速率的低估。对乙炔抑制法的上述潜在缺点, 很多文献进行了理论上的定性分析[35-37]。近年来, 一些学者进一步用直接定量氮气的方法对乙炔抑制法的系统误差进行了定量研究。2013年, Qin等[38]发现在厌氧条件下不同土壤基本理化性质之间的差异对乙炔抑制法的系统误差也会造成影响,高浓度乙炔(>0.1 kPa)无法完全抑制土壤氧化亚氮还原酶活性, 导致乙炔抑制法测定的土壤反硝化潜势比直接定量氮气法偏低8%~98%, 同时乙炔抑制法的系统误差与土壤有机质与养分含量呈显著负相关关系。这些结果表明: 对于有机质与养分含量较高的土壤, 如农田土壤, 乙炔抑制法能够较准确地表征土壤反硝化潜势; 而对于有机质含量低的贫瘠土壤, 如荒漠土壤, 使用乙炔抑制法测定土壤反硝化潜势会带来较大的误差｡另外一些学者将乙炔抑制法与其他氮同位素示踪法进行了比较研究, 也发现乙炔抑制法存在负偏差[35]。上述研究表明: 乙炔抑制法虽然具有操作简单, 费用低廉的优点, 但具有难以克服的内在缺陷。在使用乙炔抑制法测定氮气通量时要慎重考虑其系统误差[39]。

2.2 氮同位素示踪法

氮同位素示踪法的原理是将15N高丰度的硝酸盐施入土壤, 利用同位素质谱仪测定经由反硝化产生的氮气同位素丰度推算土壤氮气排放通量[40-41]。氮同位素示踪法始于20世纪50年代末, 已被广泛应用于各种土壤类型的反硝化速率及氮气排放通量的测定[42-46]。氮同位素示踪法也存在一些缺点: 第一, 标记的硝酸盐很难均匀扩散到土壤中, 因此无法保证新加入的标记硝酸盐与土壤本底硝酸盐是否混合均匀[20]。但Steingruber等[47]发现标记硝酸盐和本底硝酸盐的非均匀混合对反硝化速率造成的误差不超过10%, 远远小于试验系统误差。其他研究也发现, 即使土壤中硝酸盐的标记是不均匀的, 仍可以对反硝化产生的氮气做出准确的估计[48]。第二, 土壤微生物优先利用轻质同位素, 这些因素都可能给土壤氮气排放通量的测定带来不确定性[49]。第三, 新加入的标记硝酸盐增加了土壤原有反硝化反应的底物浓度[48], 这对一些氮源缺乏的生态系统来说会显著影响该系统的氮气排放速率, 进而高估土壤氮气排放速率。因此氮同位素示踪法主要运用于氮素含量较高的生态系统, 如农田生态系统[48]。

2.3 N2/Ar比率-膜进样质谱法

N2/Ar比率-膜进样质谱法的原理是利用膜进口质谱仪测定水中的N2/Ar比率来推算覆水土壤中氮气排放速率, 如稻田与底泥中的氮气排放速率。水中的Ar浓度相对稳定, 而水中的N2浓度主要受反硝化等产氮气过程的影响[50-51]。该方法的优点是可以同时测定通过反硝化、厌氧氨氧化、共反硝化等多种途径产生的氮气总排放速率[51]。缺点是该方法难以运用于非覆水土壤中氮气排放速率的测定, 另外该方法只能运用于室内培养的原状土柱, 难以在田间原位土壤中实施[52]。Li等[53]利用N2/Ar比率-膜质谱法对水稻土施肥后0~21 d的原位氮气排放进行了测定, 发现反硝化产生的氮气损失约占氮肥投入量的4.7%。

2.4 氦环境法

该方法的原理是在密闭空间中利用惰性气体氦气置换土壤孔隙中氮气, 人为降低氮气背景浓度, 进而使用气相色谱直接测定土壤排出的微量氮气[54-59]。该方法的优点是可以直接测定土壤排出的氮气通量, 避免了人为添加抑制剂/氮源而对土壤氮气排放通量造成潜在影响[26]。另外该方法可以广泛运用于湿地与旱地原状土柱经由多种途径(反硝化、厌氧氨氧化、共反硝化)产生的氮气通量。该方法的缺点是需要建造高密闭性的培养装置[54], 该装置的气密性直接决定该方法的测定精度。

2.5 田间原位无扰动土壤氮气通量测定方法

目前大部分土壤氮气排放的测定方法, 如乙炔抑制法只局限在室内测定[24]; 氮同位素示踪法适合本底氮素含量较高的土壤[48], 如农田土壤; N2/Ar比率法只适用于具有覆水的条件, 无法测定无覆水条件的湿地或旱地土壤的原位氮气排放速率[53,60]。田间原位土样采集后如何避免溶解态的氮气不释放到气相中以及气相中的氮气不污染水相中的溶解态氮气是需要注意的问题。另外该方法无法测定通过非溶解途径(如气泡)排出土体的氮气, 可能低估氮气排放通量。传统的原状土柱-直接定量氮气法, 由于装置气密性要求较高, 难以测定大直径原状土柱原位反硝化通量[54]。最近秦树平等[61]将原状土柱-直接定量氮气法加以改进, 利用两层密封罐体之间的无氮气夹层来抵消大气中的高浓度氮气的泄露, 将罐内土柱中的气体置换成无氮气的人工合成气(79%氦气, 21%氧气), 最后利用Robot系统测定罐内氮气、氧化亚氮与一氧化氮的浓度变化, 使大直径原状土柱原位氮气通量测定成为可能(双密闭原状土柱-直接定量氮气法)。然而, 双密闭原状土柱-直接定量氮气法也有不可克服的缺点: 首先需要将土柱中的气体置换成低浓度无氮气的人工合成气体(如氦/氧混合气), 这一操作过程可能会对土壤团聚体结构、含水量等因素造成一定潜在影响; 另外密闭环境下会造成培养体系不能与外界进行物质交换, 如: 土柱周围硝酸盐无法扩散进入密闭土柱, 密闭土柱产生的气体无法扩散到大气中, 造成产物的累积, 进而影响土壤氮气排放速率测定的准确度。对于硝酸盐含量较高、厌氧程度较高的湿地土壤, 一种基于氧化亚氮15N同位素自然丰度的方法已经被证明能够用于原位无扰动湿地生态系统的氮气排放通量的测定。Qin等[62]利用自然丰度15N同位素分馏理论与He/N2置换技术, 集成创新了土壤反硝化测定方法体系。该方法的原理是利用在氧化亚氮还原为氮气过程中的自然丰度同位素分馏原理, 将15N同位素丰度值与N2O/N2排放比率之间建立经验函数关系, 利用氧化亚氮通量与N2O同位素的值来推算田间原位无扰动氮气排放通量。该方法的优点是测定过程对被测定系统的扰动很小, 测定值反映田间实际氮气排放情况。缺点是无法测定除反硝化途径以外其他途径产生的氮气;15N同位素丰度值与N2O/N2排放比率建立的经验函数随不同土壤类型及环境条件有一定变异性, 会对试验结果造成一定的误差; 最后, 氧化亚氮稳定同位素自然丰度法目前只证明适用于受氮素面源污染的湿地与河流底泥、稻田等厌氧程度较高, 氮素含量较高的土壤类型, 而对于旱地土壤硝化—反硝化过程来说, 这两种途径产生的氧化亚氮具有不同的氮同位素值范围, 当这两种途径对氧化亚氮产生量的贡献发生变化时, 理论上会导致氧化亚氮15N同位素丰度值产生变化, 进而影响15N同位素丰度值与N2O/N2排放比率建立的经验函数的变化, 从而导致试验误差的出现。

3 土壤氮气排放的环境和微生物调控机制

3.1 氧气

在反硝化、厌氧氨氧化与共反硝化这3种途径中, 反硝化是最早被确认的氮气排放途径, 也是研究最多的一条途径。由于氧化亚氮还原酶需要在厌氧条件下才能保持活性, 因此厌氧环境被认为是反硝化发生的必要条件。传统土壤学理论认为反硝化过程是一个严格的厌氧过程[63], 但是相关研究表明: 在土壤中存在氧气/硝酸盐共呼吸(co-respiration)现象[64], 陆续从土壤中分离到了一些能在好氧条件下产生氮气的反硝化微生物菌株[65-67]。1984年, Robertson等[68]在反硝化处理系统中首次分离出好氧反硝化菌, 并发现了好氧反硝化酶系的存在, 同时证实了在脱氮副球菌()生长过程中, 如果氧气和硝酸盐共同存在, 其生长速率会比二者单独存在时高[68-70]。目前, 越来越多的证据表明: 土壤微生物可能在好氧条件下进行反硝化脱氮[65,71-75]。由于土壤中存在团聚体等厌氧微域, 土壤中的好氧反硝化作用还一直没有被证实。Qin等[76]最近利用低温低速离心, 将土壤微生物与土壤颗粒相分离, 排除潜在的厌氧微域后, 利用前期改进的反硝化研究新方法证实: 土壤微生物能通过好氧反硝化作用产生氮气。上述结果表明: 厌氧环境可能不是土壤反硝化脱氮的必要条件, 反硝化脱氮可能广泛存在于好氧与厌氧环境中。具体的好氧反硝化氮气排放机制还有待于进一步研究。

厌氧氨氧化被认为在高度厌氧的土壤中对氮气排放具有一定作用, 最高可占到氮气排放量的70%[77-78]。Shan等[79]综合运用氮同位素示踪法与N2/Ar比率法测定了中国11种典型水稻土的厌氧氨氧化速率, 发现厌氧氨氧化占总氮气损失比率的4.48%~9.23%。厌氧氨氧化氮气排放速率与土壤可溶性有机碳和硝酸盐含量呈显著正相关关系, 而在旱地土壤中, 厌氧氨氧化对氮气排放的贡献普遍认为比在湿地土壤中低(约0.3%~37%)。值得注意的是, 以往的研究只是测定了土壤通过厌氧氨氧化排放氮气的潜力, 由于培养条件与田间实际条件差异巨大, 因此上述厌氧氨氧化在氮气总排放量中所占的比率可能与田间实际存在较大差异。

3.2 可溶性有机碳与硝酸盐

可溶性有机碳被认为是异养反硝化微生物提供自身增值所需的有机碳源, 同时为微生物提供电子供体进行反硝化作用[80]。Qin等[81-82]通过研究硝酸盐、可溶性有机碳与氧气含量等关键环境因素对土壤反硝化速率及产物构成的调控机制, 发现碳源增加显著提升土壤反硝化速率, 同时降低了反硝化产物中N2O/N2的比率。在深层土壤, 特别是植物根层以下土壤中, 可溶性有机碳含量普遍较低, 可溶性有机碳匮乏而不是反硝化微生物数量是限制深层土壤反硝化氮气排放速率的关键因素。最近又有研究表明: 除可溶性有机碳以外的其他反硝化电子供体, 比如通过电极直接提供电子, 可以强化深层缺碳土壤反硝化氮气排放速率[83]。

硝酸盐是反硝化作用的底物, 大部分文献认为反硝化速率与土壤硝酸盐浓度呈正相关关系。但是高浓度硝酸盐会抑制氧化亚氮还原酶活性[84-86], 导致反硝化产物中N2O/N2比率升高。

3.3 微生物群落结构与功能基因

反硝化其实是由硝酸还原酶基因(、)、亚硝酸还原酶基因(、)、一氧化氮还原酶基因(、)和氧化亚氮还原酶基因()驱动将硝酸盐转换为亚硝酸盐、一氧化氮、氧化亚氮和氮气的过程, 长期以来一直以为氧化亚氮还原酶由典型反硝化细菌具有的基因编码[87]。然而, 近几年却发现土壤中存在大量未知的非典型反硝化细菌, 这些细菌不具有完整的反硝化能力, 只具有氧化亚氮还原能力, 是全世界巨大的氧化亚氮汇[88-89]。2014年, Jones等[89]发现欧洲土壤中的非典型反硝化菌的数量和系统发育多样性决定了欧洲陆地生态系统的氧化亚氮汇的容量。非典型反硝化细菌的基因(CladeⅡ)与典型反硝化细菌的基因(CladeⅠ)在系统发育方面距离比较远, 同时在基因结构和调控方面差异也较大。目前还没有系统地开展土壤氧化亚氮还原细菌生态学的研究, 对典型农田土壤基因CladeⅠ和CladeⅡ群落组成和丰度知之甚少, 土壤氧化亚氮还原菌与氧化亚氮汇效应的关系尚不明确。在草地生态系统中, 土壤真菌可以通过共反硝化作用产生氮气[1,17], 但是目前农业生态系统中真菌共反硝化研究的认知还未完全清晰, 需要更深层次的研究。

4 结论

目前对典型生态系统(如旱地农田、稻田、森林、草地与湿地)的土壤氮气排放通量仍然缺乏田间原位观测数据, 基于室内间接测定的氮气排放通量具有较大不确定性, 与原位通量存在较大差异。土壤氮气排放的研究仍然受制于研究方法的限制, 虽然近年来发展了一些新的技术, 仍然缺乏广谱、可靠与简便的土壤原位氮气排放测定方法。研发新的田间原位无扰动测定技术仍然是土壤氮气排放研究的首要问题。就目前的技术水平来说, 多种方法联合运用是提高土壤氮气排放通量测定精度的一种有效策略。近年来发现的土壤氮气排放新途径(如厌氧氨氧化、共反硝化与好氧氮气排放过程)对土壤氮气排放的相对贡献及相关的微生物学机制是目前研究的热点。典型生态系统土壤氮气排放定量与模拟、未来环境变化(如氮沉降增加、土壤酸化、温度升高等)对土壤氮气排放的影响机制是本领域的重要研究方向。

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Advance in soil dinitrogen emission*

ZHANG Zhijun1, QIN Shuping1**, YUAN Haijing1, ZHANG Yuming2, HU Chunsheng2

(1. Key Laboratory of Soil Environment Health and Regulation in Fujian Province / College of Resources and Environment, Fujian Agricultural and Forestry University, Fuzhou 350002, China; 2. Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050022, China)

The amount of applied nitrogen fertilizer has increased dramatically since the invention of the industrial ammonia synthesis in the early 20th century. In some countries or regions, the amount of nitrogen fertilizer input has exceeded crop nitrogen demand. This has led to a large amount of nitrogen losses to the environment, causing environmental pollution such as ammonia volatilization, nitrous oxide emission and groundwater contamination. Soil microbes can transform active nitrogen into inert dinitrogen and consequently remove superfluous nitrogen from soil via denitrification and anammox. Direct and precise measurement of soil denitrification has been a continuous challenge due to high background concentration of atmospheric dinitrogen, which has hindered progress in research on soil dinitrogen emissions. This paper reviewed the main pathways of soil dinitrogen emission [denitrification, dissimilatory nitrate reduction to ammonium (DNRA) and co-denitrification] and their contributions to soil dinitrogen emission. It also covered the methods of soil dinitrogen flux determination (acetylene inhibition technique,15N tracing method, N2/Ar membrane-inlet mass spectrometry, helium environment method and natural abundance15N2O isotopic method) and their advantages, disadvantages. The key factors regulating soil dinitrogen emission (oxygen, dissolved organic carbon, nitrate, microbial community structure and functional gene expression) and the related mechanisms were also summarized. In conclusion, it was essential to develop new methods fordinitrogen flux determination in undisturbed soils. More studies were needed to quantify soil dinitrogen flux in typical ecosystems (such as dryland, farmland, forest, grassland and wetland), clarify microbial mechanism involved, and simulate and predict the responses of soil dinitrogen emission to global change.

Soil;Dinitrogen emission; Denitrification; Anaerobic ammonia oxidation; Nitrous oxide emission; Nitrogen loss

, E-mail: qinshuping@sjziam.ac.cn

Nov. 21, 2017;

Dec. 4, 2017

10.13930/j.cnki.cjea.171070

X51; S154.1

A

1671-3990(2018)02-0182-08

秦树平, 主要从事土壤反硝化方法学及反硝化脱氮机理研究。E-mail: qinshuping@sjziam.ac.cn 张志君, 主要研究方向为好氧条件下土壤性质对乙炔抑制法测定误差的影响。E-mail: 15895217170@163.com

2017-11-21

2017-12-04

* This study was supported by the National Natural Science Foundation of China (41530859, 41771331, 41571291).

* 国家自然科学基金重点项目(41530859)与国家自然科学基金面上项目(41771331, 41571291)资助