Disentangling Immobilization of Inorganic Nitrogen by Fungi and Bacteria in Soil with Adapting a Novel Amino Sugar-based Stable Isotope Probing Approach
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摘要: 土壤微生物对无机氮的固持作用是构成土壤保氮机制的重要组成。作为土壤微生物的两大主要类群，真菌和细菌是微生物固持无机氮作用的主要参与者。然而，由于土壤微生物的高度复杂多变性，如何有效区分和量化土壤中真菌和细菌各自对无机氮的固持作用是个难题。针对该问题，本文采用“氨基糖稳定同位素探针（AS-SIP）”技术来区分和表征土壤中真菌、细菌各自对无机氮的固持速率。基于此进一步揭示了农业利用和外源碳输入分别对土壤真菌、细菌各自固持硝态氮作用的影响及原因，构建了土壤中真菌、细菌各自固持无机氮实际速率的估算模型，为区分和量化土壤中真菌、细菌各自对无机氮的实际固持速率提供了更为可信的新方法。本文介绍了AS-SIP 技术原理、主要技术优势、应用案例、不足之处以及改进对策，以期推进该方法的应用和发展。
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关键词:
- 微生物无机氮固持 /
- 氨基糖稳定同位素探针 /
- 真菌 /
- 细菌
Abstract: Microbial inorganic nitrogen (N) immobilization is an important mechanism in the retention of N in soils. Due to the high diversity and complexity of soil microorganisms, how to distinguish and quantify the inorganic N immobilization by fungi and bacteria is a challenging issue. The amino sugar-based stable isotope probing (AS-SIP) approach has been used to indicate the respective inorganic N immobilization rates of fungal and bacterial communities in soils. The effects of agricultural land-use and exogenous organic carbon input on the respective fungal and bacterial nitrate immobilization in soil were unraveled for the first time. Furthermore, a mathematical framework, combining the experimentally measurable gross inorganic N immobilization rate and proxies for fungal and bacterial inorganic N immobilization rates, was proposed to quantify the respective immobilization rates of inorganic N by fungal and bacterial communities in soil. This paper introduces the principle, advantages, case studies, limitations, and coping strategies of this novel AS-SIP approach, aiming to promote its development and application. -
无机氮在土壤中不稳定性高，易于损失，例如淋洗、氨挥发、反硝化等。因此，农业土壤中无机氮（尤其是硝态氮）的过度累积是导致氮素损失风险提高、氮肥利用率低下的重要原因之一[1-7]。无机氮的微生物固持作用指土壤微生物利用无机氮作为氮源并转化为自身有机体组成部分。该微生物有机体氮可以经短期储存后进行再矿化产生铵态氮或者进入土壤稳定有机氮库长期保存。因此，微生物对无机氮的固持功能有助于减少土壤氮素损失，是构成土壤保氮机制的重要组成[8-13]。
真菌和细菌作为土壤微生物的两大主要类群，是微生物固持无机氮作用的主要参与者[14-16]。由于在土壤中的生理、形态、生存方式和数量等特性迥异[17-20]，真菌、细菌固持无机氮作用对于环境条件改变的响应也可能有所不同[14,16,21]。然而，由于土壤微生物的高度复杂多变性，如何真实有效地区分并量化两者各自对无机氮的固持作用是个难题。
微生物选择性抑制剂法是传统用来区分真菌、细菌各自固持无机氮潜力的唯一方法[16]。但是，选择性抑制剂具有特异性不高、用量难以控制等缺陷，直接导致该方法所获结果可信度低[14,22]。近两年，“氨基糖稳定同位素探针”技术（Amino Sugar-based Stable Isotope Probing，简称AS-SIP）被用来区分和表征更加接近于真实情形的土壤中真菌、细菌各自对无机氮的固持速率，有望在土壤微生物氮素利用领域引领出新的学科增长点[21-24]。
本文介绍了AS-SIP新用法的技术原理、主要优势以及应用案例，讨论了AS-SIP新用法的不足之处以及未来发展方向，以期推进该方法的应用和发展。
1. AS-SIP新用法的技术原理
氨基糖作为微生物细胞壁的重要组成物质，是土壤微生物同化碳、氮的储库[25-26]。在可测的土壤氨基糖中，胞壁酸（MurN）唯一来源于细菌，氨基葡萄糖（GluN）主要来源于真菌 [25-27]。另外，氨基糖在土壤中较难分解，主要以微生物死亡残体的形式存在，一般与土壤有机碳、氮含量具有极强的正相关性 [28-30]。基于氨基糖的异源性和稳定性特点，国内外学者自2000年以来建立稳定同位素标记（15N或13C）色谱/质谱联机测定技术（即氨基糖稳定同位素探针技术，AS-SIP）实现了对土壤中新合成和原有氨基糖的区分，从而使得微生物参与的土壤有机碳、氮周转研究取得突破性进展[31-35]。
不同于以往把AS-SIP用于揭示土壤氨基糖库自身周转动态来反映土壤有机氮周转动态的常规用法，本文作者将AS-SIP发展来区分和表征土壤中真菌、细菌各自对无机氮的固持速率[21-24]。方法原理：以土壤中氨基糖具有的异源性和稳定性两个基本特性为切入点，特别是稳定性特点（现有研究报道氨基糖在土壤中的平均周转时间至少大于2年，远长于活体微生物）[36-38]，由此推知微生物新合成的氨基糖短期内大多积累在土壤中，包括活体微生物及其死亡残体中[39-40]。基于此，利用添加15N标记无机氮后短期培养内新合成真菌、细菌来源氨基糖（15N-GlcN和15N-MurN）各自的累积速率来分别表征土壤真菌、细菌固持无机氮的速率。
AS-SIP的新用法突破了常规采用选择性抑制剂的固有方法框架，开拓了一条探究土壤中真菌、细菌各自固持无机氮功能的新途径，赋予了AS-SIP技术一个新的研究应用领域，即区分和表征真菌、细菌固持无机氮的速率。与选择性抑制剂法相比，由于无需额外添加真菌或细菌选择性抑制剂，AS-SIP新用法所获结果更加接近于实际情形。此外，由于氨基糖同时含碳原子，新方法对于研究土壤微生物对含碳底物的固持功能也具有重要借鉴意义。
2. 方法应用案例及发展
基于新的AS-SIP用法，作者进一步揭示了农业利用和外源碳输入对土壤真菌、细菌各自固持硝态氮作用的影响及原因[21-24]。具体如下：
案例一：土壤微生物固持硝态氮作用下降是导致亚热带农业红壤硝酸盐累积、氮素损失风险提高的重要原因[10-12]。然而，作为土壤微生物的主要类群，真菌、细菌各自固持硝态氮作用对于农业利用如何响应还未知。
基于AS-SIP新用法，比较了华南地区不同土地利用方式下红壤真菌、细菌各自固持硝态氮速率的表征值，同时结合土壤理化因子测定，发现：农业利用导致森林红壤真菌、细菌固持硝态氮作用均显著下降，并且真菌固持硝态氮作用的降幅（81%）高于细菌（61%）；与细菌相比，真菌固持硝态氮作用的下降不仅与农业利用所导致的土壤有机碳和碳氮比下降有关，而且还与pH和有效磷升高显著相关（图1）。
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图 1 农业利用降低土壤微生物固持硝态氮功能的机制[21]短期培养内新合成真菌、细菌来源氨基糖（15N-GlcN和15N-MurN）各自的累积速率分别表征土壤真菌、细菌固持NO3−的速率。Figure 1. Mechanisms of decreasing soil microbial nitrate immobilization in agricultural land-use [21]Note: The cumulative amounts of newly formed 15N-GluN and 15N-Mur at the end of incubation were used as proxies of fungal and bacterial NO3− immobilization rates.该工作的主要科学意义在于提出AS-SIP的新用法，基于此区分并表征了农业利用对于土壤真菌、细菌各自固持硝态氮速率的影响，进一步揭示了农业利用导致红壤微生物固持硝态氮作用下降的内在机制，有助于认清农业土壤硝酸盐累积的原因，进而制定合理应对措施[21]。
案例二：农作物秸秆作为农业生产中重要的副产品，一般具有较高的碳、氮含量，且碳氮比一般高于微生物体，是重要的有机物料来源。施加具有较高碳氮比的有机物料被普遍认为可以有效提高土壤微生物固持硝态氮作用[41-43]。这恰好与案例一所发现的有机碳和碳氮比下降可能是导致农业利用红壤真菌、细菌固持硝态氮作用下降的主要原因相互印证。然而，真菌、细菌各自固持硝态氮作用对于外源碳添加的响应还未知。
同样基于AS-SIP新用法，以南方典型坡地果园红壤为研究对象，通过设置不同梯度的常见坡地覆盖植物残体添加实验，同步结合土壤微生物群落组成测定。结果发现：植物残体添加同时提高了土壤真菌、细菌固持硝态氮作用，并且对于前者的提高作用更大；真菌、细菌生物量分别可以解释各自固持硝态氮作用变化的70%和32%；真菌、细菌生物量比值可以解释真菌、细菌固持硝态氮作用相对变化的49%。
该方法的主要科学意义在于最先区分并表征了外源碳输入对于土壤真菌、细菌各自固持硝态氮作用的影响，在此基础上初步阐明了外源碳输入、微生物群落结构改变（即：土壤真菌、细菌数量及真菌细菌比同时增加）、微生物固持硝态氮功能提升（即：土壤真菌、细菌固持硝态氮功能及二者相对优势同时增加）和土壤持留硝态氮能力提高四者之间的内在关联，进一步揭示了外源碳输入对于土壤微生物固持硝态氮作用的影响机制，为合理利用氮肥及有机物料、解决农业土壤硝酸盐累积问题提供了科学依据（图2）[24]。
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然而，由于土壤微生物种类复杂多样且组分各异，土壤中真菌、细菌有机体的氨基糖含量以及微生物体不同含氮组分（包括氨基糖）的周转速率难以测定 [40,44-45]。这导致无法由AS-SIP所获取的土壤真菌、细菌各自固持无机氮速率的表征值（即：真菌、细菌来源新合成氨基糖的积累速率）来推知二者固持无机氮的实际速率，也就无法比较两类微生物对于无机氮固持作用的实际贡献大小。
针对该问题，作者采用逆向思维模式绕开了AS-SIP的上述方法瓶颈，结合可以通过实验测定的真菌、细菌各自固持无机氮速率的表征值（采用AS-SIP测定）和土壤微生物无机氮固持初级速率（例如采用15N稀释技术测定），构建出线性回归估算模型，利用最小二乘法原理反推出土壤中真菌、细菌各自固持无机氮速率的表征值和各自实际固持速率之间的转换系数，最终估算出真菌、细菌各自固持无机氮实际速率[23]。
作者利用公开发表数据对估算模型的应用进行了演示[23]。具体而言：土壤微生物固持硝态氮初级速率和真菌、细菌各自固持硝态氮速率的表征值分别源于Zhang et al. (2013)[10]和Li et al. (2019)[21]。该两项研究均报道了我国亚热带地区森林转变为农田对红壤微生物固持硝态氮作用的影响，发现农业利用导致微生物固持硝态氮作用下降，且真菌、细菌固持作用均下降，前者降幅大于后者。然而，真菌、细菌固持硝态氮的相对贡献及其对于土地利用方式改变的响应还未知。基于新的估算模型，利用以上发表数据计算得出：真菌不仅在不同土地利用方式下红壤微生物固持硝态氮作用中起主导作用，而且对农业利用所导致的红壤微生物固持硝态氮作用下降也负主要责任。
3. 问题与展望
同位素示踪技术在土壤学研究中的应用需要注意示踪元素（例如15N）的微生物循环再利用问题，这可能导致测定结果与实际情形产生偏差。以AS-SIP技术为例，特定类群微生物同化利用15N标记无机氮生成的化合物可能快速被其他土壤生物所同化利用[46]，最终影响测定结果的可信性。鉴于该不足之处，未来可以考虑结合现有相关方法的优缺点，联合运用多种方法来提高结果的可信度，例如基于AS-SIP技术结合估算模型获得的真菌、细菌固持无机氮速率可以与同步采用14C标记前体分子结合微生物标识物分析法测定的真菌、细菌生长速率进行互相对比及验证[47]。
AS-SIP的新用法为区分和量化土壤中真菌、细菌各自对无机氮的固持功能提供了更加可信的解决途径，有望初步定量揭示土壤微生物群落结构和无机氮固持功能之间的内在关联，从而显著推进微生物介导的土壤氮素保蓄研究。同样，由于氨基糖同时包含碳元素，新方法对于量化土壤微生物对含碳底物的固存功能也具有重要借鉴意义。因此，AS-SIP新用法有望催生土壤微生物碳、氮利用研究领域新的学科增长点。
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图 1 农业利用降低土壤微生物固持硝态氮功能的机制[21]
短期培养内新合成真菌、细菌来源氨基糖（15N-GlcN和15N-MurN）各自的累积速率分别表征土壤真菌、细菌固持NO3−的速率。
Figure 1. Mechanisms of decreasing soil microbial nitrate immobilization in agricultural land-use [21]
Note: The cumulative amounts of newly formed 15N-GluN and 15N-Mur at the end of incubation were used as proxies of fungal and bacterial NO3− immobilization rates.
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