• 关键词：反硝化厌氧甲烷氧化菌  旱地小麦田  丰度  活性

• 基金项目：国家自然科学基金（No.41322012，21277156）；中国科学院战略性先导科技专项（No.XDB15020303）；中国科学院青藏高原环境变化与地表过程重点实验室开放基金（No.TEL201304）；环境模拟与污染控制国家重点联合实验室专项经费项目（No.14L01ESPC）；中国科学院饮用水科学与技术重点实验室2015年度自由研究课题（No.15Z07KLDWST）；洪堡奖学金（No.1152633）；中国科学院青年创新促进会资助项目

• 周利
• 青岛理工大学环境与市政工程学院, 青岛 266033

• 李怡潇
• 1. 青岛理工大学环境与市政工程学院, 青岛 266033;2. 中国科学院生态环境研究中心饮用水科学与技术重点实验室, 北京 100085

• 庄林杰
• 中国科学院生态环境研究中心饮用水科学与技术重点实验室, 北京 100085

• 刘璐
• 中国科学院生态环境研究中心饮用水科学与技术重点实验室, 北京 100085

• 张坤
• 中国科学院生态环境研究中心饮用水科学与技术重点实验室, 北京 100085

• 祝贵兵
• 中国科学院生态环境研究中心饮用水科学与技术重点实验室, 北京 100085

• 摘要：在厌氧条件下，以亚硝酸盐作为电子受体将甲烷氧化的反硝化厌氧甲烷氧化反应（nitrite-dependent anaerobic methane-oxidizing，n-damo）的发现，彻底颠覆了人们对甲烷循环的传统理解.通过分子生物学方法及¹³C和¹⁵N稳定同位素示踪技术，对河北省北澧河附近的旱地农田土壤（0~1 m）中n-damo菌的群落结构、丰度和活性进行了研究，深入探究了n-damo菌的亚硝酸盐底物来源.结果显示，n-damo菌更多存在于旱地浅层土壤中，并且随季节变化分布在不同深度的土壤中.针对其pmoA基因的系统发育分析显示，旱地土壤中n-damo菌的群落结构具有明显的空间异质性，来自土壤0~20 cm和40~60 cm土层的序列完全分开，处于系统发育树不同分枝.针对其16S rRNA基因的实时荧光定量PCR结果显示，n-damo菌丰度随土壤深度的增加而降低，夏季丰度（1.44×10⁴~1.02×10⁵ copies·g^-1）低于冬季（3.66×10⁴~2.67×10⁵ copies·g^-1）.在浅层土壤（0~20 cm）中，硝化反应和反硝化反应共同为n-damo菌提供亚硝酸盐底物来源；而在深层土壤（60~80 cm）中，亚硝酸盐底物主要来源于硝化反应.n-damo菌的活性（0.18 nmol·g^-1·d^-1，以CO₂计）只能在夏季表层土壤（0~20 cm）中检测到，其余深度均未检测到其活性.在旱地农田土壤中，反硝化厌氧甲烷氧化菌对农田碳循环的影响可能不大.

• Abstract：The discovery of nitrite-dependent anaerobic methane oxidation (n-damo) with nitrite as the electron acceptor has changed the understanding about methane oxidation. In this study, the unique distribution characteristics and activity of n-damo bacteria were investigated in upland soil cores (1 m depth) from an agricultural upland at the Beili River in Hebei Province, China. Meanwhile, nitrite source for n-damo bacteria was also studied. Evidence was obtained using molecular biology methods and isotope-tracing technique. Results showed that n-damo bacteria were detected only in the shallow upland soil and distributed at various depths in different seasons. Phylogenetic analysis of pmoA genes showed apparent spatial heterogeneity of n-damo bacteria, and the sequences from 0~20 cm and 40~60 cm were completely separated in different branches of the phylogenetic tree. The abundance of n-damo bacteria decreased with soil depths, and the abundance in summer (1.44×10⁴~1.02×10⁵ copies·g^-1) was lower that in winter (3.66×10⁴~2.67×10⁵copies·g^-1). In this study, we found that the NO₂^- source for n-damo process came from both nitrification and denitrification processes in surface soils (0~20 cm), while in deep soils (60~80 cm) it mainly came from the nitrification process. Moreover, the n-damo activity (0.18 nmol CO₂·g^-1·h^-1) was only observed in surface soil (0~20 cm), and activity at other depths could not be detected. The influence of n-damo bacteria in the agricultural upland soil may be limited.

• Key words：nitrite-dependent anaerobic methane-oxidizing(n-damo)  agricultural upland soi  abundance  activity

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