氮沉降增加影响温带森林土壤甲烷氧化的微生物机制

To date there have been still many inconsistent results about the effects of increased atmospheric nitrogen on the atmospheric methane consumption by forests, yet the key microbial mechanisms involving the soil methane consumption under the disturbance of long-term nitrogen deposition are extremely unclear at present. In the light of long-term atmospheric nitrogen deposition simulation experimental platform within a mature temperate Korean pine and broadleaf forest at the foot of Changbai mountains, the microbial responses mechanisms of forest soil methane consumption to increased nitrogen deposition including nitrogen forms will be studied by using microbial molecular ecology techniques, such as real-time PCR, terminal restriction fragment length polymorphism (T-RFLP), cloning and sequencing, and in combination with a series of incubation experiments and measurements of soil basic properties. In this study, we try to systematically find out the seasonal changes in methanotroph abundance and community structure and methane oxidation potential and their respective responses to long-term nitrogen deposition including nitrogen forms, and their inter-coupling relationships by taking into account the changes in soil basic properties. The results obtained can indeed provide a vital scientific basis for fully understanding the key microbial mechanisms involving the microbial methane oxidation in temperate forest soil under the stresses of increasing atmospheric nitrogen deposition in the future.

目前关于大气氮沉降增加对森林土壤甲烷氧化影响的研究结果存在着不一致性，但对于其中关键的微生物作用机制仍不太清楚尤其是在长期氮沉降增加条件下的。因此，本项目基于长白山温带阔叶红松林长期氮沉降模拟试验平台，拟采用微生物分子生态学方法（包括实时荧光定量PCR、末端限制性片段长度多态性（T-RFLP）和克隆文库构建等技术），并结合实验室的培养实验和土壤属性测定，来研究长期施加不同形态氮时森林土壤甲烷氧化菌的数量和群落组成随季节变化的特征及其与土壤甲烷氧化能力和土壤基质属性的耦合关系，从而揭示长期氮沉降增加影响温带森林土壤甲烷氧化的微生物机制，为深入认识温带森林土壤甲烷氧化能力应对未来大气氮沉降增加胁迫时的响应特征和作用机理提供坚实的科学依据。

尽管有研究报道氮沉降量增加对森林甲烷吸收量的影响存在季节性差异，但是有关微生物驱动机制仍缺乏研究。本文基于长白山典型温带森林长期氮沉降模拟实验平台样地，采用定量PCR和克隆测序技术结合土壤属性测定，研究了长期施加不同形态氮处理的森林土壤甲烷氧化菌的数量和群落结构组成随季节变化的特征。结果表明，夏季，森林土壤甲烷氧化菌pmoA基因丰度在不同施氮处理之间无显著性差异（每克干土1.54×106 -3.20×106拷贝数）；秋季，pmoA基因丰度在施加NH4Cl和(NH4)2SO4处理小区（每克干土1.93×105_7.6×105拷贝数）显著低于对照（每克干土4.03×106±1.2×106拷贝数）和KNO3处理小区（每克干土1.70×106±1.51×106拷贝数）；无论夏季和秋季，施加不同形态氮处理土壤样品甲烷氧化菌均以Type I型为主（相对丰度在70.6—85.4%之间），并以Type I_Clade A为优势类群，占Type I型的55.1－91.7%；Type I_Clade A的相对丰度夏季在施加不同形态氮的土壤样品中无显著差异，但秋季样品中在施加(NH4)2SO4 (52.7% ± 6.52%)和NH4Cl（56.1%±8.89%）的处理显著低于对照和KNO3土壤, Type I_Clade B的相对丰度则在(NH4)2SO4和NH4Cl处理土壤中高，尤其在秋季样品中表现出显著差异，与Type I_Clade A呈现出彼消此长的趋势。这些结果表明铵态氮肥添加对温带森林土壤甲烷氧化菌的生长和活性具有抑制作用，受夏季温度和水分的影响，这种抑制作用在秋季表现更明显，而NO3--N添加对土壤甲烷氧化菌的活性无显著影响。这些结果解释了以往观测到的施铵态氮肥显著降低秋季温带林地土壤甲烷净吸收量，而在夏季无显著影响的观测结果，解释了长期氮沉降影响森林土壤甲烷吸收的微生物机制。