垦殖与自然恢复黑土反硝化作用活性及其微生物菌群结构研究

In agroecosystems, denitrification is an important biogeochemical process that removes N from soil via microbial stepwise reduction of NO3- to gaseous products （NO, N2O, N2）. N2O is an important greenhouse gas that also contributes to the destruction of the ozone layer. Therefore, in depth knowledge of the activity of denitrification and communities of denitrifiers is essential for reducing the negative impact of N2O on the environment and increasing the availability of N fertilizer. A suit of environmental factors can influence denitrification, including soil management practice, fertilizer and season. In this project, soil denitrifiers in black soil （0-20 cm） with different fertilizing treatments [no fertilizer （NoF）, chemical fertilizer （CF） and chemical fertilizer plus manure （CFM）] and in a neighboring plot of natural restoration （NR） in Northeast China were compared during different seasons. PCR, DGGE, clone, qRT-PCR and sequencing methods targeting the functional genes of denitrifiers will be used to investigate how soil management practices influence the communities and quantities of denitrifiers. Meanwhile, phylogenetic affiliation of culturable denitrification bacteria isolated by culture-dependent approach will be subjected to in this study. Through this project, the denitrifier communities, quantities and phylogeny will be clarified. It also provide important information for the maintenance of soil fertility quality, protecting the black soil resources, reducing the amount of greenhouse gas emissions and establishing the mechanical theory of denitrification.

反硝化作用是指反硝化微生物将硝酸盐还原为NO、N2O和N2的过程，是导致温室效应和氮肥损失的原因之一。土壤反硝化作用与土地利用方式、施肥和季节等关系密切，研究土壤反硝化作用活性及其菌群结构可以为温室气体减排和肥料利用率的提高提供科学依据。本研究基于海伦农业生态试验站长期定位试验，以不同季节垦殖不同施肥处理和自然恢复黑土为研究对象，测定土壤反硝化作用活性；以nirK和nirS为靶基因，采用PCR-DGGE、克隆、测序和qRT-PCR技术，解析反硝化微生物群落结构和丰度变化，揭示反硝化作用活性与其菌群之间的关系。同时也将对可培养反硝化细菌进行分子鉴定和功能基因的序列分析，以期进一步明确垦殖与自然恢复黑土反硝化细菌组成，明确何种细菌具有何种反硝化功能基因。该项目的研究，不仅揭示出土地利用方式改变后黑土反硝化作用活性和菌群结构的动态变化，而且也将为保护黑土资源、明确黑土区反硝化作用机制奠定理论基础

本项目基于中国科学院海伦农业生态试验站长期定位试验，对比研究了于不同季节采集的垦殖不同施肥处理与自然恢复黑土反硝化细菌群落结构和丰度的季节变化。高通量测序结果表明占据垦殖与自然恢复黑土中反硝化细菌成员比例最高的细菌是来源于Betaproteobacteria纲、Rhodocyclales目、Rhodocyclaceae科的Aromatoleum和Rhodanobacter属。NMDS和PCA结果表明，长期的施肥处理改变了黑土区反硝化细菌的群落结构，而不同季节对反硝化细菌的群落结构影响不明显，同时以施肥和采样时期作为影响因子，对反硝化细菌OTU的相对数量进行的ANOVA分析，进一步说明了肥料是影响黑土区反硝化细菌的主要因素。另外，RDA结果表明本研究所选取的土壤理化因子中对反硝化细菌群落结构的影响比较大的环境因子有TC、TN和pH。针对于nirS基因的荧光定量PCR结果表明，4种施肥处理8月份采集的样品反硝化细菌的丰度最高，与其他3个时期采集的样品相比差异显著，其余样品在不同时期反硝化细菌的丰度没有明显规律。本研究不仅明确了土壤反硝化细菌群落与土地利用方式、施肥和肥料利用率之间的关系，同时也为解析土壤反硝化作用和提高肥料利用率提供了科学依据。