摘要:华北平原农田由于长期过量施用氮肥，造成了土壤硝酸盐累积，导致地下水硝酸盐污染日趋严重。微生物的反硝化作用可将土壤中累积的硝酸盐或亚硝酸盐还原为气态产物，是消减厚包气带土壤累积的硝酸盐的重要途径。因此筛选高效反硝化微生物资源，对人工强化厚包气带土壤反硝化脱氮，阻控地下水硝酸盐污染具有重要作用。基于此，本研究采集位于华北平原的中国科学院栾城农业生态系统试验站长期施氮[施氮量为600 kg（N）?hm-2?a-1]定位试验0~150 m农田厚包气带及含水层土壤样品，从中筛选到62株细菌。16S rRNA基因序列分析表明这62株菌株与变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、厚壁菌门（Firmicutes）中的9个属具有较高的同源性。根据系统发育树的结果，挑选7株亲缘关系较远的菌株进行反硝化潜势试验，结果表明，菌株L71、L13和L103具备反硝化产气能力。电镜观察结果表明，这3株菌均为无鞭毛的杆状细菌，其长度分别为1.0 μm、1.5 μm和1.5 μm，只有L103具有运动能力。此外，菌株L103具有完全反硝化能力，且脱氮能力受到pH的影响，在本试验条件下，菌株L103的反硝化速率高达1.62~2.36 g（KNO3）?d-1?L-1，具备实际应用潜力。本研究表明华北平原厚包气带土壤中存在完全反硝化微生物，并可为人工强化治理厚包气带土壤硝酸盐污染提供菌种资源和理论依据。
Abstract:The long-term excessive use of nitrogen (N) fertilizer in agricultural regions has increased soil nitrate and residual N accumulation and poses a threat to groundwater quality. Nitrate leaching into the vadose zone is becoming a global concern as the N stock in this habitat comprises a significant portion of N budgets. The vadose zone is also an essential channel for the conversion and reduction of nitrate. Therefore, the elimination of nitrate accumulation in the vadose zone is significant for maintaining groundwater safety. Microbial denitrification is the reduction of nitrogen nitrate (NO3--N) to gaseous nitric oxide (NO), nitrous oxide (N2O), or dinitrogen (N2). This mechanism is essential for removing excess nitrate in the subsoil before it leaches into the groundwater and saturates deep soil zones or discharges into ground aquifers through subsurface drainage. Therefore, isolating and screening bacteria with strong denitrification abilities may strengthen the vadose zone and aquifer microbial denitrification process, preventing groundwater nitrate pollution. In this study, 62 microbial denitrifiers were isolated from the 0-150 m vadose zone in a position experiment of long-term nitrogen application in the Agricultural Ecosystem Experimental Station of Luancheng, Chinese Academy of Sciences, located the North China Plain. 16S ribosomal RNA (16S rRNA) gene sequence analysis showed that the isolated denitrifiers had high homology with nine genera, belonging to the phyla Proteobacteria, Actinobacteria, and Firmicutes. Of the 62 denitrifiers, seven strains (L37, L71, L96, L103, L104, L133, and L13) were selected for denitrification potential experiments based on the phylogenetic tree results. Gas kinetics under anoxic incubations showed that three strains (L71, L13, and L103) could reduce nitrate substrates to nitrous oxides, such as N2O and N2, in anaerobic conditions. Electron microscopy showed that the denitrifying strains were 1.0 μm (L71), 1.5 μm (L13), and 1.5 μm (L103) long rod-shaped bacteria. Strain L103 had motile and complete denitrification abilities, and the denitrification rate was between 1.62 and 2.36 g(KNO3)?d-1?L-1, indicating a high potential for use in agricultural practices. Furthermore, the denitrification ability of strain L103 was inhibited in acidic conditions, suggesting that pH also affects the microbial denitrification potential. Bacterial denitrifiers that reduce nitrate to N2 in hypoxic/anoxic conditions exist in the deep vadose zone of the North China Plain. The denitrification potential of these strains is important for understanding how microorganisms contribute to the soil nitrate accumulation self-remediation process.
?Equivalent contributors
注释:
1) ?同等贡献者: 赵会成, 主要研究方向为微生物分子生态学, E-mail:hczhao@sjziam.ac.cn;刘梦帅, 主要研究方向为微生物分子生态学, E-mail:lms9095@163.com
PDF全文下载地址:
http://www.ecoagri.ac.cn/article/exportPdf?id=07ea7a65-314c-4a41-9120-671a20ff0f20
