双氰胺对北方棕壤土壤微生物代谢α-酮戊二酸的影响

Dicyandiamide is a worldwide used nitrification inhibitor. It could prolong the nitrification, reduce the nitrogen loss, enhance the nitrogen efficiency. Now, assessments of the genes abundances of ammonia monooxygenase are often used to evaluate the effects of nitrification inhibitors on ecological risk and economic efficiency, but there were few assessments of nitrification inhibitors on microbes’ community level physiological profile in soil. Our preliminary research findings showed that metabolism potentials of α-ketoglutaric acid in brown soil were reduced significantly by the application of DCD in both microcosm study and field study, which speculated this phenomenon was the consequence of soil microbe community to application of DCD in soil. In this project, brown soil in northern China will be studied to investigate the soil microbial community metabolism mechanism under DCD treatment. In this project, ion chromatography, MicrorespTM and real-time PCR were used to evaluate the nodes of carbon metabolism which were sensitive to DCD and investigate the α-ketoglutaric acid (as well as other low molecular organic acids) metabolic features in the soil microbial community. After the application of DCD, the soil microbial community metabolic functions in physiological and genetic levels were analyzed to provide support for comprehensively evaluating the ecological risk and economic efficiency.

双氰胺（Dicyandiamide，DCD）是一种被广泛应用的硝化抑制剂，其可以延长土壤硝化作用，减少氮素的损失，提高氮肥的利用率。目前，常采用定量土壤氨氧化基因丰度的方法，评估硝化抑制剂的施用效果和生态学风险，而从微生物群落代谢功能角度来评估硝化抑制剂对环境影响的研究工作鲜有报道。本课题组前期研究发现，在室内模拟实验和田间实验中，DCD均可使土壤分解α-酮戊二酸的潜力显著降低，推测这是土壤微生物群落氮循环受到人为干预后作出的响应。本项目拟将北方棕壤作为研究对象，研究DCD对土壤微生物群落代谢功能的影响机制。项目采用Microresp和实时定量PCR技术，判断受到DCD扰动较大的碳代谢节点；采用离子色谱技术研究α-酮戊二酸等低分子量有机酸的微生物群落代谢特征，旨在研究土壤微生物群落代谢功能在生理和基因水平上对DCD干扰的响应机制，为全面评估DCD的施用效果和风险提供理论依据。

双氰胺（DCD）是一种低毒的人工合成的硝化抑制剂，部分研究显示DCD还可以对土壤呼吸作用产生一定影响。本项目研究了施用DCD对北方棕壤CO2排放量和微生物三羧酸循环重要中间底物α-酮戊二酸的代谢能力的影响。2018-2020年年玉米田地田间观测结果显示，施用氮素量2%的DCD可以减排28.1%，39.2%，和39.3%的N2O排放量。因降水较少，2018和2020年玉米田地土壤通气良好，使DCD较尿素处理年可以减排17.1%和13.9%的CO2排放量，暗示DCD减排CO2和土壤通气量有关，即DCD的减排CO2效果源自有氧呼吸。实验显示α-酮戊二酸的呼吸作用对土壤微生物呼吸贡献最大，是影响其呼吸的主要中间底物。2020年玉米田间实验多次采样点的土壤呼吸能力分析显示， DCD可以抑制北方棕壤α-酮戊二酸的代谢能力。DCD实现的70%以上减排N2O和CO2效果发生在培养初期，且在这期间微生物代谢 α-酮戊二酸能力同时会受到DCD抑制，土壤可溶性有机碳消耗减慢，β-D-葡萄糖苷酶活性受到DCD抑制，且对微生物群落和相关功能基因没有影响。据此，初步推测DCD产生的CO2减排效果可能源自微生物代谢 α-酮戊二酸能力受到抑制。项目测试了三种模式生物大肠杆菌、酵母和硅藻对不同浓度DCD的响应情况，确定DCD抑制的作用点在α-酮戊二酸脱羧阶段。本项目考察了推荐用量DCD可促进玉米幼苗生长，减排幼苗产生的CO2和N2O。而高浓度DCD的毒性其提高根尖果胶酶活使细胞间隙加大，影响正常组织结构。综上，本项目研究结果推测DCD对生物有机体呼吸作用的靶点是α-酮戊二酸的脱羧过程。施用DCD可以有效减排各种环境下不同生物体产生的N2O，可以在好氧条件下，减排微生物和植物产生的CO2。这些研究结果可以有效拓展DCD的应用范围，提供切实可行和符合经济可持续发展的减排农田温室气体的方法，为对抗全球变暖和更好地减排农田温室气体提供部分理论依据。