基于稳定性同位素技术研究丙酸类手性除草剂对映体选择性降解机制

Pesticides are applied extensively and cause many eco-environmental problems, therefore, it is important to understand their degradation-being the only process that actually clears pesticides from the environment. Application of stable isotope probing (SIP) and compound-specific isotope analysis (CSIA) to unveil enantioselective biodegradation of chiral pesticides are now coming to light. In this study, we are going to asymmetric synthesis 13C-labelled chiral phenoxypropionic acid herbicide enantiomers, and to investigate active microorganisms in soil microscosms by using DNA-SIP and pyrosequencing. Incubation conditions on enantioselectivity will be also studied. On the other hand, making use of both enantioselective degradation and isotope fractionation as indicators, herbicides and their intermediates will be ananlyzed to characterize degradation kinetics and pathways. Based on isotop fractionation factors, in situ biodegradation processes will be tested qualitatively and quantitatively. The results will provide scientific data for evaluation of eco-environmental risks of chiral pesticides and further environmental friendly remediation of pesticide polluted sites.

农药大量使用带来了一系列生态环境安全问题，弄清农药在环境中唯一真正清除途径的降解机制显得尤为重要。稳定性同位素探针和单体同位素分析技术为深入阐明农药、特别是手性农药对映体选择性降解提供了契机。本项目首先通过手性不对称合成制备13C标记的丙酸类除草剂对映体，采集典型土壤在实验室进行微生态培养，结合稳定性同位素探针和454测序，揭示选择性降解农药对映体的功能微生物，并研究培养条件对选择性的影响。同时，根据手性农药在微生物降解过程中的对映体选择性和同位素分馏现象，分析农药母体及其代谢中间产物的降解动力学、对映体比和同位素富集系数，解析对映体选择性降解的规律与途径。并根据实验室得到的同位素分馏系数，研究农药对映体在实际环境中的降解。从而更加全面地评价手性农药的污染风险，也为今后的环境友好修复提供科学依据。

本项目开展了DCPP和MCPP在土壤中选择性降解和对映体转化的研究， DCPP和MCPP在土壤中的消减和对映体转化主要由微生物引起，外消旋体半衰期分别在10.5-19.8天和10.3-22.2天之间，与除草剂初始浓度和培养条件密切相关，DCPP和MCPP的残留浓度与对映体比显著相关（p < 0.01）。DCPP和MCPP均有显著的同位素富集现象，表观动力学效应常数AKIA在1.008-1.032之间，表明降解初始反应为C-H键的断裂。对映体和同位素分析技术都能准确定量评估DCPP和MCPP在土壤中的生物降解。添加除草剂土壤中sdpA基因相对丰度均表现为先增加后降低，最高值在半衰期附近。Illumina MiSeq高通量测序结果表明，添加除草剂促进了Proteobacteria（β-Proteobacteria）相对丰度均显著增加和相关降解菌的生长，同时土壤微生物多样性先下降后又恢复到接近初始状态。除菜地土外，DCPP处理土壤中Comamonadaceae随培养时间而增加，森林土壤中Sphingobium 和 Flavobacterium 显著增加；MCPP处理土壤中Methylophilaceae增加最为显著，除桑园土外，其余三个土壤中Sphingomonadaceae显著增加。sdpA基因测序结果显示，大多数是未知物种，主要已知降解菌株为S型优先降解菌Sphingobium herbicidovorans，与降解实验S-DCPP/MCPP优先降解的结果一致。添加DCPP的一个土壤中Nitrospira的丰度显著增加，施加DCPP很有可能影响土壤中的氮循环，这个现象值得进一步研究。