抗生素-重金属复合污染对土壤微生物的生态毒理效应及其作用机制研究

With the rapid development of livestock and poultry industry in China, residues of veterinary antibiotics (Such as suflonamides, tetracyclines, etc) and heavy metals (such as Cu, Zn, etc ) in animal manure eventually get into soils, which pose a serious potential threat to soil ecology and human health. Considering their persistence in soils and associated impacts on soil ecosystem, it is necessary to investigate the ecotoxicology of binary antibiotics and heavy metal mixtures on soil microbial communities and function. .Nitrification is a key process in the soil nitrogen (N) cycle. Ammonium-oxidizing microbes (AOM), including AOB and AOA, have been identified as the key group that controls the rate-limiting step of nitrification, which is catalyzed by the ammonia monooxygenase (AMO). Moreover, it was reported that the abundance of AOB and AOA could serve as a relevant and cost-effective bio-indicator for soil monitoring. Therefore, it is significant to research the effect of sulfonamides and Cu on ammonia oxidizing microbes (AOB and AOA) and their activities using functional amoA gene..In this study, we selected Copper, sulfadiazine (SDZ) and sulfamethazine (SMA) as the tested toxic chemicals; we select two type soils sandy soil and clay soil as the tested soils, which are the typical soils in China. The soils will be collected from eight locations, which have different characteristics in terms of pH, texture, and organic matter content, and nitrate and ammonium level. All fields were used for wheat and corn cropping and subjected to (1:1) crop rotation. we evaluate the toxic effects of Cu, SDZ/SMA and their binary mixtures using two novel molecular techniques: 1) an assay based on reverse-transcription qPCR, which quantifies the abundance of amoA functional gene transcripts, and 2) an assay evaluating the diversity of bacterial communities. For functional genes, we quantified amoA transcripts from both bacteria and archaea, and for diversity, we evaluated the Miseq sequencing of the V3 region of the 16S ribosomal RNA (rRNA) gene—in both cases in response to treatment with the Cu, SDZ/SMA and their binary mixtures. Moreover, we will measure key soil chemical parameters and soil nitrification potential activities. The final aims is 1) to build a large dataset suitable for assessments of soil functioning in agricultural soils, and to provide a tool to establish the NOR of soil functioning, allowing the detection of soils undergoing disturbances; 2) to determine the joint toxic effects of Cu-SDZ/SMA on ammonia oxidizing communities and their function at molecular level. This study will provide a theoretical basis for ecological risk assessment of contaminated soils with heavy metals and veterinary antibiotics, and the reference for the ecological risk evaluation and soil pollution control with field application of manures.

近年来，随着畜禽粪便的频繁大量农用，土壤中抗生素与重金属等化合物的复合污染问题日趋严重。这将对土壤生态系统稳定性及其功能造成严重威胁。为此，本项目拟在前期研究的基础上，以土壤氨氧化菌群功能基因amoA为生物标志物，以典型土壤功能NOR值的确定、氨氧化微生物对Cu-SDZ/SMA复合污染响应的分子生态特征研究为主线，采用定量PCR、Miseq测序、mRNA转录等技术手段，在分子水平上分析Cu和典型的磺胺类抗生素的复合污染生态毒理效应及其联合作用机制；建立抗生素和重金属复合污染的土壤生态毒理效应诊断的指标体系，为土壤抗生物-重金属污染复合污染的生态风险评价以及畜禽粪便的科学管理提供理论支持。

随着社会经济发展，农田土壤重金属-抗生素复合污染问题日趋严峻。本课题以磺胺嘧啶（SDZ）和铜（Cu）为典型污染物，以我国不同地理区域典型农田土壤为供试土壤，在土壤微生物群落及功能的地理区域性及季节性差异分析的基础上，研究了SDZ和Cu单一及复合污染的土壤微生物群落结构、功能活性及其联合作用机制，并在此基础上，探讨了生物炭对二者复合污染的修复作用。本课题研究结果对重金属-抗生素污染土壤生态风险分析、评价及管理具有重要的意义。主要研究结果如下：.1、不同地理区域农田土壤微生物群落及功能的自然波动变化.尽管各地农田土壤微生物群落在门（phylum）和属（genus）水平上存在差异不明显，但其优势种群组成差异显著，这导致土壤微生物的代谢活性存在显著的区域性差异，且不同土壤潜在硝化活性（PNR）变化范围在1.84 mg/kg/d~10.71 mg/kg/d，脲酶、磷酸酶活性也呈倍数关系。.2、不同地理区域的农田土壤微生物对重金属和抗生素复合污染的效应差异.土壤PNR对Cu单一污染较为敏感，五种土壤的EC50（Cu）值分布在150~300 mg·kg-1之间，但SDZ对五种土壤PNR的EC50值均远高于其土壤检出浓度（1.93 ~ 760.09 μg·kg-1），即PNR不适于作为土壤SDZ污染风险评价指标； Cu和SDZ单一及复合污染均可显著改变AOA 和AOB的种群占比以及其种群多样性，二者存在协同抑制作用，且与区域土壤地理环境因子密切相关； 但是不同土壤中AOA amoA基因丰度或优势种群对污染胁迫的响应较为一致。.3、季节变化对重金属-抗生素复合污染的土壤微生物效应的影响.土壤生物指标对Cu和SDZ单一及复合污染胁迫的响应与季节变化密切相关，即土壤酶及群落代谢水平在生长季对各污染胁迫的响应较非生长季敏感；尽管不同地区土壤氨氧化菌群及其功能对季节变化的响应差异显著，但是其优势菌群在不同作物生长季节对土壤污染胁迫的响应与土壤PNR保持了较高的一致性。