莫能菌素在鸡粪中的降解规律及其对鸡粪堆肥微生物群落的影响

Degradation regulation of monensin in chicken manure and its impact on microbiota in chicken manure

【目的】明确鸡粪中莫能菌素的降解规律及其在环境中的趋向,为评估莫能菌素用作饲料药物添加剂的生态毒性效应提供理论依据,也为畜禽粪便的无害化处理提供技术支撑。【方法】建立快速测定粪便莫能菌素残留的高效液相色谱串联质谱法(LC-MS/MS),并构建莫能菌素鸡粪模型,提取鸡粪总DNA,通过Illumina NovaSeq 6000平台完成高通量测序,使用Trimmomatic(v0.33)处理测序结果,然后分别进行OTU聚类分析、物种注释、Alpha和Beta多样性分析、组间差异显著性分析及相关分析,以预测莫能菌素残留对鸡粪堆肥微生物群落的影响。【结果】LC-MS/MS检测鸡粪中莫能菌素残留的准确度和精密度良好,检出限为0.10μg/kg,定量限为0.25μg/kg。莫能菌素在低(40μg/g)、中(80μg/g)、高(120μg/g)浓度下的降解半衰期分别为111.95、287.55和374.59 d,即莫能菌素浓度越高,在鸡粪中的降解半衰期越长。在鸡粪中加入莫能菌素,厚壁菌门相对丰度随时间的推移呈极显著的降低趋势(P<0.01);莫能菌素的存在也改变了鸡粪堆肥中优势菌属的相对丰度,初期导致鸡粪微生物群落的物种丰度及其多样性降低,但随着时间的推移鸡粪微生物群落物种丰度及其多样性不断升高。化学异养是鸡粪微生物群落的主要代谢,其次是需氧化学异养、碳氢降解、发酵及硝化等;莫能菌素的加入能促使盐单胞菌属改变反硝化细菌组成,降低反硝化作用。【结论】以分散固相萃取结合LC-MS/MS测定鸡粪中莫能菌素残留具有快速、灵敏、可靠的特点,可应用于实际样品检测。莫能菌素在鸡粪中的降解时间与浓度呈正相关,其浓度越高,在鸡粪中的半衰期越长。莫能菌素能改变鸡粪堆肥过程中的微生物群落组成,进而影响鸡粪堆肥肥力。

【Objective】The purpose of the study was to reveal the regulation of degradation of monensin in chicken manure and its tendency in the environment,which would provide theoretical basis for evaluating the ecotoxic effect of monensin used as a feed drug additive,and the support for the harmless treatment of livestock and poultry manure.【Method】A high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)method was established for the rapid determination of manure monensin residue,and a model of chicken manure for monensin was constructed.Total DNA of chicken manure was extracted,and high-throughput sequencing was completed by the Illumina NovaSeq 6000 platform,and the results of the sequencing were processed by using Trimmomatic(v0.33).Then,the OTU cluste-ring analysis,species annotation,Alpha and Beta diversity analysis,significance of differences between groups and correlation analysis were performed to predict the effect of monensin residue on the microbiota in chicken manure compost.【Result】The accuracy and precision of LC-MS/MS for the determination of monensin residue in chicken manure were well,with a limit of detection of 0.10μg/kg and a limit of quantification of 0.25μg/kg.The degradation half-lives of monensin in low(40μg/g),medieum(80μg/g),high(120μg/g)concentrations were 111.95,287.55 and 374.59 d,that was,the higher the concentration of monensin,the longer the half-life of the degradation in chicken manure.The relative abundance of the Firmicutes showed extremely significant decrease(P<0.01)with time when monensin was added to chicken manure.The monensin also altered the relative abundance of dominant genera in chicken manure.The species abundance and diversity of manure microbiota decreased initially,while these indicators increased over time.Chemical heterotrophy was the main metabolism of the chicken manure microbiota,followed by oxidative heterotrophy,hydrocarbon degradation,fermentation and nitrification.Monensin induced Halomonas to change the composition of denitrifying bacteria and decrease denitrification.【Conclusion】The determination of monensin residue in chicken manure by dispersive solid-phase extraction coupled with LC-MS/MS is rapid,sensitive and reliable,which can be applied to the detection of real samples.The degradation of monensin in chicken manure is positively correlated with its concentration,and the higher the concentration,the longer the half-life in chicken manure.Monensin can change the composition of microbiota in chicken manure composting process,then affect the fertility of chicken manure compost.