土壤环境科学实验室知识库

        抗生素耐药性是21 世纪最 严重 的公共卫生挑战之一。抗生素广泛用于预防或治疗疾病并维持或 促进食用动物的生长，尤其是在猪场和鸡场中 此外 抗生素的 使用增加 了细菌的选择性压力。 对动物蛋白需求的增长推动了全球畜牧业的蓬勃发展。 食品 动物 数量 的增加导致每年畜禽粪便 产 量 的增加。随着畜禽粪便治污减排力度的加大，带动和促进了商品有机肥 的生产和销售。全球范围内对有机食品消费需求的增加， 使得 畜禽粪便来源的商品有机肥的 施用 越来越多，尤其是在中国。目前，农业行业标准（ NY525 2012 ）对商品有机肥的 pH 、 粪大肠菌群数等做出要求，但并不监测其中的抗生素、 抗生素 抗性基因等。 因此， 本研究探讨重复施用商品有机肥后土壤中抗生素的出现以及归趋，表征 抗生素 抗性基因的出现及归趋 ，此外研究土壤细菌群落组成的变化，并确定 抗生素 抗性基因组成与微生物群落组成的 关联 。最后， 本 研究提供了一种环境 友好 且 经济 可行 的技术，即采用 热裂解炭化技术将猪粪堆肥制备成生物炭 ，以缓解 抗生素 抗性基因从畜禽粪便向环境的传播。
        1）本实验采集了我国 9 个省份的 10 个 畜禽粪便来源的 商品有机肥样品 。通过土壤微宇宙实验 研究重复施用鸡粪或猪粪来源的商品有机肥对土壤抗生素和抗生素 抗性基因的影响。 结果表明 施用无害化处理的商品有机肥导致 土壤抗生素和 抗生素 抗性基因的普遍增加。这些抗生素在土壤中持续存在 4 个月，并且重复施用商品有机肥导致抗生素的累积。然而，第一次 施肥 后 60 天和第二次施肥后60 天，大多数土壤中 抗生素 抗性基因的丰度差异并不显著。
       2）在以上实验的基础上，选择 4 种商品有机肥施用的土壤， 使用高通量荧光定量 PCR 和 Illumina 测序 研究抗生素抗性组 和 细菌群落组成的变化 。 结果表明 商品有机肥的施用增加了土壤中广泛的 抗生素 抗性基因和可移动基因元件的多样性和丰度。 第一次施肥和第二次施肥促进了土壤中 β 内酰胺类和万古霉素类抗性基因的富集。 Procrustes 分析和 mantel 检验显示 抗生素 抗性基因组成和细菌群落组成显著 正 相关 ，表明微生物群落组成可能是土壤 抗生素 抗性基因组成的决定因素。 网络分析进一步证明 抗生素 抗性基因和可移动基因 元 件 显著正相关 ，暗示 商品有机肥施用的土壤中 抗生素 抗性基因 存在 较高的水平转移潜力。
        3)本实验研究与施用堆肥相比，将猪粪堆肥转化为生物炭是否可以降低 抗生素 抗性基因向土壤的传播。 通过 盆栽实验种植小白菜， 使用 两种猪粪堆肥和这两种 猪粪 堆肥烧制的生物炭作为有机肥料。 高通量 荧光 定量 PCR 和 Illumina 测序评估种植期间土壤中抗生素抗性组 、 可移动基因 元 件和细菌群落组成的分布 。在小白菜种植期间 施用堆肥烧制的生物炭的土壤中 所有 抗生素 抗性基因和可移动基因 元 件的丰度显著低于施用堆肥的土壤 。 施用生物炭的土壤中 所有抗生素抗性基因和可移动基因 元 件的丰度与对照土壤之间不存在显著差异 。 因此 将畜禽粪便转化成生物炭可以有效缓解 抗生素抗性基因向环境的传播。

         Antibiotic resistance is one of the most important public health challenges of the 21st century. A ntibiotics are widely used to prevent or treat diseases and maintain or promote the growth of food animals, increasing selection pressure on bacteria to become resistant , particularly in swine and chicken farms Growth in demand for animal protein has driv en the bloom of the global livestock industry. Accordingly, large amounts of manure are produced every year. In recent years, a nationwide campaign against pollutants from intensive livestock industry has driven the production and sale of commercial organic fertilizers (COFs) from animal farms.
        With rising consumer demand for organic food in the world, the use of manure based COFs is increasing, especially in China. At present, the agricultural industr y standards NY525 2012 ) govern the acceptable pH, and number of fecal coliforms of manure based COFs, but antibiotics and antibiotic resistance genes ( ARGs are not regulated in China. This study was to explore the occurrence and fate of antibiotics in soils following the repeated use of COFs , characterize the occurrence and shift of the ARG content s after repeated applications of COFs , and investigate change s in the composition of bacterial communities and deter mine any association s with the ARG content s In addition, this study provided an environmentally and economically sustainable technology , t urning composted manure into biochar to mitigate dissemination of ARGs from animal waste to the environment .
        1)Ten manure based COF samples were obtained from nine provinces of China. This study examined the effects of repeated applications of chicken or swine manure based COFs on the fate of antibiotics and ARGs in soil by conducting a soil microcosm experiment. T he study demonstrated that the application of COFs which have been harmlessly disposed still resulted in general increases of antibiotics and ARGs in soils. T hese antibiotics persisted in COF amended soils for 4 months, and they accumulated with repeated a pplications. However, the relative abundance of ARGs did not differ significantly in most of the COF amended soils between the first application and the repeated application.
        2)Four COF amended soils were chosen to investigate the shift of the antibiotic resistome and change s in the bacterial community composition , using h igh throughput quantitative PCR and Illumina sequencing . A pplication s of COFs increased the diversity and abundance of a wide spectrum of ARGs and mobile genetic elements (MGEs) in the so il. The first and second COF applications enhanced the proliferation of genes encoding resistance to beta lactam and vancomycin. Procrustes analysis and mantel test revealed that ARG profiles were significantly correlated with bacterial community compositi on, indicating that the microbial community composition might be a determinant of the soil resistome. Network analysis further demonstrated significant associations between ARGs and MGEs, highlighting the likelihood of the horizontal gene transfer of ARGs in the COF amended soils.
        3) This study investigated whether converting composted pig manure in to biochar could reduce the dissemination of ARGs into the soil in comparison with a compost amendment. We performed a pot experiment using pakchoi Brassica chinensis )), with two pig manure based composts and the biochar derived from composted pig manure, as organic fertilizers . The distributions of the antibiotic resistome, MGEs and bacterial community composition in soil s during cultivation were evaluated by high throughput qPCR and Illumina sequencing T he total ARGs and MGEs abundance in the biochar treated soils were significant ly lower than those in the compost amended soils during cultivation. The total ARGs abundance in the biochar amended soils w as simi lar to that in the control soils during cultivation. Thus,the dissemination of ARGs from animal waste to the environment can be effectively mitigated by converting manure into biochar.