摘要

       中国是世界上最大的抗生素生产国和消费国。随着制药行业的发展,产生了大量的制药污泥,若处置不当,容易引发抗生素(antibiotic)和抗生素抗性基因(antibiotic resistance genes简称ARGs)的富集,对环境和人类健康造成潜在威胁。另一方面,人们对绿色产品的需求,特别是对有机蔬菜的需求一直在快速增长。然而,有机生产过程中引入更多的ARGs和抗生素抗性菌(antibiotic resistance bacteria简称ARB)的风险往往被忽视。本研究以青霉素制药污泥堆肥化为基础研究,同时进一步研究了施用不同污泥肥料对土壤及作物根和叶表中ARGs多样性和丰度的影响,为青霉素制药污泥资源化利用提供理论基础。主要研究结果概括如下:(1)以制药污泥为研究对象,采用葡萄糖、蔗糖、玉米秸秆粉及其混合物作为外加碳源和添加竹炭,研究不同类型的外加碳源及竹炭对堆肥系统一次发酵周期内温度、有机质等理化参数变化及青霉素的降解情况的影响。结果表明,堆体中有机质含量与外加碳源的量呈正比,堆体中有机质的质量分数随堆肥时间不断下降且趋于稳定。温度是青霉素降解的主要影响因素。外加碳源增加了堆体溶解性有机质质量分数,生物可利用碳源的增加促进了堆肥过程中微生物的转化作用,并有助于提高堆肥过程温度。在堆肥周期内外加碳源可以提高青霉素的降解速率(15 d内对照组青霉素降解率为94.44%,其他组均大于95%),且外加蔗糖与玉米秸秆粉的混合碳源处理组青霉素降解速率最快(15 d内降解率可达到99.08%)。堆肥过程中升温阶段(中温阶段和高温阶段)青霉素含量与温度呈负相关(P0.01),与溶解性有机碳呈正相关(P0.01)。15d内所有处理组青霉素降解率均可以达到90%以上,同时筛选出污泥肥料为T1-2、T2-2、T3-1。(2)基于实时荧光定量PCR的方法,对堆肥过程中各阶段堆肥样品中β-内酰胺类抗性基因(bla_(-CTX-M-1)、bla_(-CMY)、bla_(-OXA-23)、bla_(-TEM)、bla_(-NDM-1)和bla_(CEP-02)基因)进行定性和绝对定量,初步探讨制药污泥堆肥过程中β-内酰胺类抗性基因(bla_(-CTX-M-1)、bla_(-CMY)、bla_(-OXA-23)、bla_(-TEM)、bla_(-NDM-1)和bla_(CEP-02)基因)的数量变化。研究发现,高温好氧堆肥对β-内酰胺类抗性基因(bla_(-TEM)、bla_(-NDM-1)和bla_(CEP-02)基因)数量有一定的消减作用,但是对于堆肥过程中ARGs的多样性和堆肥条件的控制仍需进一步研究。(3)通过室内盆栽实验研究了施用污泥肥料对青菜非根际土、根际土以及作物根和叶表面ARGs的影响。实验结果表明,污泥肥料能够增加非根际土、根际土、根和青菜(苏州青)叶际的ARGs丰度和多样性。Veen分析发现污泥肥料、非根际土、根际土、根和叶际样品中特有和共有的ARGs,说明污泥肥料施用后的土壤是叶际的ARGs的一个主要来源。对不同处理的非根际土、根际土、根和叶际共有操作分类单元(OTUs)与其携带的ARGs进行采用Mantel test和Procrustes test的分析,说明共有的OTUs对叶际的ARGs的组成有显著的影响。综合分析,可以推断出污泥肥料中的ARGs可以以微生物为媒介,通过土壤-植物体系进行扩散,最终转移到植物叶际,而在这一过程中根发挥着桥梁纽带的作用。

       China is the world's largest producer and consumer of antibiotics. With the development of the pharmaceutical industry, a large amount of pharmaceutical sludge is produced. If it is not handled properly, it is easy to cause the accumulation of antibiotics and antibiotic resistance genes (ARGs), which poses a potential threat to the environment and human health. On the other hand, people's demand for green products, especially organic vegetables, has been growing rapidly. However, the risk of introducing more ARGs and antibiotic resistance bacteria (ARB) in the organic production process is often overlooked. This study is based on the composting of penicillin pharmaceutical sludge, and at the same time, further studies the effects of applying different sludge fertilizers on the diversity and abundance of ARGs in soil and crop roots and leaf surfaces, providing a theory for the resource utilization of penicillin pharmaceutical sludge Base. The main research results are summarized as follows: (1) Taking the pharmaceutical sludge as the research object, using glucose, sucrose, corn stover powder and their mixtures as the external carbon source and adding bamboo charcoal, to study different types of external carbon sources and the primary fermentation of bamboo charcoal in the composting system The influence of temperature, organic matter and other physical and chemical parameters in the cycle and the degradation of penicillin. The results show that the organic matter content in the pile is proportional to the amount of external carbon source, and the mass fraction of organic matter in the pile keeps decreasing and tends to be stable with the composting time. Temperature is the main factor affecting the degradation of penicillin. The addition of carbon source increases the mass fraction of dissolved organic matter in the heap, and the increase in bioavailable carbon source promotes the transformation of microorganisms in the composting process, and helps to increase the temperature of the composting process. Adding a carbon source during the composting cycle can increase the degradation rate of penicillin (the degradation rate of penicillin in the control group within 15 days is 94.44%, and that of the other groups is greater than 95%), and the degradation rate of penicillin in the treatment group treated with a mixed carbon source of sucrose and corn stover powder The fastest (the degradation rate can reach 99.08% within 15 days). During the composting process, the penicillin content was negatively correlated with temperature (P0.01) and positively correlated with dissolved organic carbon (P0.01) during the heating stage (medium temperature stage and high temperature stage). The degradation rate of penicillin in all treatment groups can reach more than 90% within 15 days, and the sludge fertilizer was selected as T1-2, T2-2, and T3-1. (2) Based on the method of real-time fluorescent quantitative PCR, the β-lactam resistance genes (bla_(-CTX-M-1), bla_(-CMY), bla_(-OXA- 23), bla_(-TEM), bla_(-NDM-1) and bla_(CEP-02) genes) were qualitatively and absolutely quantified, and the β-lactam resistance genes (bla_( -CTX-M-1), bla_(-CMY), bla_(-OXA-23), bla_(-TEM), bla_(-NDM-1) and bla_(CEP-02) genes) quantitative changes. Studies have found that high-temperature aerobic composting has a certain reduction in the number of β-lactam resistance genes (bla_(-TEM), bla_(-NDM-1) and bla_(CEP-02) genes), but it has a certain effect on the composting process. The diversity of ARGs and the control of composting conditions still need further study. (3) The effects of applying sludge fertilizers on the non-rhizosphere soil, rhizosphere soil and ARGs of crop roots and leaves were studied through indoor pot experiment. Experimental results show that sludge fertilizer can increase the abundance and diversity of ARGs in non-rhizosphere soil, rhizosphere soil, root and green vegetable (Suzhou green) leaf. Veen analysis found unique and shared ARGs in sludge fertilizer, non-rhizosphere soil, rhizosphere soil, root and leaf soil samples, indicating that the soil after sludge fertilizer application is a major source of leaf ARGs. The analysis of the non-rhizosphere soil, rhizosphere soil, root and leaf soil shared operational taxa (OTUs) and the ARGs carried by different treatments using Mantel test and Procrustes test shows that the common OTUs have a significant effect on the composition of leaf ARGs. Significant impact. Through comprehensive analysis, it can be inferred that ARGs in sludge fertilizers can use microorganisms as media, spread through the soil-plant system, and finally transfer to the leaf space of plants, and in this process the roots play a role as a bridge.

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