电离辐照去除抗生素菌渣中残留抗生素和抗性基因的作用机理研究

A large amount of residues was generated during the antibiotics fermentation production processes. The antibiotic fermentation residues were identified as hazardous wastes due to the fact that the residual antibiotics and antibiotic resistance genes (ARGs) induce and spread antibiotic resistance bacteria. It is urgent to find the safe and effective methods of treatment and disposal of the residues. Currently, most of studies focused on antibiotics removal. It is difficult to eliminate ARGs effectively by using the techniques commonly used. In the present project, we propose to apply ionizing irradiation to eliminate antibiotics and ARGs in the antibiotic fermentation residues simultaneously. The DNA in the microbial cells are destroyed by the direct effect of rays produced from ionizing irradiation and indirect reaction of the active species such as hydroxyl radicals and hydrated electrons generated by water radiolysis. As a result, the ARGs were eliminated. The main research contents include: the degradation properties of the antibiotics in the fermentation residues by ionizing irradiation; The impacts of direct and indirect effects of ionizing irradiation on ARGs removals; and the correlation between the chemical structure of antibiotics, the representative ARGs and the removal efficiencies by ionizing irradiation. The removal mechanism of antibiotics and ARGs by ionizing irradiation was elucidated. The combined methods with ionizing irradiation were proposed for the purpose to maintain the nutrition substances such as protein and polysaccharides as much as possible in addition to eliminate ARGs and antibiotics. The results of this project will provide the scientific foundation and techniques for the harmless treatment and resource utilization of antibiotics fermentation residues as well as elimination of ARGs in the environment.

抗生素类药物发酵生产过程中产生大量的抗生素菌渣。菌渣中残留的抗生素和抗性基因（ARGs）会诱发和传播耐药菌，危害生态安全，因此被定为危险废物，其安全处置是急需解决的难题。目前去除菌渣中抗生素的研究较多，但对于ARGs，常规方法难以有效去除。本项目拟采用电离辐照技术，同时去除抗生素菌渣中残留抗生素和ARGs。利用电离辐照产生高能射线的直接作用，以及激发水分子产生羟基自由基和水合电子等活性粒子的间接作用，破坏微生物细胞的DNA，消除ARGs污染。主要研究内容包括：菌渣中抗生素的辐照降解特性，电离辐照直接和间接作用对AGRs去除的影响，抗生素的化学结构、代表性ARGs与辐照降解效率的相关性。阐明电离辐照去除抗生素和ARGs的机理。探索既有效去除抗生素和ARGs，又保留菌渣的蛋白质多糖等营养成分不流失的电离辐照协同作用方法。为抗生素菌渣无害化处理和资源化利用以及环境中ARGs的去除提供科学基础。

本项目研究了电离辐照对水中青霉素G、头孢菌素C和红霉素A的辐照降解机制；建立了抗生素抑菌活性、菌渣中抗生素及抗性基因提取检测方法。选择了三种抗生素菌渣：头孢、硫红和青霉素菌渣，研究了γ射线和电子束辐照对菌渣中抗生素和抗性基因的去除效率及其对菌渣中蛋白质、多糖含量等理化性质的影响；解析了电离辐照去除菌渣中抗生素和抗性基因的作用机理和微生物种群变化规律；研究了电离辐照与低温热解、PMS和双氧水氧化等技术协同强化去除菌渣中抗生素和抗性基因；比较了电离辐照与臭氧氧化、高温热水解等技术对抗生素菌渣的处理效果；研究了辐照预处理对抗生素菌渣好氧堆肥的强化作用。在小试研究基础上，在我国西部某抗生素生产企业建立日处理量100-120吨的抗生素菌渣示范工程。为电离辐照抗生素菌渣无害化处理和资源化利用提供科学技术支持。.研究结果表明：电离辐照可有效去除菌渣中的抗生素和抗性基因。电离辐照对头孢菌渣中抗生素去除效果优于红霉素菌渣。在吸收剂量为50kGy，头孢菌素C的去除率可达90%，抗性基因tolC的去除率为50%左右。硫红菌渣在吸收剂量为30kGy时，抗生素和抗性基因的去除率分别为56%和90-95%。电离辐照对菌渣中抗性基因的去除作用机制为高能射线的直接辐射作用以及激发水分子产生·OH等自由基的氧化作用，抗生素的主要去除作用机制为自由基反应。菌渣中存在的蛋白质、多糖和乙酸对抗生素降解有较强的竞争抑制作用，且这种抑制作用在抗生素浓度较低时更为显著。硫红菌渣和头孢菌渣中丰度较高的细菌菌属为Clostridium, Aeromonas, Enterobacteriaceae, Morganella和Fusobacterium。抗性基因的宿主菌为Aeromonas和Enterobacteriaceae，其丰度辐照后有所降低。电离辐照过程中加入PMS或者双氧水对菌渣中抗生素和抗性基因去除有较好的促进作用。小试和示范工程运行结果表明，电离辐照与低温热处理耦合（50kGy-90℃/2h）以及高温热水解耦合（50kGy-150℃/15min），可将头孢和硫红菌渣中抗生素浓度降至液相色谱无法检出。电离辐照对抗生素菌渣的性质尤其是营养物质含量没有显著影响。电子束辐照预处理可促进菌渣好氧堆肥腐殖化进程，堆肥产品中无抗生素残留，有机质指标远高于有机肥料国家标准，可作为优质肥料进行安全再利用。