微藻-漆酶耦合体系对磺胺类抗生素的降解机理研究

Degradation and removal of antibiotics in the environment is the key step to solve the problem such as antibiotic resistance. Sulfonamide antibiotics can be stored and accumulated in the water environment for a long time, which highlights the necessity to control them from the source. Research on antibiotic degradation by laccase oxidation developed greatly in recent years. However, it often needs to add mediator materials which are often toxic to the ecosystem to the laccase oxidation system in antibiotic degradation. This situation limited the further development of laccase oxidation technology. Aiming at the situation of the frequent detection of sulfonamide antibiotics in water environment, this project is to carry out the research of sulfonamide antibiotic degradation by laccase coupled with microalgae. Firstly, the optimal conditions and parameters of sulfonamide antibiotic degradation in microalgae-laccase coupling system are to determine by orthogonal experiments to understand the degradation law in the coupling system. Then, the degradation mechanism is to clarify through two aspects: On the one hand, to verify the mediator effect of microalgae through comparing the degradation differences of microalgae and its extracellular organic matters(EOM) in coupling system and by determining the changes of reactive oxygen species(ROS) in the coupling system. And then tracking the phenolic metabolites of microalgae EOM which may act as the mediator by infrared spectroscopy and metabolomics, etc. On the other hand, to track the degradation pathway and degradation products of sulfonamide antibiotics in the coupling system by means of liquid chromatogram tandem mass spectrometry(LC-MS), etc. The results could provide scientific basis for the degradation and removal practice of sulfonamide antibiotics in the environment.

环境中抗生素的降解和去除是解决环境致病菌耐药性等问题的关键步骤。磺胺类抗生素能够在水环境中长期储存并累积，凸显了其从源头上控制的必要性。漆酶氧化降解抗生素的研究近年得到较大发展，但由于漆酶降解抗生素往往需要添加人工介体，其存在的生态毒性等不足限制了该技术的进一步发展。本项目针对水环境中磺胺类抗生素被频繁检出的现状，采用漆酶与微藻耦合进行磺胺类抗生素的降解研究。首先通过正交实验确定微藻-漆酶耦合体系中磺胺类抗生素降解的优化条件与参数，认识其作用规律；进而通过以下两方面阐明降解机理：一方面通过对比微藻及其胞外分泌物对抗生素的降解差异和测定耦合体系中活性氧自由基含量的变化来验证微藻的介体作用；进而利用红外光谱和代谢组学等方法追踪微藻可能起介体作用的酚类代谢产物；另一方面，采用液质联用等方法探明耦合体系中磺胺类抗生素的降解途径及降解产物。研究成果可为环境中磺胺类抗生素的降解去除实践提供科学依据。

本项目针对水环境中磺胺类抗生素被频繁检出的现状，采用漆酶与微藻耦合进行磺胺类抗生素的降解研究，旨在从漆酶筛选和介体筛选两方面改进漆酶-介体系统降解抗生素的技术研究与应用实践。研究首先考察了微藻-漆酶降解抗生素反应体系各要素间的相互作用。结果表明，漆酶对微藻的生态毒性效应较小，反应体系中磺胺类抗生素也不会对微藻的生长产生明显的抑制作用。本研究考察了微藻-漆酶耦合体系降解磺胺类抗生素的效果及参数优化。正交实验发现，在某些条件下（均为高漆酶浓度），磺胺类抗生素的降解率达到了40%-60%。此时抗生素降解率的主要贡献因子是漆酶，说明活体微藻胞外分泌物产生介体作用的效果并不显著。微藻胞内提取物—漆酶耦合体系对磺胺嘧啶的降解研究表明，微藻细胞内存在介体物质，与漆酶耦合对磺胺嘧啶有一定的降解能力，此时漆酶的使用量仅为活体微藻-漆酶耦合体系的1/10左右。本研究通过非靶标代谢组学分析了几种典型微藻胞外分泌物的酚酸类物质，这些酚酸类物质很可能对微藻-漆酶耦合体系产生介体作用加速体系抗生素降解。综合分析，微藻胞内或胞外分泌物产生介体作用的化合物浓度是体系抗生素降解率高低的关键因素，并与漆酶的使用量密切相关。采用非靶标代谢组学筛选对比了7种不同天然介体-漆酶降解体系对磺胺嘧啶的降解能力，并进一步深入研究了漆酶-丁香醛（介体）系统降解水产养殖水体磺胺类抗生素的参数优化及降解机理。经广泛筛选，本研究采用Novozym 51003工业化漆酶和丁香醛联合降解水产养殖污水中的磺胺类抗生素。利用响应面优化法得到了体系最佳降解参数，并探究了漆酶-丁香醛体系降解磺胺嘧啶的酶促反应动力学及降解途径和降解产物，结果可为抗生素类环境污染物的降解去除提供一种具有实践意义的技术方法。本研究还对典型环境中抗生素抗性基因的环境驱动因子开展了研究，研究表明，除抗生素外，环境中的重金属（如铜、铅等）也可能成为环境抗生素抗性基因传播扩散的重要驱动因子。