论文总字数：25332字

摘 要

近几年，我国的抗生素污染问题较为严重，其降解方案研究的重要性不言而喻。随之诞生了多种抗生素降解方法，但大部分仍存在明显缺陷。如光催化降解抗生素法，成本高、去除率低等问题仍存在；高铁酸盐化学催化法虽然去除率较高，但效率低、成本过高；而生物电化学法可同时做到电能回收与废水处理，符合绿色环保理念。这项技术成功应运化的关键之一在于高效电极生物膜的获取 。因此，若能利用稳定运行的生物电化学处理装置中的极室出水来定向筛选出抗生素高效降解菌，不仅可以解决抗生素废水的问题，还可以为能源回收技术开辟一条新的途径，在能源循环利用与菌株选择上降低成本。

由此，本论文以3个不同条件下运行的针对氯霉素处理的生物电化学装置中的阳极出水作为高效氯霉素降解菌的筛选菌源出发（菌源1、菌源2、菌源3），旨在通过定向筛选获得具有高效氯霉素降解能力的菌种为目的。实验以添加不同浓度氯霉素（30 mg/L、50 mg/L、60 mg/L）LB（Luria-Bertani）培养基作为特定选择培养基进行高效氯霉素降解菌种的分离；通过定向筛选后，在菌源1中分离到了3株不同菌落形态的菌种；在菌源2中分离到了2株不同菌落形态的菌种；在菌源3中分离到了3株不同菌落形态的菌种;随后，对这8株菌种分别进行了形态学观察、革兰氏染色、生长曲线的测定及16S rRNA分子生物学鉴定，确定此次共筛选出5株菌种（拉乌尔菌、肠杆菌、杆菌、克雷伯氏菌、假单胞杆菌）；最后，对这5株菌分别进行了氯霉素降解能力的验证，结果表明拉乌尔菌的降解能力最优。本论文将为生物电化学降解抗生素的应用提供一定的菌种来源和理论指导。

关键词：氯霉素 高效降解 菌株筛选 生物电化学法

Research on degradation of chloramphenicol-degrading bacteria

Abstract

In recent years, the problem of antibiotic pollution in China is serious, and the importance of the study of its degradation scheme is self-evident. Many antibiotic degradation methods were developed, but most of them still have obvious defects. Such as photocatalytic degradation of antibiotics, high cost, low removal rate still exist; Although the removal rate of ferrate catalysis is high, the efficiency is low and the cost is high. And the bioelectrochemical method can achieve the energy recovery and wastewater treatment at the same time, in line with the concept of green environmental protection. One of the keys to the successful application of this technology is the acquisition of efficient electrode biofilms. Therefore, if the water from the polar chamber in the stable bioelectrochemical treatment device can be used to select the bacteria with high efficiency for antibiotic degradation, it can not only solve the problem of antibiotic wastewater, but also open a new way for energy recovery technology and reduce the cost in energy recycling and strain selection.

Therefore, in this paper, the anodic effluent from three bioelectrochemical devices operated under different conditions for chloramphenicol treatment was used as the source of the screening of high-efficiency chloramphenicol degrading bacteria (source 1, source 2, and source 3), aiming to obtain the bacteria with high efficiency chloramphenicol degrading ability through directional screening. In the experiment, chloramphenicol (chloramphenicol) degrading strains were separated by adding different concentrations of chloramphenicol (30 mg/L, 50 mg/L, 60 mg/L) LB (luria-bertani) as specific selective medium. After directional screening, 3 strains of different colony forms were isolated from strain 1. Two strains with different colony morphology were isolated from strain 2. Three strains with different colony morphology were isolated from strain 3. Subsequently, morphological observation, gram staining, growth curve determination and 16S rRNA molecular biological identification were carried out on these 8 strains respectively, and it was determined that a total of 5 strains (raoulus, enterobacter, bacillus, klebsiella, pseudomonas) were screened out this time. Finally, the ability of chloramphenicol degradation of the five strains was verified, and the results showed that the degradation ability of raoulus was the best. This paper will provide some theoretical guidance for the application of bioelectrochemical degradation of antibiotics.

Key Words: Chloramphenicol High efficiency degradations Strain Screening Bioelectrochemical method

目 录

摘 要 I

Abstract II

第一章 文献综述 1

1.1 研究背景 1

1.2 氯霉素概述 2

1.2.1 氯霉素理化性质 2

1.2.2 氯霉素毒副作用 2

1.2.3 氯霉素污染现状 3

1.2.4 研究现状 3

1.3 氯霉素降解方式 3

1.3.1 传统物理法 3

1.3.2 光催化法 4

1.3.3 辐射降解法 4

1.3.4 水解法 4

1.3.5 Feton氧化法 4

1.3.6 生物降解法 4

1.4 研究内容及意义 5

1.4.1 本研究的主要内容 5

1.4.2 本研究的意义 5

第二章 材料与方法 6

2.1 实验材料 6

2.1.1 培养基 6

2.1.2 实验试剂 6

2.1.3 实验设备 6

2.2 实验方法 7

2.2.1 菌株筛选 7

2.2.2 显微镜观察 7

2.2.3 革兰氏染色 8

2.2.4 生长曲线的绘制 8

2.2.5 菌种 16S rRNA 鉴定 8

2.2.6 氯霉素降解能力验证 9

第三章 结果与讨论 10

3.1 形态学观察 10

3.1.1 各菌株形态统计 10

3.1.2 各菌属代表菌菌落形态图 10

3.2 革兰氏染色 11

3.3 生长曲线测定分析 12

3.4 菌种16S rRNA鉴定 13

3.5 氯霉素降解情况 15

第四章 结论与展望 18

4.1结论 18

4.2展望 18

参考文献 19

致谢 21

附录一 16s rRNA鉴定拼接序列 22

第一章 文献综述

1.1 研究背景

随着人类科研水平的提升,各类药品和个人护理用品(PPCPs)等生化制品使用逐年递增,他们破坏环境、对生态系统和人类本身的健康产生很大威胁。抗生素作为如今的生活必需品，在生活中产生化学方法合成的具有抗病原体的或干扰其它生物功能的化学物质[1]。世界各地的检测机构都发现了抗生素在环境中的残留，状况高低不一。尤其是水体环境中已频繁检测到该类化学药品的残留，而且残留量呈显著上升趋势[2]。由于含着许多生物毒性物质，抗生素废水具有生物化学降解的持久性，在生化处理过程中，可抑制微生物生长的活性抗生素降解情况较差，加之抗生素生产过程中废水排放的不连续性及较大的浓度波动，使降解处理难度进一步加大[3]。长期的低浓度抗生素的存在会对水体中的微生物群落产生影响，并通过食物链的传递作用影响高级生物，破坏生态系统平衡[4]。

抗生素滥用、抗生素环境污染的真正危害在于加剧细菌耐药性，引起加大恐慌的“超级细菌”[5]。抗生素刚刚研发出来的时候，是用来治疗疾病或免疫预防的，使人类医学产生了巨大变迁，但抗生素给人类带来健康便利的时候同时也带来了负面影响，由于人们盲目长时间的使用抗生素，细菌耐药性也在不断增加，抗病作用越来越小[6]。人类接下来面对的就是如何解决抗生素耐药性逐年变强的问题，今天不采取行动，明天就无药可用[7]，因此对于抗生素污染的控制极为重要。

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