生物电化学系统阴极还原降解典型抗生素研究

哈尔滨工业大学工学博士学位论文

摘要 ....................................................................................................................... I ABSTRACT ............................................................................................................ I II 第1章绪论 (1)

1.1课题来源 (1)

1.2抗生素的危害及在环境中的分布 (1)

1.2.1 抗生素的种类 (1)

1.2.2 抗生素及其抗性基因的危害 (2)

1.2.3 抗生素在环境中的分布 (2)

1.2.4 几种典型抗生素的危害及其水中残留 (3)

1.3抗生素降解研究进展 (6)

1.3.1 生物处理法 (6)

1.3.2 高级氧化法 (9)

1.3.3 含硝基或卤代类抗生素降解研究进展 (10)

1.4生物电化学系统阴极降解污染物研究进展 (12)

1.4.1 生物电化学系统原理 (12)

1.4.2 阴极还原降解污染物研究进展 (14)

1.4.3 生物阴极降解污染物研究进展 (16)

1.4.4 生物阳极反转为生物阴极研究进展 (18)

1.5本论文研究背景、目的和意义 (19)

1.6本论文研究内容和技术路线 (21)

1.6.1 主要研究内容 (21)

1.6.2 研究技术路线 (21)

第2章实验材料与方法 (23)

2.1生物电化学反应器构型及实验装置 (23)

2.1.1 生物电化学反应器构型 (23)

2.1.2 实验装置 (24)

2.2反应器的启动与运行 (25)

2.2.1 阴极电化学还原含硝基或卤代类抗生素 (25)

2.2.2 生物阴极还原降解呋喃西林 (26)

2.2.3 生物阳极反转生物阴极还原降解氯霉素 (27)

2.3实验试剂及培养液配方 (29)

2.3.1 实验试剂 (29)

2.3.2 培养液配方 (29)

2.4电化学分析方法 (30)

2.4.1 循环伏安分析 (30)

2.4.2 交流阻抗分析 (30)

2.5化学分析方法 (30)

2.5.1 抗生素还原及产物定量分析 (30)

2.5.2 抗生素还原产物定性分析 (30)

2.5.3 定量分析卤离子回收率 (31)

2.6抗生素及其还原产物抑菌活性分析 (31)

2.7生物膜扫描电镜分析 (31)

2.8微生物群落结构解析 (32)

2.8.1 生物膜样品的总DNA提取 (32)

2.8.2 Illumina MiSeq测序数据分析 (33)

2.9计算方法及统计学分析 (33)

2.9.1 阴极电流和抗生素还原降解速率 (33)

2.9.2 微生物多样性指数分析 (34)

2.9.3 Student t检验统计学分析 (34)

第3章阴极电化学还原降解典型抗生素 (35)

3.1引言 (35)

3.2五种典型抗生素紫外吸收和循环伏安特征 (35)

3.2.1 紫外吸收特征 (35)

3.2.2 循环伏安特征 (36)

3.3抗生素还原降解效能的关键影响因素 (37)

3.3.1 不同阴极电位对于抗生素还原效能的影响 (37)

3.3.2 缓冲盐浓度和种类对于抗生素还原效能的影响 (40)

3.3.3 抗生素浓度对其还原效能的影响 (42)

3.4抗生素还原产物的分析鉴定 (43)

3.4.1 呋喃西林的还原产物 (44)

3.4.2 呋喃唑酮的还原产物 (45)

3.4.3 甲硝唑的还原产物 (46)

3.4.4 氯霉素的还原产物 (47)

3.4.5 氟苯尼考的还原产物 (48)

3.5不同阴极电位下抗生素还原产物形成规律 (49)

哈尔滨工业大学工学博士学位论文

3.5.1 呋喃西林和呋喃唑酮还原产物形成规律 (50)

3.5.2 甲硝唑还原产物形成规律 (51)

3.5.3 氯霉素和氟苯尼考还原产物形成规律 (52)

3.6抗生素还原降解路径分析 (55)

3.6.1 呋喃西林和甲硝唑的还原路径 (55)

3.6.2 呋喃唑酮的还原路径 (56)

3.6.3 氯霉素和氟苯尼考的还原路径 (56)

3.7抗生素还原产物的抑菌活性分析 (58)

3.7.1 呋喃西林和呋喃唑酮还原产物的抑菌活性分析 (58)

3.7.2 甲硝唑和氟苯尼考还原产物抑菌活性分析 (59)

3.8本章小结 (60)

第4章生物阴极还原降解呋喃西林 (61)

4.1引言 (61)

4.2反应器的启动 (62)

4.2.1 降解呋喃西林富集液的驯化 (62)

4.2.2 生物阳极的启动 (63)

4.2.3 生物阴极的启动 (64)

4.3葡萄糖为碳源和电子供体下生物阴极还原降解呋喃西林 (65)

4.3.1 不同外加电压下阴极电位和电流的变化 (65)

4.3.2 不同外加电压对呋喃西林还原降解效能的影响 (66)

4.3.3 不同外加电压对呋喃西林还原降解产物生成的影响 (67)

4.4碳酸氢钠为外加碳源下生物阴极还原降解呋喃西林 (68)

4.4.1 不同外加电压下阴极电位和电流的变化 (68)

4.4.2 不同外加电压对呋喃西林还原降解效能的影响 (69)

4.4.3不同外加电压对呋喃西林降解产物的影响 (70)

4.5有机和无机碳源对生物阴极还原降解呋喃西林的影响 (72)

4.5.1 阴极电位和电流 (72)

4.5.2 还原降解速率 (73)

4.5.3 还原降解产物的生成 (73)

4.6不同外加电压影响生物阴极催化效能的电化学特征 (74)

4.6.1 循环伏安特征 (74)

4.6.2 交流阻抗特征 (75)

4.7不同外加电压下阴极生物膜群落结构分析 (76)

4.7.1 阴极生物膜的电镜观察 (76)

4.7.2 阴极生物膜群落多样性分析 (77)

4.7.3微生物群落结构分析 (79)

4.8本章小结 (81)

第5章生物阳极反转生物阴极还原降解氯霉素 (82)

5.1引言 (82)

5.2生物阳极的启动 (82)

5.3生物阳极微生物耐氯霉素的驯化 (83)

5.3.1 生物阳极微生物耐低浓度氯霉素的驯化 (83)

5.3.2 生物阳极微生物耐高浓度氯霉素的驯化 (84)

5.4阴阳极同步降解氯霉素 (86)

5.4.1 阴阳极同步降解氯霉素电位和电流变化 (86)

5.4.2 阴阳极同步降解氯霉素降解速率及产物形成规律 (86)

5.4.3 生物阳极与非生物阴极降解氯霉素的比较 (88)

5.5生物阳极反转生物阴极降解氯霉素 (89)

5.5.1 生物阳极反转生物阴极后电流的变化 (89)

5.5.2 生物阳极反转生物阴极后氯霉素的降解 (90)

5.5.3 生物阴极催化还原降解CAP及产物形成解析 (92)

5.6电极反转前后生物膜的电化学特征 (95)

5.6.1 反转前后循环伏安分析 (95)

5.6.2 反转前后交流阻抗分析 (96)

5.7电极反转前后微生物群落结构解析 (97)

5.7.1 微生物多样性指数分析 (97)

5.7.2 微生物群落结构分析 (98)

5.8小结 (101)

结论 (103)

参考文献 (106)

攻读博士学位期间发表的论文及其它成果 (124)

哈尔滨工业大学学位论文原创性声明和使用权限 (125)

致谢 (126)

个人简历 (127)

哈尔滨工业大学工学博士学位论文

Contents

Abstract (In Chinese) .............................................................................................. I Abstract (In English) ............................................................................................. I II Chapter 1 Introduction.. (1)

1.1Supporting fundings and project (1)

1.2The harm of antibiotics and distribution in environment (1)

1.2.1 The classficiation of antibiotics (1)

1.2.2 The harm of antibiotics and its resistance gene (2)

1.2.3 The distribution of antibiotics in environment (2)

1.2.4 The harm of several representative antibiotics and its distribution in

water environment (3)

1.3The research progress of antibiotics degradation (6)

1.3.1 Biological methods (6)

1.3.2 Anvanced oxidation process (9)

1.3.3 The research progress of representative antibiotics degredation (10)

1.4 The research of antibiotics degredation in bioelectrichemical system (12)

1.4.1 The principle of bioelectrochemical system (12)

1.4.2 The research progress of reductive degradation in BES cathode (14)

1.4.3 The research progress of reductive degradation in BES biocathode (16)

1.4.4 The research progress of bioanode inversion to biocathode (18)

1.5 Research background and significance (19)

1.6 Research contents and technology roadmap (21)

1.6.1 Research contents (21)

1.6.2 Technology roadmap (21)

Chapter 2 Material and methods (23)

2.1 The BES reactor configuration and other equipment (23)

2.1.1 The reactor configuration (23)

2.1.2 The used equipment (24)

2.2 The setup and performance of BES reactors (25)

2.2.1 Electrochemical reduction of antibiotics in cathode of BES (25)

2.2.2 The reduction and degredation of NFZ in biocathode (26)

2.2.3 The reduction and degredation of CAP in biocathode inversion from

bioanode (27)

2.3 Experiment reagent and medium constituent (29)

2.3.1 Experiment reagents (29)

2.3.2 Culture medium (29)

Contents

2.4 Electrochemical analytical methods (30)

2.4.1 Cyclic voltammetry (CV) (30)

2.4.2 Electrochemistry impedance spectroscopy (EIS) (30)

2.5 Chemical analytical methods (30)

2.5.1 Quantitative analysis of antibiotis and its reduced product (30)

2.5.2 Qualitative analysis reduced product of antibiotis (30)

2.5.3 Quantitative analysis of halide ion released from CAP and FLO (31)

2.6 The antibacterial activity analysis of antibiotics reduction products (31)

2.7 Analysis of the morphologyof biofilms with SEM (31)

2.8Microbial community structure and functiona genes analysis (32)

2.8.1 DNA extraction of biofilms (32)

2.8.2 Analysis of 16S rRNA genes based on Illumina MiSeq sequencing (33)

2.9Computing methods and statistical analysis (33)

2.9.1 Cathode current and CAP reductive degradation rate (33)

2.9.2 Analysis of microbial diversity indices (34)

2.9.3 Student t test statistical analysis (34)

Chapter 3 Electrochemical reduction and degradation of representative antibiotics in cathode (35)

3.1Introduction (35)

3.2 The UV absorption and cyclic voltammetry of five antibiotics (35)

3.2.1 The UV absorption of five antibiotics (35)

3.2.2 The cyclic voltammetry of five antibiotics (36)

3.3The research of efficacy on electrochemical reduction of five antibiotics (37)

3.3.1 The influence of different cathodic potential on antibiotics reduction (37)

3.3.2 The influence of different buffer solutions on antibiotics reduction (40)

3.3.3 The influence of different initial concentration on antibiotics reduction..42 3.4Analysis and identification for reduction product of antibiotics (43)

3.4.1 Reductive production of nitrofurazone (44)

3.4.2 Reductive production of furazolidone (45)

3.4.3 Reductive production of metronidazole (46)

3.4.4 Reductive production of chloramphenicol (47)

3.4.5 Reductive production of florfenicol (48)

3.5Analysis of antibiotics reduction production under different cathodic

potentials (49)

3.5.1 Analysis of reduction production of nitrofurazone and furazolidone (50)

3.5.2 Analysis of reduction production of metronidazole (51)

3.5.3 Analysis of reduction production of chloramphenicol and florfenicol (52)

3.6Analysis of reductive degradation routes for five antibiotics (55)

哈尔滨工业大学工学博士学位论文

3.6.1 Reductive degradation routes for nitrofurazone and metronidaole (55)

3.6.2 Reductive degradation routes for furazolidone (56)

3.6.3 Reductive degradation routes for chloramphenicol and florfenicol (56)

3.7 Characterization of antibacterial activity for reductive products. (58)

3.7.1 Antibacterial activity of reductive products of nitrofurazone and

furazolidone (58)

3.7.2 Antibacterial activity of reductive products from florfenicol and

metronidazole (59)

3.8 Brief summary (60)

Chapter 4 The research on reduction and degredation of nitrofurazone in biocathode (61)

4.1Introduction (61)

4.2The bioreactor setup (62)

4.2.1 The cultivation of nitrofurazone reduction enrichment (62)

4.2.2 The setup of bioanode (63)

4.2.3 The setup of biocathode (64)

4.3Nitrofurazone reduction and degredation with glucose as carbon source and

electron donor in biocathode (65)

4.3.1 The changes of potential and current in different applied voltage (65)

4.3.2 The influence of different applied voltage on the efficacy of nitrofurazone

reduction and degredation (66)

4.3.3 The influence of different applied voltage on reduction products (67)

4.4 The biocathodic reduction of nitrofurazone with NaHCO3 as carbon source 68

4.4.1 The changes of potential and current in different applied voltage (68)

4.4.2 The influence of different applied voltage on the efficacy of nitrofurazone

reduction and degredation (69)

4.4.3 The influence of different applied voltage on the reduction products (70)

4.5Comparison of biocathode with glucose and NaHCO3 (72)

4.5.1 Comparison of cathodic potential and current (72)

4.5.2 Comparison of reduction rate and efficiency (73)

4.5.3 Comparison of reduction production (73)

4.6Electrochemical characteristic for biocathode catalytic activity under different

applied voltage (74)

4.6.1 Cyclic voltammetry characteristic (74)

4.6.2 Electrochemical Impedance Spectroscopy characteristic (75)

4.7 The influence of different applied voltage on cathodophilic microbial communities (76)

Contents

4.7.1 Observation with Scanning Electron Microscope (76)

4.7.2 Diversity index analysis (77)

4.7.3 Analysis of microbial community (79)

4.8Brief summary (81)

Chapter 5 Reductive degradation of CAP with biocathode inverted from bioanode (82)

5.1Introduction (82)

5.2 The setup of bioanode (82)

5.3 The acclimiation of bioanode with chloramphenicol (83)

5.3.1 Acclimation of bioanode with low concentration of chloramphenicol (83)

5.3.2 Acclimation of bioanode with high concentration of chloramphenicol (84)

5.4 The synchronous CAP degradation with bioanode and abiotic cathode (86)

5.4.1 The changes of potential and current with synchronous CAP degradation

on anode and cathode (86)

5.4.2 The changes of degradation rate and product formation with synchronous

CAP degradation on anode and cathode (86)

5.4.3 Comparison of CAP degradation with bioanode and abiotic cathode (88)

5.5Analysis of CAP degradation with biocathode inverted from bioanode (89)

5.5.1 The changes of current at constant potential for biocathode inverted from

bioanode (89)

5.5.2 CAP degradation with biocathode inverted from bioanode (90)

5.5.3 The catalyst of biocathode on CAP reduction and production analysis (92)

5.6Electrochemical evidence for biofilm changes before and after polarity

invertion (95)

5.6.1 CV changes (95)

5.6.2 EIS changes (96)

5.7 Microbial community analysis of biocathode inverted from bioanode (97)

5.7.1 Diversity index analysis (97)

5.7.2 Analysis of microbial community (98)

5.8Brief summary (101)

Conclusions (103)

References (106)

Papers published in the period of Ph.D. education (124)

Statement of copyright and letter of authorization (125)

Acknowledgements (126)

Resume (127)

第1章绪论

1.1 课题来源

本课题来源于国家自然科学基金项目“生物电催化-水解酸化耦合强化难降解废水预处理新工艺及机制”（项目号：51078140）、国家杰出青年科学基金项目“嗜电极微生物强化污染物转化及定向代谢调控”（项目号：51225802）

1.2 抗生素的危害及在环境中的分布

1.2.1 抗生素的种类

抗生素（antibiotics）主要是由微生物在生长代谢过程中产生的次级代谢产物或发酵制品，它是具有干扰或杀死其它微生物或生活细胞的一种化学物质[1]，是主要用于治疗各种非病毒感染的药物。二战时期随着青霉素的首次应用于治疗细菌感染引起的疾病，越来越多的抗生素被研制和应用于人类健康，它的出现极大的延长了人类的寿命。目前成千上万种抗生素被研制出，应用于实际临床的也有上千种，其分类如表1-1所示[2]。而抗生素作用于细菌的机理主要有抑制DNA的合成或RNA的转录、阻碍蛋白质的表达、改变细菌细胞膜的通透性、阻碍细胞壁的形成、干扰能量代谢和叶酸代谢等。目前随着科技的发展，越来越多的新型抗生素被应用于医疗临床、畜禽和水产养殖等。

表1-1 抗生素的分类

Table 1-1 Classification of antibiotics

抗生素类别代表性抗生素

β-内酰胺类青霉素类、头孢菌素类、拉氧头孢、氟氧头孢、硫霉素、头孢西丁等

氨基糖苷类链霉素、庆大霉素、卡那霉素、妥布霉素、小诺霉素、阿米卡星等

酰胺醇类氯霉素、甲砜霉素等

大环内酯类红霉素、白霉素、乙酰螺旋霉素、麦迪霉素、阿奇霉素、罗红霉素等

磺胺类磺胺嘧啶、复方新诺明、磺胺甲噁唑、磺胺异唑、磺胺多辛等

硝基咪唑类甲硝唑、替硝唑、奥硝唑等

硝基呋喃类呋喃西林、呋喃唑酮、呋喃它酮、呋喃妥因等

喹诺酮类吡哌酸、诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、依诺沙星等

氯霉素类氯霉素、氟苯尼考

林克胺类林可霉素、克林霉素等

抗真菌类两性霉素B、制霉菌素、灰黄霉素、咪康唑、酮康唑、噻康唑等

四环素类四环素、金霉素、土霉素、多西环素、地美环素等