海岸带生物学与生物资源保护实验室知识库

铁还原菌和产甲烷古菌分布于湿地生态系统中，在其生物地球化学碳、铁循环过程中发挥着重要作用。研究发现，导电材料（如磁铁矿，活性炭和碳布等）可以促进地杆菌与产甲烷古菌之间直接的电子传递，从而在一定程度上提高甲烷产生效率。目前关于“导电材料促进微生物产甲烷机制”的发现主要基于实验室纯培养体系，然而该机制在自然环境体系中是否存在及其对生物地球化学循环的影响尚不明确。本研究以黄河三角洲滨海湿地土壤为研究对象，综合利用土壤学、微生物学、电化学等学科的研究手段，阐明了导电材料碳布对湿地铁还原菌和产甲烷古菌群落代谢活性的影响及机制；探究了新型污染物非导电材料微塑料对铁还原菌铁还原能力和电活性的影响；揭示了导电材料碳布对铁还原菌还原氯霉素的影响及机制。解析导电材料对产甲烷古菌与地杆菌生理活性的影响及机制，有助于深入认知黄河三角洲滨海湿地环境中微生物驱动的碳、铁生物地球化学循环，以及新型污染物微塑料和抗生素（氯霉素）对该过程的影响及电子传递机制，为黄河三角洲滨海湿地生态系统的保护和可持续发展提供理论支持。本研究的主要内容和结论如下：

（1）导电碳布对湿地铁还原菌和产甲烷古菌群落代谢活性的影响及机制。研究发现，导电碳布的添加，可以显著提高甲烷产量。电化学方法分析结果显示，添加碳布的实验组中电子转移速率是对照组的3倍，分别为0.0056±0.0015 s^-1和0.0017±0.0003 s^-1，表明碳布显著促进了湿地样品的电子转移速率；通过添加乙酸型产甲烷抑制剂并结合稳定同位素分析发现，碳布主要是通过促进乙酸型产甲烷途径促进甲烷产生；培养结束后，高通量测序表明，碳布的添加对菌群结构没有显著的影响，其优势细菌为Mobilitalea spp.和假单胞菌Pseudomonas spp.；古菌群落中优势菌种为甲烷八叠球菌Methanosarcina spp.。

（2）非导电材料——微塑料PBAT和PVC对地杆菌驱动的生物地球铁循环的影响。当无定形铁作为电子受体时，没有添加微塑料的对照组与添加PBAT实验组中，Fe(II)的生成量分别为16.82和16.79 mM，没有显著差异；然而在添加PVC的实验组中，Fe(II)含量仅为6.81 mM，表明PVC可以显著抑制地杆菌G. metallireducens GS15对不溶性铁的利用；而微塑料的添加对该菌还原可溶性柠檬酸铁的能力没有显著影响。此外，产电活性实验表明，PBAT和PVC的添加可以使G. metallireducens GS15产电滞后。SEM，EDS和XRD对微塑料表面形貌的表征结果发现，与添加PBAT的实验组相比，仅有少量的含铁矿物附着在PVC表面。地杆菌还原不同形态的铁时电子传递方式不同，与游离态的三价铁相比，不溶性铁氧化物不易于被微生物利用，因此PVC的添加对无定形铁的影响更为显著。

（3）导电碳布对地杆菌降解氯霉素（Chloramphenicol, CAP）的影响。循环伏安法和计时安培法证实，G. sulfurreducens PCA降解氯霉素的能力受氯霉素初始浓度、温度、导电材料、电场作用的影响。微生物活性随着氯霉素浓度升高而逐渐降低，当初始浓度大于20 mg/L时，氯霉素对G. sulfurreducens PCA的脱氯能力和生长情况具有明显的抑制作用；温度为37℃时，其对G. sulfurreducens PCA的抑制作用减弱，氯霉素的降解效率得到显著提高；同时，导电碳布具有比表面积高，导电性好等优点，其添加有助于氯霉素的去除；此外，G. sulfurreducens PCA作为电活性微生物，可利用阴极作为电子供体实现氯霉素的还原，并且阴极电压越负，氯霉素的还原效率越高；当乙酸钠和电极（-0.6 V vs SHE）同时作为电子供体时，氯霉素的还原速率进一步提高。

Iron (III) reducing bacteria (IRB) and methanogens distribute in the wetland ecosystem, involving biogeochemical carbon (C) and iron (Fe) cycle process. Extensive studies have suggested that conductive materials, such as magnetite, activated carbon and carbon cloth, are conducive to direct interspecies electron transfer (DIET) between IRB and methanogens, which could facilitate methane production. Most studies focus on the effect mechanisms of conductive materials on electron transfer and methane production via pure culture. While, it is uncertain whether these findings are appropriate for on environmental samples. In this study, to elucidate the effects of conductive materials on the physiological activities of IRB and methanogens, we investigated the effect and mechanisms of carbon cloth on methanogenic processes in wetland soil. In the meantime, the ability of iron (III) reduction and electrochemical activity as well as chloramphenicol degradation ability of IRB were evaluated in the existence of non-conductive microplastics and carbon cloth, respectively. To clarify the effects and mechanisms of conductive materials on activities of methanogens and Geobacter is conducive to enhance the further understanding of their contribution to microbial mediated C, Fe biogeochemical cycles process. Moreover, the findings about the effects of microplastics and antibiotic (CAP) on activities of IRB provide theoretical support the protection and sustainable development of the coastal wetland ecosystem in the Yellow River delta. The main conclusions are summarized as follows:

(1) In this study, the effect of carbon cloth, as a representative of conductive carbon material, on methane production with incubated wetland soil was investigated. Carbon cloth significantly promoted methanogenesis. With the application of electrochemical technology, calculation of the apparent electron transfer rate constant showed that carbon cloth significantly increased electron transfer rate (k_app) compared with the control experiment, from 0.0017 ± 0.0003 to 0.0056 ± 0.0015 s⁻¹. Results obtained from both stable carbon isotope measurements and application of specific inhibitor (CH₃F) for acetoclastic methanogenesis indicated that carbon cloth obviously promoted acetoclastic methanogenesis instead of CO₂ reduction. High-throughput sequencing showed that carbon cloth did not appear to alter the community structure of methanogenic archaea to any significant extent. Mobilitalea spp. and Pseudobacteroides spp. were the most abundant bacteria in both treatment groups and Methanosarcina spp., showed the highest abundance of methanogens.

(2) The effects of two typical microplastics, PBAT and PVC, were explored on the activity of G. metallireducens GS15 with ferrihydrite or ferric citrate as respective electron acceptors. The results showed that the iron (II) contents in PBAT and PVC treatment groups were 16.79 mM and 6.81 mM, respectively at the end of the experiment. Compared with PBAT treatment group, SEM-EDS revealed that merely small amount of iron-containing products covered the surface of PVC. Moreover, PBAT and PVC could both retard the electroactivity of G. metallireducens GS15 at the beginning of microbial fuel cell operation. Based on the results above, microplastic PVC might exhibit potential inhibition of the iron cycling process driven by G. metallireducens GS15 with ferrihydrite as the terminal electron acceptor. Geobacter spp. have evolved different mechanisms for electrons transfer with various iron (III) oxides. Compared with insoluble iron (III), microbes are prone to use soluble iron (III). Hence, the effect of PVC on ferrihydrite reduction was more obvious.

(3) As typical iron reduction bacteria, Geobacter sulfurreducens PCA was adopted to remove chloramphenicol (CAP) in the presence of carbon cloth. Cyclic voltammograms and chronoamperometry highlighted a higher peak current for CAP reduction by G. sulfurreducens PCA compared to the control without bacteria. The results showed that initial CAP concentration, temperature, the conductive material and cathode potential could influence the degradation efficiency of CAP. Microbial activity decreased with the increase in CAP concentration. There was obvious inhibition effect on Geobacter activities when the initial concentration of CAP was greater than 20 mg/L. In the meantime, the inhibition effect of CAP was attenuated with the increase of temperature and the addition of conductive carbon cloth. In addition, G. sulfurreducens PCA as a typical electroactive microorganism could reduce CAP with cathode as the terminal electron donor. Applied voltage had a significant influence on CAP reduction. The lower the cathodic potential, the higher CAP reduction efficient was achieved. The degradation rate of CAP was further improved with the addition of sodium acetate at -0.6 V vs SHE.

第1章 绪论... 1

1.1 选题背景和意义... 1

1.2 胞外呼吸菌的分布及分布... 3

1.3 微生物胞外电子传递... 3

1.3.1 电子穿梭体介导的胞外电子传递... 4

1.3.2 细胞色素c介导的胞外电子传递... 6

1.3.3 纳米导线介导的胞外电子传递... 7

1.3.4 螯合作用介导的胞外电子传递... 8

1.3.5 应电运动介导的胞外电子传递... 8

1.4 微生物种间直接电子传递... 9

1.4.1 细菌与细菌之间的电子传递... 10

1.4.2 细菌与古菌之间的电子传递... 10

1.5 导电与非导电材料介导的电子传递... 11

1.5.1 导电材料介导的电子传递... 11

1.5.2 非导电材料介导的电子传递... 12

1.6 胞外呼吸菌的应用... 12

1.6.1 介导有机污染物的降解... 14

1.6.2 介导重金属的污染修复... 15

1.6.3 介导的微生物电化学系统... 15

1.6.4 影响甲烷的产生... 16

1.7 本论文研究意义、内容与技术路线图... 17

1.7.1 研究意义... 17

1.7.2 研究内容... 18

1.7.3 技术路线... 19

第2章 导电材料对黄河三角洲湿地土壤产甲烷古菌甲烷产生途径的影响及机理.... 21

2.1 引言... 21

2.2 材料和方法... 22

2.2.1 微宇宙培养... 22

2.2.2 导电碳布电活性表面积的计算... 24

2.2.3 土壤物理性质测定... 24

2.2.4 CH₄, CO₂的测定... 24

2.2.5 气体稳定同位素比质谱仪... 24

2.2.6 有机酸的测定... 25

2.2.7 电化学活性的测定... 25

2.2.8 DNA提取和16S rRNA基因测序... 26

2.2.9 统计学分析... 27

2.3 结果与分析... 27

2.3.1 添加导电碳布对甲烷产生的影响... 27

2.3.2 导电碳布对土壤样品中电子传递的影响... 28

2.3.3 碳布对¹³C/¹²C-CH₄同位素分馏的影响... 29

2.3.4 乙酸型产甲烷抑制剂对甲烷产生的影响... 30

2.3.5 碳布对土壤样品微生物群落结构的影响... 31

2.4 讨论... 33

2.5 小结... 36

第3章 非导电材料微塑料对地杆菌介导的生物地球铁循环的影响.... 37

3.1 引言... 37

3.2 材料和方法... 39

3.2.1 菌种培养... 39

3.2.2 无定形铁的合成... 41

3.2.3 Fe(II)的测定... 41

3.2.4 G. metallireducens GS15电化学活性的测定... 42

3.2.5 微塑料表面形貌表征... 42

3.2.6 数据处理... 43

3.3 结果与分析... 43

3.3.1 微塑料对G. metallireducens GS15铁还原能力的影响... 43

3.3.2 微塑料PBAT和PVC对G. metallireducens GS15电活性的影响... 45

3.3.3 微塑料表面形态及结构表征... 46

3.4 讨论... 49

3.5 结论... 51

第4章 导电材料对地杆菌降解氯霉素的影响及机制... 53

4.1 引言... 53

4.2 材料和方法... 55

4.2.1 菌种培养... 55

4.2.2 实验设计... 56

4.2.3 G. sulfurreducens PCA电化学表征... 56

4.2.4 G. sulfurreducens PCA OD₆₀₀的测定... 57

4.2.5 氯霉素的测定... 58

4.2.6 氯霉素降解产物的测定... 58

4.2.7 微生物电解池的构建... 58

4.3 结果与讨论... 59

4.3.1 地杆菌G. sulfurreducens PCA降解氯霉素的电化学性质... 59

4.3.2 初始浓度对G. sulfurreducens PCA还原氯霉素的影响... 60

4.3.3 温度对氯霉素降解的影响... 64

4.3.4 导电材料碳布对G. sulfurreducens PCA降解氯霉素的影响... 67

4.3.5 阴极强化G. sulfurreducens PCA还原氯霉素... 69

4.4 本章小结... 75

第5章 结论与展望... 77

5.1 结论... 77

5.2 主要创新点... 79

5.3 不足与展望... 79

参考文献... 81

致谢... 103

作者简历及攻读博士期间发表的学术论文及成果... 105