IOCAS-IR
生物膜内硫酸盐还原菌代谢活性的原位实时监测及其初步应用研究
叶祥益
Subtype硕士
Thesis Advisor张盾
2020-06-05
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学硕士
Keyword硫酸盐还原菌,生物膜,代谢活性,原位检测,腐蚀 Sulfate-reducIng Bacteria, Biofilm, Metabolic Activity, In Situ Detection, Corrosion
Abstract

硫酸盐还原菌(Sulfate-reducing bacteria, SRB)作为最典型、研究最多、腐蚀性最强的微生物,其引起的腐蚀行为与其在金属材料表面形成生物膜有密切关系。本论文针对传统的生物膜内SRB活性测定方法无法区分材料表面生物膜内SRB和溶液中游离SRB的代谢活性差异,以及难以在现场大规模应用的问题,通过制备两类全固态离子选择性电化学微探针,构建了基于SRB代谢活性特征物质特异性选择识别的连续、原位检测方法,并借助有机荧光探针验证了SRB代谢活性检测的正确性和可行性,实现了惰性材料表面生物膜内外固载和游离SRB代谢活性的连续、实时、原位检测,初步揭示了惰性载体表面生物膜内外固载和游离SRB细胞代谢活性的变化规律及二者的差异特征,并实现了金属材料表面SRB生物膜形成初期膜内外固载和游离细胞代谢活性的连续、实时、原位监测。本研究的开展对揭示生物膜内外SRB代谢活性的变化规律及差异特征,以及SRB生物膜的相关腐蚀性机制具有重要意义,主要的研究结果如下:

1构建了一种对SRB代谢产生硫化物具有特异识别性的全固态硫化物选择性微探针。该微探针是以柔性银丝作为导电基底,通过电化学方法依次构建了层状还原态石墨烯和纳米结构Ag2S作为离子/电子转导层和硫化物选择性膜,优化了电化学沉积工艺,表征了相应的表面形貌,系统的研究了该全固态硫化物选择性微探针的线性范围,稳定性,选择性等性能,最后将其应用于海水和自来水样品检测。研究结果表明,全固态硫化物选择性微探针的检测范围为5×10-7-1×10-2 M,检测限达到1.78×10-7 M,对硫化物具有很好的选择性,具有良好的稳定性能。该高柔韧性的全固态硫化物选择性微探针在材料界面研究和深海环境检测中具有巨大的应用潜力

2)构建了一种全固态氢离子选择性微探针。该微探针是以柔性银丝作为导电基底,借助电化学沉积方法构建了层状还原态石墨烯离子/电子转换层,并在石墨烯层表面以三正十二胺(TDDA)作为中性载体,加入四苯基硼酸钾(KTPB)、葵二酸二辛酯(DOS)和聚氯乙烯(PVC),制备了对H+具有选择性的中性载体膜,优化了H+选择性膜的制备工艺,表征了微探针制备过程中的表面形貌的变化,系统的研究了全固态氢离子选择性微探针的线性范围,稳定性,选择性等性能,最后将其应用于海水、自来水和培养基样品的检测。研究结果表明,全固态氢离子选择性微探针对pH的响应范围达到2.5-12,选择性和稳定性能优良,展示出了在实际样品中良好的应用性能。该高柔韧性的全固态氢离子选择性微探针在材料界面研究和深海环境检测中具有巨大的应用潜力。

3)基于上述开发的选择性电化学微探针,构建了惰性表面生物膜内的SRB代谢活性的实时、原位、连续检测平台实现了惰性表面生物膜内固载SRB和溶液游离SRB代谢活性的连续监测,并借助两类有机荧光探针对生物膜内SRB代谢活性和细胞分布状态进行了解析;此外,为评估该电化学选择性微探针的实际应用性能,研究测试了D型甲硫氨基酸(D-Met)的加入对惰性材料表面SRB代谢活性的影响。研究结果表明,惰性材料表面生物膜内SRB代谢产生的硫化物浓度比溶液游离SRB0.4-0.6 mM,且溶液游离SRB的变化趋势相较于生物膜内SRB存在1-2天的滞后期;荧光探针测定结果表明,生物膜内SRB的代谢活性和细胞空间分布变化趋势非常相近,其荧光强度随时间的变化趋势与电化学选择性微探针监测的趋势一致,验证了电化学选择性微探针用于惰性材料表面生物膜内SRB代谢活性检测的可行性和准确性;此外电化学测试结果表明,D-Met的加入引起了生物膜内细胞代谢活性的降低,荧光探针的测定的结果也证实了该变化趋势。该检测平台为海洋环境中生物膜内SRB代谢活性的原位、实时检测提供了新策略。

4)基于上述开发的选择性电化学微探针,构建了金属表面生物膜内SRB代谢活性的实时、原位、连续检测平台,实现了金属表面生物膜内固载SRB和溶液游离SRB代谢活性的连续监测,并借助两类有机荧光探针对生物膜内SRB代谢活性和细胞分布状态进行了解析,验证了电化学选择性微探针检测结果的正确性。研究结果表明,金属表面生物膜内SRB代谢活性水平远高于溶液游离SRB;另外相较于惰性材料表面,金属表面生物膜内SRB代谢活性变化趋势更加复杂,且金属表面的存在显著提升了生物膜内的SRB代谢活性,荧光显微观察和荧光强度测试结果均验证了电化学监测方法的可行性和准确性。

As the typical, studied, and most corrosive microorganism, sulfate-reducing bacteria (SRB) influenced corrosion was closely related with the formation of biofilms on the surface of metal materials. Traditional methods for measuring SRB activity in biofilms cannot distinguish the metabolic activity difference between sessile SRB in the biofilm and planktonic SRB in bulk solution, and it is difficult to apply in the field application. In order to solve these problems, two types of all-solid ion selective electrochemical microprobes were prepared in this thesis, and used for construction the metabolic activity detection platforms for a continuous, in situ detection of the SRB metabolic activity in biofilms by selectively recognition the characteristic metabolic substances. In addition, the feasibility of the SRB metabolic activity detection performances were verified by two organic fluorescent probes. The metabolic activity of sessile SRB in biofilm on the surface of inert material and planktonic SRB in bulk solution were continuous measured, and the test results preliminarily revealed the changes and differences of metabolic activity between SRB cells in the biofilm and free SRB cells. Subsequently, the metabolic activity of sessile SRB in biofilm on the surface of metal material and planktonic SRB in bulk solution were continuous measured, and the results were discussed in this thesis. This study was of great significance for revealing the variation regulation and difference characteristics of SRB metabolic activity inside and outside the biofilm, and held significant meaning for the research of SRB biofilm corrosive mechanism. The main finding were as follows:

(1) An all-solid sulfide selective microprobe for specific recognition of sulfide produced by SRB metabolism was constructed. The microprobe was fabricated on a flexible silver wire as the conductive substrate, and subsequently layered reduced graphene sheets as the ion to electron transfer layer and nanostructured Ag2S as the sulfide selective membrane were successively constructed by electrochemical method. The electro- chemical deposition process was optimized and the corresponding surface morphology was characterized. The linear range, stability, selectivity, and other detection properties of the all-solid sulfide selective microprobe were systematically studied. Finally, the all-solid sulfide selective microprobe was verified in seawater and tap water samples testing. The results showed that the linearly detection range of the all-solid sulfide selective microprobe was 5×10-7-1×10-2 M, with the detection limit of 1.78×10-7 M. The highly flexible all-solid sulfide selective microprobe has good selectivity and stability, and held great application potential in material interface research and deep-sea environment detection.

(2) An all-solid hydrogen ion selective microprobe was constructed. The microprobe was fabricated on flexible silver wire as the conductive substrate, and then a layered reduced graphene sheet as the ion to electron conversion layer was constructed by an electrochemical deposition method. Next, on the surface of graphene layer, tri-n-dodecyl amine (TDDA) as a neutral carrier, was mixed with potassium tetraphenylborate (KTPB), dioctyl sebacate (DOS) and polyvinyl chloride (PVC) to fabricate a neutral carrier film for selective hydrogen ion recognition. The linear range, stability, selectivity, and other detection properties of the all-solid hydrogen ion selective microprobe were systematically studied. Finally, the all-solid hydrogen ion selective microprobe was verified in seawater, tap water and culture medium samples. The results showed that the linearly response of the all-solid hydrogen ion selective microprobe for pH was ranged from 2.5 to 12, with excellent selectivity and stability, and exhibited good application performance in practical samples. The highly flexible all-solid hydrogen ion selective microprobe has great application potential in the study of material interfaces and deep-sea environment detection.

(3) Based on the developed all-solid sulfide selective microprobe, a real-time, in situ, continuous detection platform for SRB metabolic activity in the biofilm on the surface of inert material was constructed. Consequently, successive detection of metabolic activity of sessile SRB in the biofilm and planktonic SRB in bulk solution was achieved. In addition, two types of organic fluorescent probes were used to analyze SRB metabolic activity and cell distribution in biofilms, and therefore to evaluate the practical application performance of the all-solid sulfide selective microprobe for in situ metabolic activity in SRB biofilms. The effect of addition of D-methyl sulfide amino acid (D-Met) on the metabolic activity of SRB on the surface of inert material was measured. According to the research results, the sulfide concentration produced by SRB metabolism in the biofilm on the surface of the inert material was 0.4-0.6 mM higher than that of the planktonic SRB in bulk, and the same change trend of the metabolic activity of planktonic SRB was 1-2 days delayed compared with that of in biofilm. The fluorescence measurement results showed that the metabolic activity of SRB in the biofilm and the spatial distribution of cells were very similar, and the changes trend of fluorescence intensity was consistent with that of in the all-solid sulfide selective microprobe, which verified the feasibility and accuracy of the all-solid sulfide selective microprobe in the detection of SRB metabolic activity on the surface of inert material. In addition, the results of electrochemical tests showed that the addition of D-Met caused the decrease in the SRB metabolic activity in the biofilm, which was also confirmed by the results of fluorescence probes. This detection platform provides a new strategy for in situ, real-time detection of SRB metabolic activity in biofilms in the marine environment.

(4) Based on the developed all-solid sulfide selective microprobe, a real-time, in situ, continuous detection platform for SRB metabolic activity in the biofilm on the surface of metal material was constructed. The continuous measurements of metabolic activity of sessile SRB in the biofilm on the surface of metal material and planktonic SRB in bulk solution were conducted. Next, two types of organic fluorescent probes were used to analyze SRB metabolic activity and cell distribution in biofilms. The results showed that the SRB metabolic activity in the biofilm on the metal surface was much higher than that of free SRB in bulk solution. In addition, compared with the SRB metabolic activity on surface of inert material, the variation trend of SRB metabolic activity in the biofilm on the metal surface was more complicated, and the metal material significantly improved the metabolic activity of SRB in the biofilm. The feasibility and accuracy of the electrochemical monitoring method were verified by the results of fluorescence microscopy measurements.

 

MOST Discipline Catalogue理学
Language中文
Table of Contents

1 引言... 1

1.1 SRB的生物学简介... 1

1.1.1 SRB的生物学分类... 1

1.1.2 SRB的代谢特征... 2

1.1.3 SRB的生长要素... 3

1.2 SRB生物膜的形成与腐蚀机制... 4

1.2.1 生物膜的形成与特点... 5

1.2.2 SRB的微生物腐蚀机制... 6

1.2.3 SRB致微生物腐蚀的防控... 9

1.3 SRB的检测方法概述... 11

1.3.1 SRB种群浓度测定方式... 12

1.3.2 SRB代谢活性的测定方法... 17

1.4 选题依据和研究思路... 18

1.4.1 选题依据... 18

1.4.2 研究内容... 20

1.4.3 研究方案... 22

2 全固态硫化物选择性微探针的构建及应用研究... 24

2.1 引言... 24

2.2 实验部分... 26

2.2.1 材料与试剂... 26

2.2.2 还原态石墨烯的电化学沉积... 27

2.2.3 纳米Ag2S选择性膜的制备... 27

2.2.4 全固态硫化物选择性电极的性能检测... 27

2.2.5 全固态硫化物选择性电极的实际应用性能... 28

2.3 结果与讨论... 28

2.3.1 全固态硫化物选择性电极的制备和表征... 28

2.3.2 全固态硫化物选择性电极的性能评估... 31

2.3.3 全固态硫化物选择性电极的实际应用性能... 33

2.4 本章小结... 33

3 全固态氢离子选择性微探针的制备与性能评估... 35

3.1 引言... 35

3.2 实验部分... 37

3.2.1 材料与试剂... 37

3.2.2 还原态石墨烯的电化学沉积... 37

3.2.3 氢离子选择性膜的制备... 38

3.2.4 全固态氢离子选择性电极的表征和性能检测... 38

3.2.5 全固态氢离子选择性电极的实际应用性能... 38

3.3 结果与讨论... 39

3.3.1 全固态氢离子选择性电极的制备和表征... 39

3.3.2 全固态氢离子选择性电极的性能评估... 42

3.3.3 全固态氢离子选择性电极的实际应用性能... 43

3.4 本章小结... 44

4 惰性表面生物膜内SRB代谢活性的原位监测及应用研究... 45

4.1 引言... 45

4.2 实验部分... 47

4.2.1 材料与试剂... 47

4.2.2 SRB的培养... 47

4.2.3 惰性表面生物膜内SRB生长初期代谢活性的监测... 47

4.2.4 基于荧光探针分析生物膜内SRB的代谢活性和种群数量... 48

4.2.5 SRB生物膜活性的形貌观察... 49

4.2.6 SRB生物膜活性检测平台的应用性能... 49

4.3 结果与讨论... 49

4.3.1 基于电位化学微探针原位监测惰性表面生物膜内SRB代谢活性... 49

4.3.2 惰性表面生物膜内外SRB代谢活性测试的验证... 50

4.3.3 SRB生物膜活性检测平台的应用性能... 53

4.4 本章小结... 56

5 金属表面生物膜内硫酸盐还原菌代谢活性的原位监测... 57

5.1 引言... 57

5.2 实验部分... 58

5.2.1 材料与试剂... 58

5.2.3 SRB生物膜初期代谢活性与碳钢电信号监测... 59

5.2.4 基于荧光探针分析生物膜内SRB的生长初期代谢活性和种群数量... 60

5.2.5 SRB生物膜活性的形貌观察... 60

5.2.6 激光显微镜观察金属基底形貌... 60

5.2.7 SRB生物膜活性检测平台应用性能的初步探究... 61

5.3 结果与讨论... 61

5.3.1 金属表面生物膜内SRB代谢活性的连续测试... 61

5.3.2 金属材料表面生物膜内外SRB代谢活性测试的验证... 63

5.3.3 EH 40钢的形貌观察... 66

5.3.4 D-Met对金属表面生物膜内SRB活性影响的初探... 66

5.4 本章小结... 68

6 结论与展望... 69

6.1 结论... 69

6.2 创新点... 70

6.3 展望... 70

参考文献... 72

... 86

作者简历及攻读学位期间发表的学术论文与研究成果 88

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164740
Collection中国科学院海洋研究所
海洋环境腐蚀与与生物污损重点实验室
Recommended Citation
GB/T 7714
叶祥益. 生物膜内硫酸盐还原菌代谢活性的原位实时监测及其初步应用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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