海洋环境腐蚀与生物污损重点实验室知识库

海洋资源与经济发展研究已成为近年来热点和挑战性课题。海洋环境中的金属腐蚀是发展海洋经济不可避免的一个问题。微生物腐蚀（Microbiologically influenced corrosion, MIC）防治最常用的方法之一是使用杀菌剂。微生物腐蚀与生物膜密切相关，由于生物膜的多种防御机制，生物膜中的微生物通常更难以处理。杀菌剂用量不足可能导致微生物腐蚀行为发生改变。因此，研究杀菌剂亚致死浓度下生物膜中典型微生物对金属材料的腐蚀行为影响将为MIC机理的深度解析提供参考。本论文主要通过生长曲线、生物膜观察、腐蚀形貌和腐蚀产物表征、电化学测试和转录组分析等研究了杀菌剂四羟甲基硫酸磷（THPS）耐受浓度下生物膜中典型腐蚀微生物假单胞菌Pseudomonas stutzeri和硫酸盐还原菌Desulfovibrio hontreensis代谢变化和对X70管线钢腐蚀行为影响，并研究和探讨了石油存在和耐受条件下杀菌剂THPS对Desulfovibrio bizertensis腐蚀X70管线钢的作用机制。主要结论如下： 
（1）P. stutzeri能够在X70管线钢表面形成致密的生物矿物层，这层矿物层可有效地将腐蚀介质与X70管线钢隔离开来，从而对其腐蚀过程产生抑制作用。在THPS浓度为75 ppm的亚致死浓度条件下，杀菌剂的胁迫作用促进了P. stutzeri在X70管线钢表面的附着与生物膜的形成。同时，这种胁迫还刺激了P. stutzeri的代谢活动，加剧了电子从管线钢到P. stutzeri细胞的转移过程，进而使P. stutzeri加速X70管线钢腐蚀。
（2）D. hontreensis具备在X70管线钢表面聚集并形成生物膜的能力。通过胞外电子传递机制，D. hontreensis获得X70管线钢中的电子，从而为其生命活动提供所需能量，这一过程导致了金属腐蚀现象。加入亚致死浓度75 ppm THPS后，浮游细菌的生长受到了显著抑制，细菌的转录组水平发生了明显变化，细菌通过调节鞭毛和EPS等相关基因，更倾向于在X70管线钢表面附着，并聚集形成生物膜。D. hontreensis通过提高与电子传递相关基因的表达，加速了其对金属电子的利用，导致了金属腐蚀的加剧。 
（3）加入石油后，由于能源变得充足，D. bizertensis的生长繁殖得到了显著促进，对金属中电子的依赖性减弱。石油在试片表面形成了一层保护膜，有效隔离了X70试片与氯离子、氧气、D. bizertensis等腐蚀因子，从而抑制了D. bizertensis对X70管线钢的腐蚀作用。因此，石油的加入能够在一定程度上减缓D. bizertensis对X70管线钢的腐蚀进程。当石油与杀菌剂同时存在时，X70管线钢腐蚀电位升高，D. bizertensis的对X70金属的腐蚀作用明显增加，杀菌剂的作用效果受到了显著削弱。加入亚致死浓度的杀菌剂刺激了D. bizertensis向金属表面附着和形成生物膜，进而加剧腐蚀。
（4）通过转录组学分析，发现了一系列与转录调控、趋化性、鞭毛运动、膜转运机制、ATP生物合成过程以及核糖核蛋白功能相关的功能基因，这些可能构成了微生物在暴露于亚致死浓度杀菌剂THPS应激源时的代谢适应途径。这些结果可为研究耐受浓度下微生物的腐蚀机制提供参考。

The study on marine resources and economic development has become a hot and challenging topic in recent years. Metal corrosion in marine environment is an inevitable problem for the development of marine economy. The most common method of microbiologically influenced corrosion (MIC) prevention and control is the use of biocides. MIC is closely related to biofilms, and microbes in biofilms are generally difficult to handle due to their multiple defense mechanisms. Insufficient dosage of biocides may lead to changes in microbial corrosion behavior. Therefore, the study of the effect of biocide at sublethal concentration on the corrosion behavior of typical corrosive microorganisms will provide a reference for the in-depth analysis of MIC mechanism. In this thesis, corrosion behavior on X70 steel and metabolic changes of typical corrosive microorganisms Pseudomonas stutzeri and Desulfovibrio hontreensis under the tolerance concentration of biocide THPS were studied by means of growth curve, biofilm observation, corrosion morphology and corrosion product characterization, electrochemical tests and transcriptomic analysis. The effect of THPS on the corrosion behavior of Desulfovibrio bizertensis in the presence of petroleum was discussed. Results and conclusions are as follows:

(1) P. stutzeri can form a dense biomineral layer on the surface of X70 pipeline steel, which can effectively isolate the corrosive factors from the X70 pipeline steel, thereby inhibiting its corrosion process. Under the presence of THPS at sublethal concentration of 75 ppm, the stress effect of biocide promoted the adhesion of P. stutzeri on the surface of X70 pipeline steel and enhanced the biofilm formation. At the same time, this stress also stimulated the metabolic activity of P. stutzeri, intensified the transfer process of electrons from pipeline steel to P. stutzeri cells, and then accelerated the corrosion of X70 pipeline steel.

(2) D. hontreensis had the ability to aggregate and form biofilms on the surface of X70 pipeline steel. Through extracellular electron transfer, D. hontreensis obtains electrons from X70 pipeline steel to provide the required energy for its growth and activities, which leads to metal corrosion. After adding THPS with a sublethal concentration of 75 ppm, the growth of planktonic cells was significantly inhibited, and the transcriptome level of D. hontreensis was significantly changed. By regulating related genes such as flagella and EPS synthesis, cells of D. hontreensis were more inclined to attach to the surface of X70 pipeline steel and aggregate to form biofilms. D. hontreensis accelerated the utilization of metal electrons by increasing the expression of genes related to electron transportations, leading to the intensiveness of metal corrosion.

(3) In the presence of petroleum, the growth of D. bizertensis was significantly promoted due to the sufficient energy source, and the dependence on electron from metals was weakened. Petroleum formed a protective film on the X70 steel surface. Thus, X70 steel was effectively isolated from chloride ions, oxygen, D. bizertensis and other corrosion factors. These effects together inhibited the corrosion of X70 pipeline steel by D. bizertensis. Therefore, the addition of petroleum oil can slow down the corrosion process of X70 pipeline steel by D. bizertensis to a certain extent. When oil and biocide both existed, the corrosion potential of X70 pipeline steel increased, and the corrosion effect of D. bizertensis was significantly enhanced. Consequently, corrosion of X70 pipeline steel was intensified, and the effect of biocides is significantly weakened. In addition, THPS stimulated the adhesion of D. bizertensis to the metal surface and the formation of biofilm, which accelerated the X70 steel corrosion.

(4) Through transcriptomic analysis, a series of functional genes related to transcriptional regulation, chemotaxis, flagellar movement, membrane transport mechanism, ATP biosynthesis process, and ribonucleoprotein function were detected. This may constitute the adaptive metabolic pathways of microorganisms exposed to sublethal concentrations of THPS. These results provide a reference for studying the microbial corrosion mechanism under the biocide stress.

第1章 绪论    1
1.1 引言    1
1.2 微生物腐蚀    2
1.2.1 腐蚀微生物    2
1.2.2 腐蚀微生物生物膜    2
1.3 微生物腐蚀防治措施    5
1.3.1 物理法    5
1.3.2 生物法    6
1.3.3 化学法    8
1.4 杀菌剂的分类    9
1.4.1 氧化性杀菌剂    9
1.4.2 非氧化性杀菌剂    10
1.5 选题依据与研究内容    16
1.5.1 选题依据    16
1.5.2 研究内容    16
第2章 THPS耐受浓度下Pseudomonas stutzeri对X70管线钢腐蚀机理研究    19
2.1 研究背景    19
2.2 材料与方法    20
2.2.1 菌株培养及钢片制备    20
2.2.2 实验体系    20
2.2.3 细胞计数    20
2.2.4 生物膜表征    21
2.2.5 腐蚀失重速率测试    21
2.2.6 腐蚀形貌表征    21
2.2.7 转录组分析    22
2.3 实验结果    22
2.3.1 细胞计数与pH测试    22
2.3.2 生物膜表征    23
2.3.3 腐蚀失重速率测试    24
2.3.4 点蚀观察    24
2.3.5 腐蚀形貌表征    25
2.3.6 转录组分析    26
2.4 分析与讨论    31
2.5 本章小结    32
第3章 THPS耐受浓度下Desulfovibrio hontreensis对X70管线钢腐蚀机理研究    35
3.1 研究背景    35
3.2 材料与方法    35
3.2.1 菌株培养及钢片制备    35
3.2.2 实验体系    36
3.2.3 细胞计数    36
3.2.4 生物膜表征    36
3.2.5 腐蚀失重速率测试    36
3.2.6 腐蚀形貌表征    37
3.2.7 转录组分析    37
3.3 实验结果    37
3.3.1 细胞计数    37
3.3.2 生物膜表征    38
3.3.3 腐蚀失重速率测试    38
3.3.4 腐蚀形貌表征    40
3.3.5 转录组分析    42
3.4 分析与讨论    45
3.5 本章小结    46
第4章 含石油环境中THPS耐受条件下的Desulfovibrio bizertensis对X70管线钢腐蚀机理研究    49
4.1 研究背景    49
4.2 材料与方法    50
4.2.1 菌株培养及钢片制备    50
4.2.2 实验体系    50
4.2.3 细胞计数    50
4.2.4 腐蚀失重速率测试    50
4.2.5 腐蚀形貌表征    50
4.2.6 细菌形态观察    50
4.2.7 电化学测试    50
4.3 实验结果    51
4.3.1 细胞计数    51
4.3.2 腐蚀失重速率测试    51
4.3.3 点蚀观察    52
4.3.4 腐蚀形貌表征    54
4.3.5 细菌形态观察    54
4.3.6 电化学测试    55
4.4 分析与讨论    56
4.4.1 耐受浓度的杀菌剂THPS对D. bizertensis腐蚀行为的影响    56
4.4.2 石油对D. bizertensis腐蚀行为的影响    57
4.4.3 杀菌剂THPS和石油共同作用下的D. bizertensis腐蚀行为    57
4.5 本章小结    57
第5章 结论与展望    59
5.1 结论    59
5.2 创新点    60
5.3 展望    60
参考文献    63
致  谢    75
作者简历及攻读学位期间发表的学术论文与其他相关学术成果    77