海洋环境腐蚀与生物污损重点实验室知识库

微生物腐蚀（MIC）作为一种非常重要的腐蚀形式，在海洋金属工程设施的腐蚀失效中扮有重要角色。生物膜是MIC的关键特征，调控生物膜的状态可影响MIC过程。生物膜的形成、发展和解体受到微生物严格的遗传调控，且这种调控往往通过信号分子的响应实现。因此，研究信号分子对海洋微生物生物膜的影响将为MIC机理的深度解析提供基础。本文选取D-氨基酸和群体感应信号分子作为典型信号分子，通过生物膜定量和显微结构观察等方法，研究其对在海洋环境腐蚀产物层中广泛存在的两种微生物（假交替单胞菌Pseudoalteromonas sp. SC2014（SC2014）和硫酸盐还原菌Desulfovibrio sp.（D. sp.））生物膜的影响规律；通过研究细菌表面疏水性、生物膜结构和成分变化等，揭示信号分子对生物膜的影响机制；并结合腐蚀失重法，探讨信号分子对细菌所致金属材料腐蚀的影响，建立信号分子、生物膜状态和材料腐蚀之间的相关性。主要研究结果如下：

（1）揭示了D-氨基酸对SC2014和D. sp.生物膜作用的特异性及其影响规律。在所研究的六种D-氨基酸（D-Leu、D-Met、D-Pro、D-Trp、D-Tyr和D-Phe）中，仅有D-Phe可在不影响细菌生长的情况下抑制细菌生物膜的形成，但其对两种细菌生物膜发展过程的影响规律不同。对SC2014来说，D-Phe对细菌在316L不锈钢表面的初期附着无显著影响，且对生物膜解体后形成的单层结构无明显作用，不影响细菌与材料之间的相互作用。但是D-Phe可抑制生物膜由单层结构发展为成熟的多层结构，并使已形成的生物膜结构解体，抑制和破坏细菌之间的相互作用。对D. sp.来说，D-Phe不但可显著抑制细菌在316L不锈钢表面的初期附着，还可抑制生物膜的进一步发展，既阻碍细菌与材料之间的相互作用又影响细菌间的相互作用。

（2）解析了D-Phe对SC2014和D. sp.生物膜的作用机制，主要探讨了D-Phe对细菌本身表面性质和生物膜的组成、成分分布以及结构的影响。D-Phe可使SC2014的表面疏水性由27.44 ± 1.35 º变为34.21 ± 2.79 º，通过改变细菌表面成分的种类或数量，并抑制胞外多聚物在材料表面的聚集来影响细菌之间的相互作用，从而使细菌在316L不锈钢表面只能形成单层生物膜而无法形成成熟的多层生物膜。对D. sp.来说，一方面，D-Phe使细菌的形状由单一的杆状变为不均一的纤维状或球状，并改变细菌表面成分的种类或数量；另一方面，D-Phe显著抑制细菌和胞外多聚物在材料表面的聚集。最终使D. sp.在Q235碳钢表面所形成的生物膜结构由分布不均匀的蘑菇状变为分布相对均匀的网状，且使生物膜成分分布的相对层次性（蛋白质在最外侧，细菌、脂质与多糖位于内侧）消失。

（3）揭示了金属材料腐蚀与生物膜状态的相关性。对SC2014来说，虽然D-Phe可抑制细菌在Q235碳钢表面形成生物膜，但其对细菌所致材料的腐蚀失重无明显影响。对D. sp.来说，D-Phe可使细菌所致Q235碳钢的腐蚀失重由0.844 ± 0.076 mg/cm²降为0.552 ± 0.048 mg/cm²；使材料表面最大坑深达5 µm，坑宽达41.5 µm，密度为251 ± 118 mm^-2的腐蚀坑减少。D-Phe通过抑制D. sp.在Q235碳钢表面形成生物膜来抑制细菌对材料的腐蚀失重，而生物膜结构和分布的均一性使细菌所致材料的腐蚀更加均匀。

（4）确定了SC2014和D. sp.的群体感应系统，并解析了其与细菌生物膜和腐蚀的相关性。SC2014具有LuxR/LuxI型群体感应系统，不具有LuxS/AI-2型群体感应系统，信号分子C6-AHL和AI-2对细菌的生物膜形成和生长均无影响。而D. sp.不具有所用报告菌株能检测到的LuxR/LuxI型群体感应系统，具有LuxS/AI-2型群体感应系统，信号分子AI-2可通过抑制细菌、多糖和脂质在316L不锈钢表面的聚集来抑制细菌在其表面形成生物膜，且主要是抑制生物膜的后期生长。但是，AI-2对D. sp.所致Q235碳钢的腐蚀失重及在其表面所形成的生物膜无显著影响。

As a very important form of corrosion, microbiologically influenced corrosion (MIC) plays an important role in the corrosion failure of marine metal engineering facilities. Biofilm is a key feature of MIC, and regulating the state of it can affect the MIC. The formation, development and disassembly of biofilms are strictly controlled by microorganisms, and these regulations are often achieved through the response of signal molecules. Therefore, studying the effect of signal molecules on biofilms will provide a basis for in-depth analysis of MIC mechanism. In this paper, D-amino acids and quorum sensing signal molecules are selected as typical signal molecules. By means of biofilm quantification and microstructure observation, the effects of D-amino acids and quorum sensing signal molecules on the biofilm formation of two microorganisms Pseudoalteromonas sp. SC2014 (SC2014) and Desulfovibrio sp. (D. sp.) which are widely found in the corrosion product layer of the marine environment were studied. By studying the surface hydrophobicity of the bacteria, and the structure and compositional changes of biofilms, the influence mechanism of signal molecules on biofilms is revealed. In combination with the corrosion weight loss method, the effect of signal molecules on the corrosion of metals caused by bacteria was explored, and the correlation between signal molecules, biofilms and corrosion of the metals was established. Main results are shown as follows:

(1) The specificity and effect of D-amino acids on biofilms of SC2014 and D. sp. were revealed. Among six D-amino acids (D-Leu, D-Met, D-Pro, D-Trp, D-Tyr and D-Phe), only D-Phe could inhibit the formation of biofilms without affecting the growth of bacteria, but its influences on the development of biofilms formed by these two bacteria were different. For SC2014, D-Phe had no significant effect on the initial adhesion of bacteria to the surface of 316L stainless steel, and had no significant effect on the monolayer biofilms left after the disassembly of the mature biofilms, and did not affect the interaction between the bacteria and the metal. However, D-Phe could inhibit the development of biofilms from a monolayer structure into a mature multilayer structure, and disintegrate the mature biofilms. D-Phe could inhibit and break the interaction between bacteria. For D. sp., D-Phe not only inhibited the initial adhesion of bacteria to the surface of 316L stainless steel, but also prevented the further development of biofilms. D-Phe not only hindered the interaction between bacteria and metals, but also affected the interaction between bacteria.

(2) The effect mechanism of D-Phe on SC2014 and D. sp. biofilms were investigated. Effects of D-Phe on the surface properties of bacteria and the composition and structure of biofilms were discussed. D-Phe could change the surface hydrophobicity of SC2014 from 27.44 ± 1.35 º to 34.21 ± 2.79 º, by changing the type and quantity of the surface composition of bacteria, and inhibiting the aggregation of extracellular polymetic substances (EPS) on the surface of the metal to influence the interaction between bacteria. Thereby, bacteria could only form a monolayer biofilm on the surface of 316L stainless steel, and could not form a mature multilayer biofilm. For D. sp., on the one hand, D-Phe altered the shape of the bacteria from rod to inhomogeneous fibrous or spherical, and affected the type or amount of bacterial surface components. On the other hand, D-Phe inhibited the aggregation of bacteria and EPS on the surface of the metal significantly. Consequently, the structure of biofilms formed on the surface of Q235 carbon steel by D. sp. changed from mushroom shapes with uneven distribution to network structure with relative uniform distribution, and the relative layering of the distribution of biofilm components (proteins on the most outside, and bacteria, lipids and polysaccharides on the inside) disappears.

(3) The correlation between corrosion of metals and the state of biofilms was revealed. For SC2014, although D-Phe could inhibit the formation of biofilms on the surface of Q235 carbon steel, it had no significant effect on the weight loss of the metal caused by bacteria. For D. sp., D-Phe could reduce the weight loss of Q235 carbon steel from 0.844 ± 0.076 mg/cm² to 0.552 ± 0.048 mg/cm², and made corrosions pits with maximum depth of 5 µm, width of 41.5 µm and density of 251 ± 118 mm^-2 decrease. D-Phe inhibited the weight loss of the metal caused by D. sp. by inhibiting the formation of biofilms on the surface of Q235 carbon steel, and the uniformity of the structure and distribution of biofilms made the corrosion of the metal more homogeneous.

(4) The quorum sensing systems of SC2014 and D. sp. were determined, and the correlation between the quorum sensing system, biofilm and corrosion of metals was analyzed. SC2014 had LuxR/LuxI quorum sensing system, and did not have LuxS/AI-2 quorum sensing system. Signals C6-AHL and AI-2 had no effect on its biofilm formation and growth. D. sp. did not have LuxR/LuxI quorum sensing system which could be detected by the reporter strain, but had LuxS/AI-2 quorum sensing system. AI-2 inhibited the formation of biofilms on the surface of 316L stainless steel by inhibiting the aggregation of bacteria, polysaccharides and lipids, and mainly inhibited the later development of biofilms. However, AI-2 had no effect on the weight loss of Q235 carbon steel caused by D. sp., and biofilms formed on the surface of it by the bacteria.