Development of extracellular polymeric substance-derived protective films against microbiologically influenced corrosion by Desulfovibrio vulgaris
It is widely accepted that microorganisms can play a pivotal role in corrosion by not only accelerating (microbiologically influenced corrosion, MIC) but also by inhibiting (microbio-logically influenced corrosion inhibition, MICI) the (electro)chemical degradation processes. Both phenomena are caused by biofilms accommodating immobilised, often complex microbial consortia embedded in a matrix of extracellular polymeric substances (EPS). EPS are a heterogeneous mixture of (mainly) polysaccharides, proteins, lipids, and nucleic acids and greatly influence interfacial processes (and, hence, MIC and MICI). In this study, Desulfovibrio vulgaris was used as a model organism for examination of MIC of steel caused by sulphate-reducing bacteria (SRB). A novel combination of scanning Kelvin probe force- and epifluorescence microscopy (SKPFM & EFM) was developed and evaluated to visualise localised effects of the bacterial cells on the surface potential of alloyed steel and pure iron in high resolution. EPS from cells grown in the presence of both working materials were extracted using DOWEX mar-athon C cation exchange resin. The strictly intracellular enzyme G6PDH was measured to ensure that extraction was not effected by cell lysis. Polysaccharides, proteins, uronic acids, humic acids, lipids, and nucleic acids from loosely and tightly bound EPS fractions as well as the TOC were quantified. Additionally, the polysaccharide and lipid moieties of the EPS were qualitatively analysed by gas chromatography. To mimic MICI, 25 differently functionalised cyclodextrins were used as EPS-analogue substances. Their protective ef-fect was determined from MIC simulations with Desulfovibrio vulgaris under anaerobic conditions. Corrosion rates were determined gravimetrically after up to 21 d. The stability of the cyclodextrin films and their impact on biofilm formation was assessed by fluores-cence microscopy. Results from the SKPFM & EFM measurements showed that the surface potential of Desulfovibrio vulgaris cells was approximately 25 mV higher compared to the surrounding alloyed steel surface. After removal of the cells, the same spot showed a negative shift in surface potentials of approximately -25 mV compared to the surrounding surface. In agreement with observations reported in other studies which demonstrated changes in the same order of magnitude between austenitic and ferritic phases and at areas with defects of the passivating oxide layer, these results provide strong evidence for a direct microbial influence on the passivating oxide layer of stainless steel. For EPS analysis, 7- 15 % of the loosely bound and 39 – 64 % of the tightly bound fraction were identified by colorimetric methods. TOC measurements indicated that the remaining proportion is of inorganic nature. G6PDH activity was not detectable in any of the ex-tracts. The total amount of polysaccharides, proteins, and lipids is greatly influenced by the working material present. However, a significant influence on the qualitative composition of polysaccharides and lipids could not be proven within this context. The strongest effect was observed for uronic acids with a significant increase to approximately 63 µg mg-1 EPS (compared to 19 µg mg-1 EPS in the controls without metallic substrata). Considering a 4-fold higher amount of Fe(III) ions in the EPS of cells grown in the presence of metallic substrata, a ratio of 2 moles glucuronic acids to 1 mole Fe(III) ions was calculated, indicating the formation of organometallic complexes and suggesting that uronic acids represent an important factor in MIC by SRB. Out of the 25 differently functionalised varieties, (polymerised) carboxy(m)ethylated cy-clodextrins showed the highest protective effect against MIC with 77 % lower corrosion rates after 21 d incubation. CLSM images show dense, up to 10 µm thick films clearly sep-arating biofilms from the material surface. An influence on the biofilm formation itself was not found, suggesting that the cyclodextrin films work as a passive barrier against the microorganisms.