Applied DNA HCR-FISH for Biofilm Distribution Imaging on Stainless Steel in Brackish Seawater

Microbially-induced corrosion or biocorrosion driven by microorganisms in brackish seawater has been associated with the destruction of the passivation layer on stainless steel. This has been especially linked to the metabolisms of sulphate-reducing bacteria (SRB). In recent years, various methods have been employed to investigate the phenomena, including open circuit potential (OCP) and anodic cyclic polarization methods, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) analyses, as well molecular biological methods such as enumeration by quantitative polymerase chain reaction (qPCR) and characterization of biofilm microbial communities by amplicon sequencing.Hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) is a bio-imaging technique that provides unique microbial distribution maps of multispecies biofilms on the steel surface, supporting current methods employed for a biocorrosion study. The technique uses DNA nucleotide probes labelled with fluorescence dyes, binding to the 16S ribosomal RNA (16S rRNA) to visualize simultaneously targeted multispecies microbes at the single-cell level. HCR-FISH protocol introduced by Yamaguchi et al. (2015a and 2015b) has been further adapted for bio-imaging marine sediment and seawater in recent years due to its simplicity, and high efficiency. We modified the standard HCR-FISH protocol to be applicable directly on stainless steel surfaces to study the biocorrosion of austenitic stainless steel EN 1.4404 that had been exposed to natural brackish seawater circulated in a lab-scale loop. HCR-FISH enabled simultaneous visualization of two microbial groups forming biofilm (bacteria and archaea or, bacteria and SRB). In addition, HCR-FISH was counterstained with 4,6-diamidino-2-phenylindole (DAPI), a cell-permeable fluorescent stain binding all double-stranded DNA.The modified HCR-FISH protocol produced promising results for the studied environmental mix species biofilms on stainless steel but requires further method development. The targeted cell detection was clear, specified, and intensive, resulting in high-contrast epifluorescence microscopy images which were applicable and supportive for biocorrosion investigation