Suggested improvement of HCR-FISH in biofilm bioimaging of stainless steels in biofouling studies

The sulfide derivatization resulting from the microbial sulfate-reduction processes and ennoblement of corrosion potential are two microbiological processes frequently discussed in literature concerning biofouling and microbially induced corrosion (MIC). These phenomena have been investigated for their initial stages of microbial colonization and effects on the ennoblement of commonly used stainless steel (SS) industrial equipment, particularly in the brackish Baltic Sea.

This study employed a bioimaging method of hybridization chain reaction fluorescence in situ hybridization (HCR-FISH), within a comprehensive assessment pipeline to evaluate biofouling and ennoblement phenomena. A modified protocol of multi-labeling HCR-FISH was directly applied to two surface types of SS grade EN 1.4404 to identify localized bacteria, archaea, and sulfate-reducing bacteria (SRB). This involved targeting bacterial and archaeal 16S rRNA genes and dissimilatory sulfite reductase (dsrB) genes. Particularly, surface finishes 2R (cold rolled and bright annealed surface) and finish 1D (hot rolled, heat treated, and trimmed surface) were selected for examination.

Our methodology encompassed validating HCR-FISH with controlled microbial pure cultures and steel materials in laboratory settings. Subsequently, the technique was employed in a biofouling study, utilizing samples immersed in aquariums containing flowing seawater from the Baltic Sea. The study findings revealed the efficacy of multi-labeling HCR-FISH, which targeted bacteria, archaea, and SRB, in facilitating the detection of microbial features with enhanced interactive distinguishability within biofilm compositions. This method offers insights into the varied extent and nature of biofilms and deposits on the alloy surfaces.

However, the presence of natural diatoms on the SS surface, particularly during the spring bloom in the Baltic Sea, introduced challenges of nonspecific binding signals in HCR-FISH. Diatoms, a group of photosynthetic algae, possess pigments of diatoms that enable a broad range of light absorption across the visible spectrum. This spectrum overlaps with the emission and excitation spectrum of the fluorescent dyes utilized in FISH, leading to unintended signals.

While exploring diatom’s impacts on organic fluorescence dyes remains limited, our recent study has shed light on their potential influence on MIC bioimaging. Therefore, we propose the adoption of fluorescence nanoparticles bioconjugated with FISH molecules to target DNA sequences within microbial cells. This approach aims to supplant organic fluorescence dyes, mitigating the issues of false signaling induced by environmental organisms. By doing so, it enhances the effectiveness of bioimaging in MIC studies. Furthermore, this proposed engineering solution holds promise for fostering collaboration across diverse research disciplines.