Bivalent cations are known to affect the structural and mechanical properties of biofilms. In order to reveal the impact of Fe2+ ions within the cultivation medium on biofilm development, structure and stability, Bacillus subtilis biofilms were cultivated in mini-fluidic flow cells. Two different Fe2+ inflow concentrations (0.25 and 2.5 mg/L, respectively) and wall shear stress levels (0.05 and 0.27 Pa, respectively) were tested. Mesoscopic biofilm structure was determined daily in situ and non-invasively by means of optical coherence tomography. A set of ten structural parameters was used to quantify biofilm structure, its development and change. The study focused on characterizing biofilm structure and development at the mesoscale (mm-range). Therefore, biofilm replicates (n = 10) were cultivated and analyzed. Three hypotheses were defined in order to estimate the effect of Fe2+ inflow concentration and/or wall shear stress on biofilm development and structure, respectively. It was not the intention to investigate and describe the underlying mechanisms of iron incorporation as this would require a different set of tools applied at microscopic levels as well as the use of, i.e., omic approaches. Fe2+ addition influenced biofilm development (e.g., biofilm accumulation) and structure markedly. Experiments revealed the accumulation of FeO(OH) within the biofilm matrix and a positive correlation of Fe2+ inflow concentration and biofilm accumulation. In more detail, independent of the wall shear stress applied during cultivation, biofilms grew approximately four times thicker at 2.5 mg Fe2+/L (44.8 µmol/L; high inflow concentration) compared to the low Fe2+ inflow concentration of 0.25 mg Fe2+/L (4.48 µmol/L). This finding was statistically verified (Scheirer-Ray-Hare test, ANOVA) and hints at a higher stability of Bacillus subtilis biofilms (e.g., elevated cohesive and adhesive strength) when grown at elevated Fe2+ inflow concentrations.

Microbes interact in natural communities in a spatially structured manner, particularly in biofilms and polymicrobial infections. While next generation sequencing approaches provide powerful insights into diversity, metabolic capacity, and mutational profiles of these communities, they generally fail to recover in situ spatial proximity between distinct genotypes in the interactome. Hi-C is a promising method that has assisted in analysing complex microbiomes, by creating chromatin cross-links in cells, that aid in identifying adjacent DNA, to improve de novo assembly. This study explored a modified Hi-C approach involving an initial lysis phase prior to DNA cross-linking, to test whether adjacent cell chromatin can be cross-linked, anticipating that this could provide a new avenue for study of spatial-mutational dynamics in structured microbial communities. An artificial polymicrobial mixture of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli was lysed for 1-18 h, then prepared for Hi-C. A murine biofilm infection model was treated with sonication, mechanical lysis, or chemical lysis before Hi-C. Bioinformatic analyses of resulting Hi-C interspecies chromatin links showed that while microbial species differed from one another, generally lysis significantly increased links between species and increased the distance of Hi-C links within species, while also increasing novel plasmid-chromosome links. The success of this modified lysis-Hi-C protocol in creating extracellular DNA links is a promising first step toward a new lysis-Hi-C based method to recover genotypic microgeography in polymicrobial communities, with potential future applications in diseases with localized resistance, such as cystic fibrosis lung infections and chronic diabetic ulcers.