Community structure of biofilms on ennobled stainless steel in Baltic Sea water

Stainless steel samples (AISI 316) were ennobled in a laboratory simulator with natural Baltic Sea water. After completion of ennoblement (increase of open circuit potential of ca 400 mV), the biofilm on the steel surface was characterized using confocal laser scanning microscopy (CLSM) in combination with functional and phylogenetic stains. The biofilm consisted of microbial cell clusters covering 10-20% of the surface. The clusters were loaf-formed, with a basal diameter of 20-150 mu m, 5-20 per mm(-2), each holding >10(4) cells in a density of 1-5 x 10(7) cells mm(-3). The typical cluster contained mainly small Gram-negative bacteria (binding the EUB338 probe when hybridized in situ on the steel surface), and often carried one to three spherical colonies, either homogeneously composed of large Gram-negative cocci or more often small bacterial rods in high density, 10(8)-10(9) cells mm(-3). The clusters in live biofilms contained no pores, and clusters over 25 mu m in diameter had a core nonpenetrable to fluorescent nucleic acid stains and ConA lectin stain. Fluorescently-tagged ConA stained cells at a depth of <5 mu m, indicating the presence of cells with alpha-D-mannosyl and alpha-D-glucosyl residues on surfaces, Ethidium bromide (log K-ow-0.38) penetrated deeper (17 mu m in 15 min, corresponding to >10 cells in a stack) into the cluster than did the less polar dyes SYTO 16 (log K-ow 1.48) and acridine orange (log K-ow 1.24), which stained five cells in a stack. Fluorescent hydrophobic and hydrophilic latex beads (diameter 0,02, 0.1 or 1.0 mu m) coated patchwise the cluster surface facing the water, but penetrated only to depths of less than or equal to 2 mu m indicating a permeability barrier. About 1/3 of the stainable cells hybridized in situ with Alf1b, while fewer than 1/7 hybridized to GAM42, probes targeted towards alpha- and gamma-Proteobacferia, respectively. Our results represent a microscopic description of an ennobling biofilm, where the ennoblement could follow the sequence of redox events as suggested by the model of Dickinson and Lewandowski (1996) for the structure of corrosive biofilms on a steel surface.