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 combinati on with functional and phylogenetic stains. The biofilm consisted of microb ial cell clusters covering 10-20% of the surface. The clusters were loaf-fo rmed, 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 c ontained mainly small Gram-negative bacteria (binding the EUB338 probe when hybridized in situ on the steel surface), and often carried one to three s pherical colonies, either homogeneously composed of large Gram-negative coc ci 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 2 5 mu m in diameter had a core nonpenetrable to fluorescent nucleic acid sta ins and ConA lectin stain. Fluorescently-tagged ConA stained cells at a dep th of <5 mu m, indicating the presence of cells with alpha-D-mannosyl and a lpha-D-glucosyl residues on surfaces, Ethidium bromide (log K-ow-0.38) pene trated deeper (17 mu m in 15 min, corresponding to >10 cells in a stack) in to the cluster than did the less polar dyes SYTO 16 (log K-ow 1.48) and acr idine orange (log K-ow 1.24), which stained five cells in a stack. Fluoresc ent 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 onl y to depths of less than or equal to 2 mu m indicating a permeability barri er. About 1/3 of the stainable cells hybridized in situ with Alf1b, while f ewer than 1/7 hybridized to GAM42, probes targeted towards alpha- and gamma -Proteobacferia, respectively. Our results represent a microscopic descript ion of an ennobling biofilm, where the ennoblement could follow the sequenc e of redox events as suggested by the model of Dickinson and Lewandowski (1 996) for the structure of corrosive biofilms on a steel surface.