IN-SITU CHARACTERIZATION OF BIOFILM EXOPOLYMERS INVOLVED IN THE ACCUMULATION OF CHLORINATED ORGANICS

The chemical nature and spatial arrangements of exopolymers in a degra dative biofilm community were studied using a panel of fluorescein iso thiocyanate-and tetramethyl rhodamine isothiocyanate-conjugated probes . Image analysis and dual channel imaging, in conjunction with scannin g confocal laser microscopy, allowed detection and quantification of l ectin binding to a variety of glycoconjugates. Relative abundance of t hese components varied between 0 and 67% of biofilm area at any depth. Lectin binding sites were distributed nonuniformly, both horizontally and vertically, within the >30-mu m thick biofilms when the herbicide diclofop methyl was provided as the sole carbon source. A more unifor m distribution of lectin binding sites was formed by the same biofilm community, when grown on a labile medium. Diclofop and its metabolites accumulated in extracellular polymers when biofilms were grown with d iclofop as the sole source of carbon and energy, but not in the presen ce of the labile carbon source. There was a nearly 1:1 correspondence between the distribution of regions that accumulated diclofop (and oth er chlorinated ring compounds) and regions with binding sires for the alpha-L-fucose-specific Ulex-europaeus Type I lectin. These regions al so bound polyanionic and cationic fluor-conjugated dextrans, and a hyd rophobic-specific dye, demonstrating the nonuniform distribution of ch arged and hydrophobic regions in the biofilm matrix. Hydrolytic enzyme s, some of them selected for their specificity against residues identi fied by the lectin assay, had no effect on either structural integrity or diclofop binding. The distribution of diclofop binding, lectin bin ding, and charged regions observed in these biofilms indicated a degre e of spatial organization and differentiation within the biofilm commu nity. In addition, based on cell morphology and fluorescent gram react ion, these regions were primarily associated with one community member , a Bacillus coagulans strain.