Various biofilm structural forms were observed and characterized in sand columns simulating groundwater aquifer conditions. The mixed microbial community utilized toluene as a sole carbon source. Sand column experiments were conducted with a linear flow velocity of 5.3 m/d and toluene feed concentration of 6 mg/L. Sand columns attained a pseudo steady-state profile for bulk liquid toluene concentration prior to biofilm characterization. Profiles of viable biomass, carbohydrate, and bulk liquid toluene concentration were obtained at 5 cm intervals from 0 to 15 cm depth. Biofilm structural forms were observed using scanning confocal laser microscopy in flow cells that duplicated conditions in the sand columns. The following classes of viable biofilm structures were observed: thin uniform films, protrusions extending from the biofilm, and thin strands of cell aggregates bridging sand grains. Viable biomass profiles indicated approximately 45 percent of the biomass was weakly attached to sand grains versus firmly attached growth held on sand grain surfaces. Biofilm structural forms provided insight into distribution of viable bacteria during aquifer bioremediation and identified opportunities for engineered processes to modify biofilm structures for the optimization of hydrophobic organic compound bioremediation in groundwater aquifers.

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