Metabolic Differentiation in Biofilms as Indicated by Carbon Dioxide Production Rates

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO₂ production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O₂ flux, with CO₂ production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO₂ production rate of cells from this region increased within 0.25 h from 9.45 ± 5.40 fmol of CO₂·cell⁻¹·h⁻¹ to 22.6 ± 7.58 fmol of CO₂·cell⁻¹·h⁻¹ when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.