Metabolic differentiation in biofilms as indicated by carbon dioxide production rates

Abstract

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 x cell(-1) x h(-1) to 22.6 +/- 7.58 fmol of CO2 x cell(-1) x 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.

FIG. 1.

A schematic diagram of the experimental setup showing the flow cell and the CMR.

FIG. 2.

The average whole-biofilm CO₂ production rate (μmol of CO₂ produced·h⁻¹) as measured for six biofilms (24 to 120 h) and three biofilms (122 h and 124 to 192 h) using the open-loop system configuration. Up until 120 h, the average CO₂ production for six biofilms was determined. After the steady-state measurement of the respiration rate was taken at 120 h, an air bubble was introduced into the each flow cell to remove the shear-susceptible biofilm region. Three of the biofilms were sacrificed at this stage, and open-loop measurements continued on the three remaining flow cells for a total of 192 h.

FIG. 3.

The number of cells produced by the biofilms and released into the bulk liquid was enumerated from the flow cell effluent with direct fluorescent counts and plate counts by sampling the effluent at 24-h intervals (n = 6 biofilms from 24 to 120 h, and n = 3 biofilms from 144 to 192 h).

FIG. 4.

Single CLSM micrographs taken at 24-h intervals at random locations of the gfp-labeled Pseudomonas sp. CT07 biofilms cultivated in conventional flow cells under continuous-flow conditions: 24 h (A), 48 h (B), 72 h (C), 96 h (D), and 120 h (E). (F) The single cells at the glass surface 1 h after the bubble perturbation. The biomass along the edge of the flow cell where the glass coverslip meets the Plexiglas is shown 1 h after the perturbation (G), 24 h after the perturbation (144 h) (H), and 48 h after the perturbation (168 h) (I). Micrographs shown in panels F and G were not included in the image analysis with COMSTAT.

FIG. 5.

COMSTAT image analysis of four biofilm parameters was averaged for three biofilms cultivated under the same conditions, as described previously. The average amount of biofilm biomass at the surface (μm³·μm⁻²) and average biofilm thickness (μm) are plotted in panel A, and the roughness coefficient and surface area-to-biovolume ratio (μm²·μm⁻³) are plotted in panel (B). The absence of sufficient biofilm biomass at the glass surface after the bubble perturbation at 121 h (Fig. 4F) did not allow the capture of images suitable for analysis, and hence this data point could not be included.

FIG. 6.

The average rate of CO₂ production per cell was compared between planktonic cells in the exponential phase of growth, biofilm-derived effluent cells, the shear-susceptible biofilm, and the non-shear-susceptible base biofilm layer. Activity of the shear-susceptible biofilm layer was determined in situ (as part of the biofilm) and as suspended cells after removal from the biofilm.