Roles of ionic strength and biofilm roughness on adhesion kinetics of Escherichia coli onto groundwater biofilm grown on PVC surfaces

Abstract

Mechanisms of Escherichia coli attachment on biofilms grown on PVC coupons were investigated. Biofilms were grown in CDC reactors using groundwater as feed solution over a period up to 27 weeks. Biofilm physical structure was characterized at the micro- and meso-scales using Scanning Electron Microscopy (SEM) and Optical Coherence Tomography (OCT), respectively. Microbial community diversity was analyzed with Terminal Restricted Fragment Length Polymorphism (T-RFLP). Both physical structure and microbial community diversity of the biofilms were shown to be changing from 2 weeks to 14 weeks, and became relatively stable after 16 weeks. A parallel plate flow chamber coupled with an inverted fluorescent microscope was also used to monitor the attachment of fluorescent microspheres and E. coli on clean PVC surfaces and biofilms grown on PVC surfaces for different ages. Two mechanisms of E. coli attachment were identified. The adhesion rate coefficients (kd) of E. coli on nascent PVC surfaces and 2-week biofilms increased with ionic strength. However, after biofilms grew for 8 weeks, the adhesion was found to be independent of solution chemistry. Instead, a positive correlation between kd and biofilm roughness as determined by OCT was obtained, indicating that the physical structure of biofilms could play an important role in facilitating the adhesion of E. coli cells.

Fig. 1

SEM images of (A) PVC, (B) 2-week old biofilm, (C) 4-week old biofilm, (D) 16-week old biofilm, (E) 24-week old biofilm, and (F) 27-week old biofilm at magnification of 10,000×. All biofilm samples were fixed, dried, and coated with Au before imaging.

Fig. 2

OCT biofilm characterization data calculated by using algorithm in Matlab. A) absolute biofilm roughness (μm), B) relative biofilm roughness coefficient, C) mean biofilm thickness (μm).

Fig. 3

Cluster analysis of T-RFLP for biofilm community diversity.

Fig. 4

Electric surface charge properties of E. coli S17 (square), 2-week old biofilm (triangle), 4-week old biofilm (open star), 6-week old biofilm with 5-min sonication (open circle), 6-week biofilm with 30-min sonication (closed star), 8-week old biofilm (diamond), 27-week old biofilm (cross) as a function of ionic strength (KCl) at pH 8.2–8.5. Zeta potential was calculated from experimental electrophoretic mobility using Smoluchowski equation.

Fig. 5

Adhesion rate coefficient (k_d) of A) E. coli S17, and B) CML on clean PVC and biofilm surface grown at different times as a function of ionic strength (KCl) at pH 8.2–8.5 and at 25 °C. The error bars correspond to 95% confidence intervals.

Fig. 6

Adhesion rate coefficient (k_d) of E. coli S17 as a function of biofilm relative roughness coefficient at different biofilm ages. Adhesion experiments were carried out at 10 mM KCl, pH 8.2–8.5 at room temperature. For 2-week old biofilm, the biofilm roughness coefficients were undetected because the biofilms were too thin for OCT imaging.