Spatial distributions of copper in microbial biofilms by scanning electrochemical microscopy.

The spatial distribution of Cu was determined in Escherichia coli PHL628 biofilms using a scanning electrochemical microscope (SECM) consisting of a microelectrode in conjunction with a piezoelectric micropositioning system. Aqueous labile copper species were determined using voltametric stripping after reductive deposition of Cu for 4 min on the microelectrode at -0.7 V (vs Ag/AgCl). The position of the bulk solution-biofilm interface was determined from the change in current produced by 0.4 mM hydroxymethyl ferrocene that was added as a redox indicator. After a 2 h exposure to 0.2 mM copper, Cu was located in the upper region of the biofilm with a penetration depth less than 150 microm. A one-dimensional diffusive transport model adequately described the spatial distribution of copper in the biofilm, but the Cu retardation factor in the biofilm was more than 6-fold larger than that calculated from the isotherm for Cu binding to suspensions of E. coli PHL628 cells. There are several possible reasons for this difference, including an increase in the amount of extracellular polymer per cell within the biofilm and/or tortuosity that might hinder Cu transport into biofilms. The SECM technique in combination with model calculations provides direct evidence in support of the concept that formation of a biofilm may confer resistance to transient spikes in the bulk solution concentration of toxic metal species by retarding metal diffusion and reducing the metal exposure of cells within the biofilm.