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Previous Article | Next Article 
Applied and Environmental Microbiology, September 2006, p. 6364-6366, Vol. 72, No. 9
0099-2240/06/$08.00+0 doi:10.1128/AEM.00442-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Center for Biofilm Engineering and Department of Chemical and Biological Engineering, Montana State University—Bozeman, Bozeman, Montana 59717-3980
Received 23 February 2006/ Accepted 20 June 2006
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ABSTRACT |
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INTRODUCTION |
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Another means of generating antimicrobial agents at a surface is through electrolysis. Electrolysis of water can generate local pH changes which may affect bacteria or their matrix polymers. When chloride ions are present, electrolysis can generate free chlorine, a powerful antimicrobial agent. Matsunaga and coworkers have demonstrated the potential of this approach to remove and inactivate marine biofouling bacteria (2-5). Their results also show that biofouling control can be achieved without perturbing the bulk fluid pH or imparting a chlorine residual to the bulk fluid.
The purpose of the research reported in this article was to investigate the effects of electrolysis on biofilms formed by Staphylococcus epidermidis. This microorganism was chosen because of its medical relevance and because it forms thick biofilms in in vitro reactor systems. Experiments were conducted in the presence of chloride ion to allow for the electrolytic generation of free chlorine at the cathode.
Biofilms of Staphylococcus epidermidis ATCC 35984 were grown on stainless steel coupons in drip flow reactors (7, 12) at 37°C with provision of 1/10-strength tryptic soy broth at 10 ml h–1. Two-day-old biofilms were removed from the drip flow reactor chamber, placed in either of two vessels, and incorporated into an electrical circuit (Fig. 1). The first vessel used was a 500-ml beaker filled with sterile 10 g liter–1 sodium chloride so that the metal coupons were completely submerged. The circuit was completed by clamping the other ends of the cables to their respective positions on a 6-volt battery for a certain length of time, between 10 s and 5 min. The biofilm-covered coupon was connected as the anode (to the negative terminal of the battery) in some experiments and as the cathode (to the positive terminal of the battery) in other experiments. At the end of the treatment period, the biofilm-covered coupon was lifted from the NaCl solution with ethanol-sterilized tweezers and rinsed by slowly pipetting 2 ml of deionized water over each side of the coupon. The specimen was then either scraped and plated or stained and examined microscopically as described below.
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Viable cells were enumerated by serial dilution and plating. Intact biofilms were stained for respiratory activity with a 0.2% solution of 5-cyano-2,3-ditolyltetrazolium chloride (CTC), counterstained with a 10 µg ml–1 solution of 4',6'-diamidino-2-phenylindole (DAPI), cryoembedded, sectioned, and examined by epifluorescence microscopy with a Nikon E800 microscope using a 10x dry objective and a 450/50-nm excitation filter, a 480-nm dichroic mirror, and a 630/30-nm emission filter for CTC and a 360/40-nm excitation filter, a 400-nm dichroic mirror, and a 460/50-nm emission filter for DAPI.
Staphylococcus epidermidis formed dense, cream-colored biofilms in the drip flow reactor after 2 days of growth. These biofilms contained approximately 2 x 109 viable cells per cm2 prior to treatment. Untreated biofilms were 360 ± 80 µm thick, as measured from microscopic examination of several frozen sections.
When a biofilm-covered steel slide was connected as the anode in an electrical circuit with a 6-V potential, biofilm rapidly sloughed from the surface. Biofilm sloughing was accompanied by the copious evolution of gas bubbles from the metal surface (visit http://www.erc.montana.edu/Res-Lib99-SW/Movies/2006/06-M001.htm to view a video of this treatment). After approximately 30 seconds of treatment, most of the biofilm had been visibly removed from the surface. The log reduction in the number of surface-associated viable cells after 30 seconds of treatment as the anode corresponded to a decrease of 4 orders of magnitude (Fig. 2). Extending the treatment period did not further decrease the number of viable cells. The mean log reduction for treatments of 0.5 to 5 min was 3.99 ± 0.64. The decrease in viable cells, comparing controls (n = 6) and treatments (n = 7), was statistically significant (P = 3 x 10–6).
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These effects on the biofilm were qualitatively confirmed at the microscale by staining biofilms with the respiratory indicator CTC and examining frozen sections microscopically. This stain deposits red fluorescent crystals at locations of active respiration. Untreated control biofilms exhibited intense red staining (Fig. 3a). Biofilms treated as the anode were largely removed from the surface, leaving only a trace of biomass (Fig. 3b). Some of the residual biomass in these specimens did stain red, indicating respiratory potential. In biofilms treated as the cathode, thick regions of biomass remained intact and could be seen in frozen sections (Fig. 3c). These specimens had much less red color than the untreated control, indicating a significant loss of respiratory activity. What residual respiratory activity could be visualized seemed to be localized to an interior stratum just below the surface of the biofilm. Cells at the base of the biofilm were mostly inactive, as were cells in the layer adjacent to the bulk fluid interface.
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2OH– + H2. Thus, at the anode, one predicts an increase in pH and the evolution of hydrogen gas. At the cathode, the important reaction is likely to be H2O + Cl–HOCl + H+ + 2e–. At the cathode, one predicts a decrease in pH and the generation of the potent antimicrobial hypochlorous acid. We hypothesize that the increase in pH near the anode leads to alkaline hydrolysis of the polysaccharide matrix of the biofilm. Removal of biofilm from the surface is probably aided by hydrogen gas bubbles physically pushing biomass away from the substratum. The pH increase may also kill some of the bacteria. Dilute sodium hydroxide has been reported elsewhere to weaken and remove biofilms formed by S. epidermidis (11).
We hypothesize that the generation of hypochlorous acid at the cathode is responsible for most of the loss of viable cells that occurs in biofilms treated as the cathode. We further suggest that a reduction in pH is not conducive to hydrolysis of the particular polymers that comprise the matrix of these biofilms. This feature, along with less gas production at the cathode, could explain why less biofilm is removed than when a specimen is treated as the anode.
A decrease in pH is probably responsible for the obvious corrosion occurring at the cathode. The discoloration of the solution that was observed in our experiments was likely due to the release of Fe2+ and Fe3+ from the metal substratum. Corrosion is an issue that would need to be addressed to make this technology practicable. Potential solutions include plating the cathode with an inert metal (e.g., gold) or using conductive polymers or paints.
Our results are in general agreement with those of Matsunaga and coworkers (2-5) and of van der Borden et al. (8). The electrical enhancement of antibiotic action against biofilms, dubbed the "bioelectric effect," must be distinct from the results reported in this article, because the bioelectric effect produces no inactivation or removal of biofilm in the absence of added antimicrobial agent (1).
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ACKNOWLEDGMENTS |
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We thank Haluk Beyenal for his review of the manuscript.
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FOOTNOTES |
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REFERENCES |
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). The error indicated is the standard deviation.