Direct measurement of chlorine penetration into biofilms during disinfection

Abstract

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.

Analysis of biocide transport limitation in an artificial biofilm system

“…Such transport limitation of biocide influx into the biofilm has been convincingly demonstrated for free chlorine at neutral pH. The profoundly retarded penetration of chlorine has been experimentally determined using a chlorine-sensitive micro-electrode in natural (de Beer et al 1994) and artificial Xu et al 1996) biofilm systems. The penetration time of free chlorine can be hundreds of times longer than for a similarly sized non-reactive solute.…”

“…Biocides are widely used to control the detrimental formation of microbial biofilms, but their efficacy is always disappointing when compared with disinfection assays performed with planktonic micro-organisms (Costerton et al 1987;Brown and Gilbert 1993). The mechanisms by which biofilms resist disinfection are not all elucidated, but over the past few years, progress has been made in describing and quantifying the extent to which incomplete biocide penetration impacts biocide performance (de Beer et al 1994;Stewart and Raquepas 1995;Chen and Stewart 1996;Xu et al 1996). Retarded delivery of biocide into the biofilm occurs when the biocide is neutralized by reaction with biofilm constituents faster than it diffuses into the biofilm.…”

Anti-biofilm agents: recent breakthrough against multi-drug resistantStaphylococcus aureus

“…In addition, the EPS matrix may act as a diffusion barrier to delay the infiltration of some antimicrobial agents (Xu et al, 2000). The reactive chlorine species in a number of these agents may be deactivated at the surface layers of the biofilm before they are able to disseminate into the interior of the biofilm (de Beer et al, 1994). A recent study showed that oxacillin, cefotaxime, and vancomycin had reduced penetration throughout S. aureus and S. epidermidis biofilms (Singh et al, 2010).…”

Biofilm formation and the food industry, a focus on the bacterial outer surface

“…Biofilm resistance to chlorine is still incompletely understood, but is at least partly because of hindered penetration of the biocide into the biofilm (De Beer et al 1994;Chen and Stewart 1996;Xu et al 1996). Active chlorine concentrations as high as 1000 ppm are necessary for a substantial reduction in bacterial numbers in multispecies biofilms (formed by L. monocytogenes, Ps.…”

“…pneumoniae and Ps. aeruginosa were typically only 20% or less of the concentration in the bulk liquid (De Beer et al 1994). The slow or incomplete penetration of the biocide into the biofilm is partly because of diffusion limitation in the exopolymeric matrix, but primarily because of neutralization of the active compound in the outermost regions of the matrix.…”