Chlorine penetration into artificial biofilm is limited by a reaction-diffusion interaction

The retarded penetration of chlorine into artificial biofilms of Pseudomonas aeruginosa entrapped in agarose gel slabs was investigated experimentally and shown to be consistent with an unsteady reaction-diffusion model. A chlorine microelectrode was used to measure transient chlorine concentration profiles in artificial biofilms in a flow cell. While chlorine penetrated relatively quickly into pure agarose films (similar to 15 min), its penetration into biofilms was greatly retarded when cells were present. The degree of retardation was proportional to the initial cell density in the biofilm. After 3 h of treatment with a flowing chlorine solution, the chlorine concentration at the substratum under a 526 mu m thick biofilm containing 14 400 mg L(-1) cell mass had only risen to 10% of the bulk solution value. A mathematical model of the transient reaction-diffusion interaction correctly captured the qualitative behavior of experimentally measured chlorine concentration profiles. Parameter values for the simulations were obtained from the literature and from independent investigations of biomass-chlorine reactions using well-mixed suspensions. Kinetic and stoichiometric coefficients for the reactions of agarose and cell mass with chlorine were obtained by fitting a simple first-order (in both reactants) kinetic model to chlorine versus time data. The reaction rate coefficient for chlorine-cell reaction (1.1 x 10(-3) L mg(-1) s(-1)) exceeded that of chlorine-agarose reaction (3.7 x 10(-6) L mg(-1) s(-1)) by 2 orders of magnitude. The yield coefficient relating the amount of cell mass consumed to the amount of chlorine consumed ranged from 0.6 to 4.3 mg mg(-1), depending on the duration of the experiment. This study shows that the reaction rate of chlorine with cellular biomass is fast enough that diffusion of this disinfectant into the biofilm readily becomes rate limiting. This reaction-diffusion interaction affords an excellent explanation for the poor efficacy of chlorine when used against biofilm microorganisms.