Integration of raman microscopy, differential interference contrast microscopy, and attenuated total reflection fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in candida albicans biofilm

Abstract

Factors limiting hydrogen sulfide production were identified in a two-species biofilm containing sulfate-reducing bacteria (Desulfovibrio desulfuricans) and nonsulfate-reducing bacteria (Pseudomonas fluorescens). Profiles of hydrogen sulfide (H2S) concentration, pH, local mass-transport coefficient, local flow velocity, and local relative effective diffusivity in the biofilm were measured using microelectrodes. Biofilms had a heterogeneous structure consisting of cell clusters separated by voids. Typically, the H2S concentration was lower in the voids than in the adjacent cell clusters, demonstrating that the voids acted as transport channels for removing H2S from cell clusters. The extent of biofilm heterogeneity was directly correlated with the flux of H2S from cell clusters. At flow velocities below 2 cm/s, the flux of H2S from cell clusters depended on the flow velocity. We concluded that at these flow velocities the H2S production rate was limited by the delivery rate of sulfate ions to the biofilm. At flow velocities above 2 cm/s, the H2S production rate was nearly constant and did not depend on the flow velocity. At high flow velocities (<2 cm/s) the H2S production rate was limited by metabolic reactions in the biofilm. Local intrabiofilm flow velocity profiles were influenced strongly by biofilm heterogeneity without significant pH variation within biofilms. Surprisingly, profiles of local relative effective diffusivity indicated that the biofilm was made up of two layers, which could be related to the specimen with a two-species biofilm.