Synergistic disinfection and removal of biofilms by a sequential two-step treatment with ozone followed by hydrogen peroxide

Abstract Synergistic disinfection and removal of biofilms by ozone (O 3 ) water in combination with hydrogen peroxide (H 2 O 2 ) solution was studied by determining disinfection rates and observing changes of the biofilm structure in situ by confocal laser scanning microscopy (CLSM) using an established biofilm of Pseudomonas fluorescence . The sequential treatment with O 3 , 1.0–1.7 mg/L, followed by H 2 O 2 , 0.8–1.1%, showed synergistic disinfection effects, while the reversed treatment, first H 2 O 2 followed by O 3 , showed only an additive effect. The decrease of synergistic disinfection effect by addition of methanol (CH 3 OH), a scavenger of hydroxyl radical ( OH), into the H 2 O 2 solution suggested generation of hydroxyl radicals on or in the biofilm by the sequential treatment with O 3 followed by H 2 O 2 . The primary treatment with O 3 increased disinfection rates of H 2 O 2 in the secondary treatment, and the increase of O 3 concentration enhanced the rates. The cold temperature of O 3 water (14 °C and 8 °C) increased the synergistic effect, suggesting the increase of O 3 adsorption and hydroxyl radical generation in the biofilm. CLSM observation showed that the sequential treatment, first with O 3 followed by H 2 O 2 , loosened the cell connections and thinned the extracellular polysaccharides (EPS) in the biofilm. The hydroxyl radical generation in the biofilm may affect the EPS and biofilm structure and may induce effective disinfection with H 2 O 2 . This sequential treatment method may suggest a new practical procedure for disinfection and removal of biofilms by inorganic oxidants such as O 3 and H 2 O 2 .