The effects of damaged cells on biofouling formation in a reverse osmosis (RO) membrane system were investigated. Using Pseudomonas aeruginosa PAO1 as a model bacterium, damaged cells were artificially made by autoclaving at 121 degrees C for 15 min, resulting in 97% dead cells and 3% live cells. The physiological characteristics of damaged cells were analyzed by comparing the cell wall hydrophobicity, surface charge, and carbohydrate concentrations of extracellular polymeric substance (EPS) to those of live cells. Biofouling formation was then observed in a lab-scale RO system under the one-time inoculation of live or damaged cells or two inoculations of live/live (LL), live/damaged (LD), damaged/live (DL), and damaged/damaged (DD) cells. The damaged cells showed higher hydrophobicity and carbohydrate concentrations, though a lower negative cell surface charge than live cells. When respective damaged and live cells were inoculated, the flux decline percentages were 74% and 65%. In addition, the flux declines under LL, LD, DL, and DD conditions were 63.2%, 62.5%, 57.7%, and 81.0%, respectively. The inoculation of damaged cells after a live cell injection increased fouling and decreased the water flux, with an increase in biomass in both the feed water and on the membrane surface. Thus, this work shows that damaged cells have the potential to cause severe biolouling on RO membranes. (C) 2015 Elsevier B.V. All rights reserved.