Injury of drinking water organisms in biofilms and effects upon ecology and physiology

Analysis of polar lipid fatty acids has revealed that unique oxirane fatty acids are detected from chlorine exposed bacteria. These compounds are heretofore unrecognized in bacteria. Oxirane fatty acids of 16 and 18-carbon chain length were detected in water distribution system biofilms, monoculture biofilms and sloughed cells of Sphingomonas paucimobilis, and batch cultures of Escherichia coli. In control populations unexposed to chlorine oxirane fatty acids were not detected. Biofilm populations not exposed to chlorine had higher molar percentages of monounsaturated fatty acids. For S. paucimobilis a decrease from 92% to 7-12% monoenoic molar percentages was observed. The relative decrease in monounsaturated fatty acids correlated with formation of oxirane fatty acids. Exposure to increasing concentrations of residual free chlorine was followed by increased molar percentages of oxirane fatty acids in all experiments. Seasonal comparisons of PLF A from distribution system biofilms showed higher molar percentages of oxiranes in summer compared to winter (30% versus 20%). The maximum residual chlorine concentration in summer was 1.6 parts per million (ppm) compared to 1.2 ppm in winter. This evidence supports the hypothesis that oxirane fatty acids are formed by chlorine exposed bacterial cells. If this hypothesis is accepted, oxirane fatty acids could be used as biomarkers of chlorination in water distribution systems even where unculturable cells are present. Effects of chlorination upon biofilm ecology were also examined using confocal scanning laser microscopy. Biofilms of S. paucimobilis and Pseudomonas aeruginosa were formed under conditions mimicking those found in water distribution systems and chlorination effects were investigated. Order of inoculation of the two species was shown to affect distribution of cells within microcolonies. When P. aeruginosa was inoculated first, primarily homogeneous (single species) microcolonies were observed. Upon addition of chlorine to the bulk fluid, nearly all cells and microcolonies of that species detached. P. aeruginosa microcolonies and cells associated with S. paucimobilis microcolonies typically remained attached longer than unassociated single cells or microcolonies. When S. paucimobilis was inoculated first, heterogeneous (mixed species) microcolonies were observed. S. paucimobilis was shown to harbor P. aeruginosa in biofilms after chlorine exposure.