Interaction of Introduced Biological Agents with Biofilms in Water Distribution Systems

Abstract

Basic research is needed to better understand the potential risk of dangerous biological agents that are unintentionally or intentionally introduced into a water distribution system. Included in this research is an assessment of how bio-pathogens will interact with biofilms growing on water-distribution system pipe-wall surfaces. Experiments are being run at Sandia National Laboratories to better understand the impact of biofilms on pathogen dissemination through water distribution systems, both before and after decontamination of the system. The hope is that these data can be fed into numerical tools that quantify the risk and associated uncertainty of pathogenic biological agents being introduced into water distribution systems and the effects of biofilms on this risk. In addition these data could be used in numerical tools that assess decontamination methods for water distribution system networks. Reproducible Pseudomonas fluorescens biofilms were grown in annular reactors with plate counts on the order of 10 5 and 10 6 CFU/cm 2 . A series of pathogen-introduction experiments, where both 1-μm-diameter fluorescently labeled polystyrene microspheres and Bacillus cereus (as a surrogate for B. anthracis ) were introduced in the annular reactor after the P. fluorescens biofilms have reached stationary phase. Integration of the pathogens in the biofilms was monitored by gram-specific plating techniques and epifluorescent microscopy. Fluorescent nucleic-acid stains were used to visually differentiate the gram-positive ( B. cereus ) and gram-negative ( P. fluorescens ) organisms. Fluorescence spectrophotometry was used to measure the microsphere concentrations. Variables examined in the experiments include initial pathogen concentration, rotation speed of the inner cylinder of the annular reactor (shear force), and biofilm plate counts prior to the start of the experiment. Chlorine was added to the system after the pathogen had at least 14 days in contact with the biofilms and the impact of the chlorine on pathogen concentration in the biofilms was monitored. Pathogens are observed to get integrated in the biofilms at a relatively constant concentration until the start of the chlorine treatment. The higher the initial pathogen concentration and the higher the initial biofilm plate counts prior the pathogen introduction, the more pathogen integrated into the biofilms. A decrease in pathogen concentration to below the detection limit in the biofilms and the reactor water was observed during the chlorine treatment. From the results of one experiment, it appears that the pathogens do not recover after termination of the chlorine treatment. However, more data need to be collected to confirm these results. These results indicated that biofilms may act as a safe harbor for bio-pathogens in drinking water systems, making it difficult to decontaminate the systems. Chlorination of the system appears to be effective in decontaminating the system in the annular reactor geometry at the concentrations that we used. Additional experiments are being run to further test this observation. As these experiments cannot assess the effectiveness of chlorination at pipe junctions and because annular reactors have a higher surface area to volume ratio than pipe systems, the results from these experiments should also be tested in a system with a more complicated geometry that more closely matches water distribution systems. This paper was presented at the 8th Annual Water Distribution Systems Analysis Symposium which was held with the generous support of Awwa Research Foundation (AwwaRF).