Evaluation of the Factors Controlling the Time-Dependent Inactivation Rate Coefficients of Bacteriophage MS2 and PRD1 ROBERT ANDERS* ,†,‡ AND CONSTANTINOS V. CHRYSIKOPOULOS †,§ Department of Civil and Environmental Engineering, University of CaliforniasIrvine, California 92717, USA, U.S. Geological Survey, 4165 Spruance Road, Suite 200, San Diego, California 92101, USA, and Department of Civil Engineering, University of Patras, Patras 26500, Greece Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first- order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air- solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater. Introduction Sources of pathogenic viruses to drinking water supplies include septic tanks, broken sewer lines, improperly con- structed landfills, open dumps, or intentional groundwater recharge and crop irrigation with treated municipal waste- water (1, 2). In all these cases, viruses released into the subsurface environment may remain infective and survive for a considerable period of time (i.e., weeks to months). As a consequence, these microorganisms can infiltrate through the unsaturated zone and, upon reaching the water table, can continue to migrate downstream to points of withdrawal. The most important processes controlling the fate and transport of viruses in the subsurface are sorption and inactivation (3, 4). Virus sorption is affected by several factors including viral surface properties, groundwater quality, and soil surface charges (5). The process of virus inactivation can result from alteration of the surrounding protein coat contained in the viral capsid. Early studies determined that the inactivation of viruses in groundwater appears to be influenced by a number of factors including virus type, soil characteristics, groundwater quality, and the presence of microorganisms (6-9). However, published results on the significance of some of these factors on virus inactivation are not consistent. The only factor found to consistently control the inactivation of viruses in groundwater is tem- perature (10). Viruses and viral nucleic acids are presumably inactivated by a first-order process, and the reaction is strictly dependent on temperature (i.e., the reaction appears to be monomolecular) (11). However, various attempts to predict virus inactivation in groundwater as a function of temperature by using constant inactivation rate coefficients have been of limited success (12, 13). One possible explanation for these differences might be the presence of two (biphasic) or more (multiphasic) viral subpopulations undergoing sequential inactivation with different inactivation rate coefficients (14- 17). Therefore, accurate prediction of virus inactivation should account for temporal variation of the inactivation rate coefficients. In the present investigation, the inactivation process is represented by a pseudo-first-order expression with a time- dependent rate coefficient. Static and dynamic batch experi- ments were performed to compare the effects of temperature and the presence of sand on the time-dependent rate coefficient. Finally, the ability to more accurately predict the inactivation of viruses in groundwater by using thermody- namic parameters obtained from time-dependent inactiva- tion rate coefficients is discussed. Materials and Methods Bacteriophage Assays. The single-stranded RNA male- specific coliphage, MS2, and the double-stranded DNA somatic Salmonella typhimurium phage, PRD1, were used as model viruses in this study. These bacteriophages have been used extensively in virus inactivation studies and are considered to be good model viruses because they behave more conservatively (lower sorption) than many pathogenic viruses and are capable of surviving for significant periods of time in groundwater (18-21). Bacteriophage concentra- tions were measured by using the double-agar-layer assay method (22). PRD1 bacteriophage was analyzed by using Salmonella typhimurium LT2 (ATCC no. 15277) as the host bacterium. For the analysis of MS2 bacteriophage, E. coli HS(FFamp)R (ATCC no. 700891) were used as the host bacterium with the same concentration of antibiotics, as described by Debartolomeis and Cabelli (23). The double-agar-layer assay method was performed by preparing a mixture of 1.0 mL of host bacteria, 4.0 mL of molten soft agar (50 °C), and 1.0 mL of sample. To ensure that all host bacteria were in log-phase growth, 0.1 mL of prepared stocks of each host were transferred to a test tube containing 10 mL of trypticase soy broth (Difco) and placed in a shaker at 37 °C for 4 h or until cultures were visibly turbid. The mixture was gently vortexed and poured onto the appropriate plating medium. Plates were incubated at 37 °C for 24 h. The concentrations were determined by counting the number of plaques on each plate. Plates were made in duplicate, and the concentration was averaged from the plaques counted on each plate. The double-agar-layer assay used for this study was estimated to have a detection limit of 0.5 plaque-forming units (PFU)/mL. Static and Dynamic Batch Experiments. A low-ionic- strength phosphate buffered saline (PBS) solution was prepared with 0.25 mM Na 2HPO4, 1.2 mM NaCl, and 0.037 * Corresponding author e-mail: randers@usgs.gov. † Department of Civil and Environmental Engineering, University of CaliforniasIrvine. ‡ U.S. Geological Survey. § Department of Civil Engineering, University of Patras. Environ. Sci. Technol. 2006, 40, 3237-3242 10.1021/es051604b CCC: $33.50  2006 American Chemical Society VOL. 40, NO. 10, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3237 Published on Web 04/13/2006