Sunlight-Mediated Inactivation Mechanisms of Enteroccocus faecalis and Escherichia coli in Waste Stabilization Ponds

Sewage treatment is a pressing global problem, with both environmental as well as human health impacts. In areas where piped sewerage systems are viable, waste stabilization pond systems are one possible method for the treatment of wastewater. Pond systems are often advantageous due to ease of construction and operation as well as their low cost and high effectiveness. With regards to human health, the most important aspect of sewage treatment is the removal and inactivation of pathogens. Pathogen inactivation in pond systems has been attributed to multiple factors including sedimentation, predation, temperature, pH, and sunlight. This research investigates the sunlight-mediated inactivation of pathogens in surface waters, with a primary focus on wastewater treatment pond systems. Two common fecal bacterial indicator organisms were studied: the gram-positive Enterococcus faecalis and gram-negative Escherichia coli. Laboratory experiments were conducted in 150-mL batch reactors that were filled with different aqueous solutions, including deionized water, D2O, waste stabilization pond water, or combinations of these waters. These solutions were spiked with E. faecalis or E. coli, exposed to simulated sunlight, and loss of culturability was monitored over time. Temperature, pH, dissolved oxygen concentration, and irradiance were carefully controlled and monitored. Furthermore, some experiments involved the addition of photosensitizers or quenchers of radical oxygen species (ROS), while in others we measured the concentrations of ROS in the reactors. We began by identifying and describing the role of oxygen, exogenous sensitizers, light intensity and light wavelength upon the sunlight-mediated inactivation of E. faecalis and E. coli. We found that, while in DI water the presence of UVB wavelengths increased inactivation of both E. faecalis and E. coli, in pond water, the presence of the UVB wavelengths did not increase the rate of E. faecalis inactivation. Pond water constituents played a dual role, as either photosensitizers increasing inactivation rates (E. faecalis) or as light-attenuators, decreasing inactivation rates (E. coli). Inactivation rates of both E. faecalis and E. coli were correlated to dissolved oxygen concentrations, though differently. Decreasing dissolved oxygen below air saturation always decreased inactivation rates, however increasing above air saturation was not as straightforward. In waste stabilization pond water, where oxygen increases during the day due to algal photosynthesis, raising dissolved oxygen above air saturation increased inactivation of E. coli slightly, however E. faecalis inactivation remained constant. Next we characterized the effects of pH on the sunlight-mediated inactivation of E. faecalis and E. coli, studying inactivation in the absence and presence of sunlight. E. coli inactivation in pond and DI water had the same trend, with small changes in inactivation rates between more moderate pH, but above and below extremes (pH<4 or pH>9) sharp increases in inactivation rates. E. faecalis, however, was more sensitive to small changes in pH, particularly in pond water. We then investigated which ROS were involved in inactivation of E. faecalis and E. coli. Photosensitizers produce reactive oxygen species following the absorption of light and the transfer of light-energy to oxygen through a number of different pathways. Although our focus was upon exogenous mechanisms, endogenous mechanisms could not be excluded because in the presence of exogenous sensitizers damage by both exogenous and endogenous mechanisms can occur simultaneously. While we found evidence for the involvement of 1O2 in endogenous inactivation of both E. faecalis and E. coli, the importance of other species, while likely, remains unclear. As expected, no evidence was found to support a role for ROS produced exogenously in the inactivation of E. coli. A combination of quencher and D2O experiments, together with ROS measurements in pond water, provided strong evidence for the importance of exogenous 1O2 in E. faecalis inactivation. In addition, E. faecalis was significantly more sensitive than E. coli to inactivation by 1O2 produced by the synthetic sensitizer rose bengal, which followed the same pattern as their sensitivity to pond water constituents. To apply our results from laboratory microcosms to actual treatment systems, we developed a simple model based on our empirical data. The model takes into account the effects of dissolved oxygen concentration, pH, and pond water sensitizers as well as the effects of light attenuation on sunlight mediated inactivation. We applied the model to scenarios that might occur in natural settings, and discuss the strengths and limitations of our approach. Three mechanisms have been proposed to describe sunlight-mediated inactivation: direct UVB damage to DNA, indirect endogenous inactivation caused by UVB light, and indirect exogenous inactivation involving all wavelengths of sunlight up to 550 nm. While the first mechanism is a likely part of sunlight-mediated inactivation, it did not dominate the inactivation of either bacteria in this study. The second mechanism, while previously attributed to UVB light, should be expanded to include UVA and visible wavelengths, as these caused E. faecalis inactivation in our DI water wavelength experiments. The third mechanism dominated E. faecalis inactivation and was driven by the UVA and visible wavelengths, though E. faecalis inactivation occurred through all three mechanisms. E. coli, on the other hand, was not subject to the third, exogenous mechanism in our microcosms. Instead E. coli inactivation was dominated by endogenous mechanisms, and these mechanisms were driven by the UVB and UVA wavelengths. A final mechanism was described that does not fit into the three mechanisms above; exogenous production of H2O2, which then crosses into cells and causes endogenous microbial damage. This mechanism was not important in our pond water microcosms because H2O2 concentrations were low due to high scavenging by pond water constituents, but it may be important in other waters. Only E. coli appears susceptible to such a mechanism