Sunlight inactivation of fecal indicator bacteria in open-water unit process wetlands

Constructed wetlands harness natural processes to provide a low cost and energy technology for wastewater treatment. Although not the primary goal of wetland design, wetlands may remove and inactivate pathogens via a number of mechanisms including sunlight inactivation, predation, and sorption coupled to sedimentation. The contribution of sunlight inactivation is generally insignificant in vegetated wetlands due to light screening by emergent macrophytes and deep water. A novel wetland design, termed open-water wetlands, utilizes geotextile fabrics on the wetland bottom to prevent the growth of emergent vegetation. This allows sunlight to penetrate the shallow water column (<30 cm), thereby enhancing sunlight inactivation of pathogens. The goal of this research was to evaluate the potential of open-water wetlands to inactivate pathogens in secondary municipal wastewater effluent. To this end, indicator organism inactivation was monitored in a pilot-scale open-water wetland. In addition, to gain insight into inactivation mechanisms (e.g., endogenous versus exogenous inactivation), laboratory experiments were conducted using photosensitizer-free water, wetland water and simulated sunlight. These studies resulted in a descriptive model that predicts inactivation rates of bacteria in open-water wetlands and other sunlit water bodies. As a first step towards developing a complete photoinactivation model for bacteria, a model was developed for a simpler organism (MS2 coliphage) considering only endogenous inactivation (Chapter 2; published in Environmental Science and Technology, 2014). The objective of the model was to account for wavelength specificity of photoinactivation, using a photoaction spectrum (PAS), and accounting for changes in the light spectrum due to season and attenuation in the water column. Experiments were set up to measure inactivation rates of MS2 in photosensitizer-free water in under natural sunlight in different seasons (one summer and one winter day), and in a wetland water column under simulated sunlight. The PAS model successfully predicted MS2 inactivation rates based on natural sunlight irradiances throughout the year, as well as in different water column depths. One of the challenges in developing and applying the PAS model was the uncertainty in measuring and predicting (using a radiative transfer model) sunlight irradiance in the range of 280 - 300 nm. The possibility of using total UVB as a simplified model parameter for estimating rates of endogenous inactivation was also tested using the experimental results. Although the total UVB model has a major limitation (it values each wavelength in the range of 280 - 320 nm equally), this simplified model can be used for estimating endogenous inactivation rates of organisms whose photoaction spectra have not yet been measured. To evaluate the performance of open-water wetlands in removing pathogens, fecal indicator bacteria concentrations were monitored in a pilot-scale open-water wetland over a one- year period (Chapter 3). The pilot-scale wetland provided effective inactivation of fecal indicator bacteria (up to 3-log and 2-log removal of E. coli and enterococci, respectively), primarily via sunlight inactivation. A novel model to predict endogenous and exogenous inactivation rates of fecal indicator bacteria in the wetland was developed and validated using data from laboratory experiments and field data. Endogenous inactivation was predicted using a total UV (sum of UVA and UVB irradiance) model. Exogenous inactivation was significant only for enterococci, and was modeled as a function of steady-state concentration of singlet oxygen in the bulk aqueous phase. The model was also applied to predict the land area required to achieve 3-log removal of fecal indicator bacteria throughout the year. Results suggested that during summer conditions in central California, open-water cells can provide greater than 3-log removal of non-pigmented fecal indicator bacteria in an area comparable to existing full-scale wetland systems.To provide further insight into the role of dissolved organic matter (DOM) in the exogenous inactivation of indicator organisms, wetland DOM isolates from different wetland types (open-water, bulrush, and cattail) were prepared using solid-phase extraction and were tested for inactivation potential (Chapter 4). Inactivation of MS2 was enhanced by all wetland DOM isolates, as well as by two standard DOM isolates [Suwannee River Fulvic Acid (SRFA) and Pony Lake Fulvic Acid (PLFA)]. The steady-state concentration of singlet oxygen in the bulk phase of the DOM isolate solutions was positively correlated with the MS2 exogenous inactivation rate. For Ent. faecalis, inactivation was enhanced in solutions of wetland DOM isolates and PLFA; however, for the SRFA solution light screening dominated any photosensitizing effect. The degree of association of Ent. faecalis with DOM varied among the isolates, and a positive trend was observed between greater association and inactivation rate. Similar inactivation rates were observed in solutions of DOM isolates and their parent whole wetland water samples for Ent. faecalis, whereas for MS2, much lower inactivation rates were observed with the isolates than in the whole wetland water samples. To quantify the effect of photosensitizer association on exogenous inactivation of fecal indicator bacteria, experiments were conducted measuring inactivation of Ent. faecalis (gram-positive) and E. coli (gram-negative) in the presence of a natural DOM and a model photosensitizer [Rose Bengal (RB)] at different Mg2+ concentrations (Chapter 5). Due to differences in cell wall structure, Ent. faecalis associated more strongly with DOM than E. coli. Inactivation of Ent. faecalis was enhanced by photosensitizer adsorption onto bacterial cells. Despite RB and EfOM association with E. coli cells, E. coli inactivation rates were not enhanced by adsorbed photosensitizers, likely due to the protection offered by the outer membrane. Inactivation of Ent. faecalis was not enhanced in irradiated solutions of polymer beads coated with RB (compared to sensitizer-free solutions), which were used to generate singlet oxygen in the absence of bacteria-sensitizer association, suggesting that association with photosensitizers is a prerequisite requirement for exogenous inactivation of Ent. faecalis. A significant output of this research is the descriptive model to predict sunlight inactivation of fecal indicator bacteria, which can be used to evaluate designs for future applications of open-water wetlands. This research also produced several important outcomes. First, the monitoring data of the pilot-scale open-water provided evidence that open-water unit process wetlands can achieve significant inactivation of fecal indicator bacteria. This finding can be used to promote the adoption of the novel open-water wetland design for pathogen removal, as a component of multi-barrier approaches to control pathogen transmission, or to reduce the use of chemical disinfectants. Second, insight into the exogenous mechanism of fecal indicator bacteria inactivation suggested that the association between photosensitizers and bacterial cells plays a key role in exogenous inactivation for the gram positive model species Ent. faecalis. Third, lab-cultured bacteria were inactivated significantly more rapidly than indigenous wastewater indicator bacteria, emphasizing that lab-cultured indicators should not be used to determine inactivation rate constants for indigenous bacteria