Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Civil and Environmental Engineering

Major Professor

James R. Mihelcic, Ph.D.

Committee Member

Sarina J. Ergas, Ph.D.

Committee Member

Valerie J. Harwood, Ph.D.

Committee Member

Mya Breitbart, Ph.D.

Committee Member

Norma Alcantar, Ph.D.


pathogens, sanitation, Sustainable Development Goals, viruses, waste stabilization ponds, water reuse


Sanitation, renewable energy, and food security are among the most pressing global development needs of the century, especially for small cities with rapid population growth. Currently, 53% of the world’s population either lacks access to improved sanitation or discharges fecal waste to the environment without treatment. Furthermore, 80% of food consumed in developing regions is produced by 500 million small farms, and while many of them are still rain-fed, irrigated agriculture is increasing. The post-2015 Sustainable Development Goals, recently adopted by the United Nations, include targets to address the water-energy-food nexus. Wastewater reuse in agriculture can be an important solution for these goals, if it is done safely. Globally, 18 – 20 million hectares of agricultural land are irrigated with wastewater, but much is untreated, unregulated, or unsanctioned, causing concerns and uncertainty about health risks.

There is a need to better understand pathogen removal in natural and non-mechanized wastewater treatment systems, such as waste stabilization ponds (WSPs) and upflow anaerobic sludge blanket (UASB) reactors, which are commonly used in small cities and towns. Riverbank filtration (RBF) is also a natural technique used by farmers in developing countries to treat surface water polluted with untreated sewage, but pathogen removal in these systems has seldom been assessed in developing countries. The focus of this dissertation is on pathogen removal in natural and non-mechanized wastewater treatment and reuse systems, to evaluate the health implications of water reuse for irrigation, with the following three objectives: 1) assess the current understanding of virus removal in WSP systems through a systematic review of the literature; 2) measure the removal of viruses and their association with particles in systems with WSPs, UASB reactors, or both; and 3) assess the fate and transport of pathogens and fecal indicators in wastewater treatment systems with direct and indirect reuse for irrigation to estimate microbial risks.

To advance the understanding of virus removal in WSP systems, a comprehensive analysis of virus removal reported in the literature from 71 different WSP systems revealed only a weak to moderate correlation of virus removal with theoretical hydraulic retention time (HRT). For each log10 reduction of viruses a geometric mean of 14.5 days of retention was required, but the 95th percentile of the data analyzed was 54 days. Also, whereas virus-particle association and subsequent sedimentation has been assumed to be an important removal mechanism for viruses in WSPs, the literature review revealed a lack of evidence to confirm the validity of this assumption.

The association of human adenovirus (AdV) with wastewater particles was assessed in five full-scale wastewater treatment systems in Bolivia, Brazil, and the United States (two with only WSPs, two with a UASB reactor and WSPs, and one with only UASB reactors). A mesocosm study was also conducted with WSP water from one of the full-scale systems, and some samples were also analyzed for pepper mild mottle virus (PMMoV), F+ coliphage, culturable enterovirus (EV), norovirus (NoV), and rotavirus (RV). Results indicate that WSPs and UASB reactors affect virus-particle associations in different ways, which may differ for different viruses. In UASB reactor effluent, PMMoV was more associated with particles <180 µm, showed no indication of settling in subsequent ponds, and appeared to degrade in pond sediments after 5 days. In contrast, AdV in UASB reactor effluent was associated with small and large particles, and in subsequent ponds, particle-associated AdV showed evidence of possible settling or more rapid decay at the water surface. AdV and culturable EV were also more volumetrically-concentrated in UASB reactor sludge than they were in untreated sewage, WSP water, UASB effluent, and WSP sediments, indicating that the reactors may cause these viruses to become entrapped and concentrated in granular sludge. Some viruses may be removed in the sludge, but others exit the reactors in solution and attached to particles. The resuspension of pellets from centrifuged UASB reactor sludge samples in an eluant buffer indicated reversible AdV association with granular sludge, but some associations with particles in solution may not be reversible.

The fate and transport of pathogens and fecal indicators was assessed in Bolivia for two WSP systems with direct reuse for irrigation, and one on-farm RBF system used to treat surface water polluted by untreated sewage. In the WSP systems, despite HRTs of 10 days, pathogen and fecal indicator removal was generally ≤1-log10, possibly due to overloading and short-circuiting from sludge accumulation. The RBF system provided removals on the order of 2-log10 for protozoan parasites, 3-log10 or more for viruses, and 4-log10 or more for bacteria. The use of RBF also reduced cumulative estimated health burdens associated with irrigated lettuce. Irrigation of lettuce with untreated river water caused an estimated disease burden that represents 37% of the existing burden from acute diarrhea in Bolivia; when RBF was used, this decreased to only 1.1%, which is not epidemiologically-significant, and complies with the World Health Organization guidelines. Ratios of concentrations of microorganisms in irrigation water to their respective concentrations in soil or crops were calculated, to assess transfer from irrigation water to soil or crops. These ratios (with units mL g-1) were generally < 0.1 mL g-1 for coliphage, between 1 and 100 mL g-1 for Giardia and Cryptosporidium, and generally between 100 and 1,000 mL g-1 for helminth eggs. Higher ratios could indicate more efficient transfer from water to soil or crops, longer persistence in soil or on crops, or slower leaching away from soil or crops.

The results from this research demonstrate that pathogen removal in full-scale natural wastewater treatment systems happens via complex mechanisms that vary with respect to pathogen type, treatment systems configuration, and other environmental and operational parameters. Future research and innovation efforts should focus on the use of a combination of natural and non-mechanized technologies, surface-flow systems (e.g., WSPs) and subsurface systems (e.g., RBF), applied at both semi-centralized (e.g., wastewater treatment plant) and decentralized levels (e.g., on farms), to evaluate how this affects the efficiency and resiliency of pathogen removal. Also, future research is needed to further elucidate reasons for the observed differences in virus-particle associations in natural wastewater treatment systems.