Date of Award


Degree Type


Degree Name

Doctor of Philosophy in Biological and Environmental Sciences


Biological Sciences

First Advisor

Jose A. Amador


Wastewater is a major source of nitrogen (N) to groundwater and coastal waterbodies, threatening both environmental and public health. Advanced N-removal onsite wastewater treatment systems (OWTS) are used to reduce effluent N concentration through biological N removal (BNR). Despite the important role that these systems play in treating nutrient- and pathogen-rich wastewater, few studies have targeted the mechanisms involved in N removal, their capacity to produce effluent to meet regulatory standards, or their impact on the atmosphere.

I evaluated effluent total N (TN) concentration, the structure and composition of N-removing microbial communities, and greenhouse gas fluxes of advanced N-removal OWTS in the town of Charlestown, Rhode Island, USA. To assess N outputs from advanced OWTS and compliance with the 19 mg N/L state regulatory standard for advanced-treated effluent, in Manuscript 1 I quantified TN concentration of effluent from 50 advanced N-removal OWTS between March 2017 and December 2019, and evaluated differences as a function of N-removal technology, home occupancy pattern (systems used seasonally vs. those used year-round), and various wastewater properties. Four N-removal OWTS technologies were included in this study: (i) Orenco Advantex® AX20 (n = 33), (ii) Orenco Advantex® RX30 (n = 9), (iii) BioMicrobics MicroFAST® (n = 3), and Norweco Singulair® (models TNT, 960, and DN; n = 5). RX30 systems produced the lowest median TN concentration (mg N/L) (13.2), followed by FAST (13.4), AX20 (14.9) and Norweco (33.8). Compliance with the state standard varied among technologies, with compliance rates of 78%, 73%, 67%, and 0% for RX30, AX20, FAST, and Norweco systems, respectively. Effluent TN concentration did not vary as a function of occupancy pattern. Ammonium and nitrate were identified as predictors for effluent TN in all technologies; temperature and pH were also part of best-fit models for FAST and Norweco systems, respectively. Systems used year-round produced a significantly higher median daily (5.3 g N/d) and annual (2.3 kg N/yr) N load than did seasonally-used systems (3.7 g N/d and 0.41 kg N/yr), likely due to differences in home usage patterns, demographics, and associated differences in system flow.

Assessment of treatment capabilities of advanced N-removal OWTS requires fast, accurate methods of monitoring performance. Regular monitoring of OWTS using in situ rapid tests can provide an inexpensive option for assessing treatment performance. In Manuscript 2 I assessed the ability of a portable photometer to accurately measure ammonium and nitrate concentrations in final effluent from 46 advanced N-removal OWTS in 2017. By comparing measurements made using the photometer with values determined by standard laboratory methods, I determined that photometer-based analysis reliably estimates inorganic N (ammonium and nitrate) concentration in field and laboratory settings. Photometer-based analysis of the sum of inorganic N species also consistently approximated the total N concentration in the final effluent from the systems. A cost-benefit analysis indicated that the photometer is a more cost-effective option than having samples analyzed by commercial environmental testing laboratories after analysis of 8 to 33 samples. These results suggest that a portable photometer can be used to provide reliable, cost-effective measurements of ammonium and nitrate concentrations, and estimates of total N levels in advanced N-removal OWTS effluent in a way that helps to identify underperforming systems.

Advanced N-removal OWTS remove N from effluent via microbial nitrification and denitrification. Despite the important role that microorganisms play in N removal, few studies have investigated the nitrifying and denitrifying microbial communities in advanced N-removal OWTS. In Manuscript 3, I used high-throughput sequencing to evaluate the structure and composition of nitrifying and denitrifying bacterial communities in 44 advanced N-removal OWTS. I sampled effluent from these systems in June and September 2017, targeting the genes encoding ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ), and assessed differences in the diversity and taxonomy of nitrifying and denitrifying communities as a function of technology, occupancy pattern, and season. Alpha diversity (species diversity at a local scale) and beta diversity (differences in species diversity among communities/sites) for amoA varied strongly as a function of season. Differences in beta diversity for nosZ were also influenced by season, although to a lesser extent than for amoA. Alpha diversity for nosZ varied among technologies. Nitrosospira and Nitrosomonas were the main genera of nitrifying bacteria in advanced N-removal OWTS. The predominant genera of denitrifying bacteria included Zoogloea, Thauera, and Acidovorax. Differences in taxonomy for each gene generally mirrored patterns observed in alpha and beta diversity, highlighting the important role that season and technology play in shaping communities of amoA and nosZ, respectively.

Microbial activity is required for the removal of N, pathogens, and organic C in advanced N-removal OWTS. However, these processes produce greenhouse gases (GHGs) including carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O). In Manuscript 4 I measured GHG emissions from 27 advanced N-removal OWTS in the towns of Jamestown (2016) and Charlestown (2018), Rhode Island, USA, and assessed differences in flux based on technology, occupancy pattern, and zone within the system (oxic vs. anoxic zone), as well as various wastewater properties. Flux values for CO2, CH4, and N2O fluxes ranged from -0.44 to 61.8, -0.0029 to 25.3, and -0.02 to 0.23 μmol m-2s1, respectively. CO2 and CH4, but not N2O, fluxes were significantly higher in the anoxic/hypoxic zone than the oxic zone. CO2 and CH4 fluxes from the anoxic/hypoxic zone were positively correlated with 5-day biochemical oxygen demand, and negatively correlated with dissolved oxygen and nitrate, suggesting that anaerobic respiration contributes significantly to CO2 and CH4 flux. CO2 and CH4 fluxes peaked at ~22 to 23oC, as expected for microbial processes. CH4 flux was positively correlated with ammonium concentration in the anoxic/hypoxic zone, likely due to inhibition of methane oxidation by ammonium. N2O flux was not significantly correlated to any wastewater parameter. I estimated that advanced N-removal OWTS contribute approximately 347 g CO2 equivalents capita-1 day-1, comparable to emissions from conventional OWTS.

My findings show that advanced N-removal OWTS in Rhode Island vary in their compliance with the state’s standard for final effluent TN, in the structure and composition of their N-cycling microbial communities, and in their contribution of GHGs to the atmosphere. Continued monitoring of effluent TN from these systems could be easily achieved using rapid tests such as a field photometer, which we found to be a reliable and cost-effective predictor of effluent TN concentrations.



To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.