Thesis advisor: Alan L. Kafka / Discerning the interrelated effects of space and time on the potential for wastewater well injection to induce earthquakes in Oklahoma is important for accurately mapping seismic hazards. This study explores how distance from wells and time after initiation of injection affect the possibility that injection activity might induce earthquakes under different conditions of operational lifetime, injection volume, and well depth. A unique feature of this study is filtering of the injection well database to isolate, as much as possible, the effect of specific well injection on the potential to induce earthquakes. The method used here is a modified version of “Cellular Seismology”, termed “Modified Cellular Seismology” (CS, MCS), where “CS Predictability” (CSP) is used as an operational definition of the extent to which injection wells are associated with earthquakes. I hypothesize that earthquakes associated with injection are most likely to occur within about 15 km of wells and within approximately the same year as active injection. Evidence shows that induced earthquake activity peaks primarily between about 2.5 and 3.5 km away from any given well, and this distance increases while CSP decreases over time. Temporal analyses suggest that CSP decreases by an average of about 5% over a period of five to seven years for any given well (or about 1% decrease per year), though there exists considerable scatter in this relationship. This change is variable across wells of different conditions, ranging from a decrease of 26% to an increase of 8% over the five to seven years covered by this study. Additionally, CSP tended to peak at least a year after injection for the most spatiotemporally isolated wells, suggesting that there may be, on average, at least a year of lag before any given well is likely to induce earthquakes. / Thesis (MS) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Earth and Environmental Sciences.

Discharging cheese production wastewater with high concentrations of organics and nutrients is detrimental to receiving aquatic systems, as the release of these deleterious substances cause oxygen depletion, and eutrophication respectively. On-site treatment of cheese production wastewater requires the removal of high concentrations of organics and nutrients with a small land footprint to meet regulations. There is therefore a critical need for compact, high-rate, cost-effective wastewater technologies such a as the moving bed biofilm reactor (MBBR). Although MBBR systems have been well established for carbon and nitrogen removal, to date only a limited number of studies have achieved enhanced biological phosphorous removal in sequencing batch moving bed biofilm reactor (SB-MBBR) systems, and only for municipal-strength wastewater. Operating SB-MBBR systems under sequencing batch mode enables the reactor operation to be well synced to the fluctuating influent concentrations and flow characteristics of cheese production wastewaters. Furthermore, cycling between anaerobic and aerobic conditions can be achieved in a single sequencing batch reactor, which can promote the proliferation of poly-phosphate accumulating organisms. The SB-MBBR is studied in this research for the removal of carbon, nitrogen, and phosphorous from cheese production wastewaters. Specifically, the effects of anaerobic staging time, aeration rate, enhanced aerobic operation, and adding a second reactor in series was studied by analyzing the kinetics, biofilm characteristics, and microbiome. Extending the anaerobic staging time was shown to achieve aerobic soluble chemical oxygen demand removal rates of 92.5±2.8 g·m⁻²d⁻¹, by selecting for a thinner biofilm with, with a lower biofilm dry-density and a more rough biofilm surface, and therefore likely a biofilm with an enhanced mass transport. A significant shift in the microbiome was observed with longer anaerobic staging times and lower aeration, whereby possible putative poly-phosphate accumulating organisms including Brachymonas, and Dechloromonas were selected for in greater relative abundances compared to anaerobic bacteria. The total phosphorous removal in the possibly resulted from enhanced biological phosphorous removal, supported by the high abundance of putative poly-phosphate accumulating organisms (43.1±8.4%), which dominated the biofilms in the SB-MBBRs with 120 and 168 minute anaerobic staging times. Finally, total ammonia nitrogen oxidation was achieved through partial nitritation with a two reactor in series configuration with a removal rate of 1.07±0.05 g-N·m⁻²d⁻¹. Two SB-MBBRs operated in series was shown to be the superior strategy for achieving TAN compared to a single SB-MBBR with extended aerobic operation. By operating two SB-MBBRs in series, competition between autotrophic nitrifiers and heterotrophs is averted, and AOB proliferate in the biofilm, achieving TAN oxidation. Since TAN oxidation is likely achieved through partial nitrification, the SB-MBBR technology may be incorporated in a deammonification treatment train. The overall study presents novel information for the SB-MBBR design and operation, along with biofilm and microbiome fundamental findings that will guide future pilot- and full-scale applications of the SB-MBBR to treat cheese production wastewater.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

Excess nitrogen and phosphorus in discharged wastewaters can lead to downstream eutrophication, ecosystem damage, and impaired water quality that may affect human health. Chemical-based and physical-based technologies are available to remove these nutrients; however, they often consume significant amounts of energy and chemicals, greatly increasing treatment costs. Algae are capable of removing these pollutants through biomass assimilation, and if harvested, can be utilized as a feedstock for biomethane or biodiesel production. Currently, difficulties in harvesting, concentrating, and dewatering algae have limited the development of an economically feasible treatment and production process. When algae are grown as surface-attached biofilms, the biomass is naturally concentrated and more easily harvested, leading to less expensive removal from treated water, and less expensive downstream processing for biofuel production. In this study, a novel algal biofilm production and harvesting system was designed, built, and tested. Key growth parameters were optimized in order to maximize biomass production and nutrient uptake from wastewater. Compared to suspended algae systems, the attached algal biofilm design of this study led to increased biomass production and greater treatment of domestic wastewater. An efficient and inexpensive algal biofilm harvesting technique was also developed in order to obtain a concentrated biosolids product, resulting in improved water quality and a feedstock suitable for further processing in the production of biofuels.

The midcontinent United States, a region which typically does not experience many earthquakes, has experienced a significant increase in the number of earthquakes over the last decade. This increase in earthquake activity has been linked to wastewater injection, a process in which large volumes of wastewater from oil and gas extraction are injected into deep (2-3 km), high-permeability sedimentary rocks, near low-permeability Precambrian (>540-million-year-old) crystalline ‘basement’ rocks. The contact between these two rock types is referred to as the Precambrian nonconformity. Injection-induced earthquakes occur on or near basement-hosted faults due to an increase in pore fluid pressures, which implies that there may be a hydrological connection between the basement-hosted faults and the injection point. We hypothesize that the Precambrian nonconformity greatly influences this hydrological connection. We investigate the geologic properties of Precambrian nonconformity zone outcrop and core analogs to examine how the geology of the nonconformity zone controls fluid flow. Methods include mapping of geological materials and deformation structures (faults and fractures), mineralogical analysis, and geochemical analysis. These data sets allow us to infer the nature of fluid flow in the past, and make predictions about fluid flow in the future. In addition, this information is used to inform hydrological models, improving the ability to predict earthquakes due to wastewater injection. We identify three main geological scenarios that are likely to be encountered at the nonconformity. These are: 1) basal conglomerate, 2) weathered/altered horizon, and 3) mineralized contacts. These scenarios, or combinations of these scenarios, may be fractured or faulted, resulting in a variety of hydrological implications. The permeability of basal conglomerates and weathered horizons at the contact depends on the textures and minerals that are present. Regolith, clast-supported granitic wash, or poorly cemented conglomeratic horizons, may act as high permeability conduits, whereas a clay-rich grus or granitic wash, or tightly cemented conglomerate, may act as low permeability barriers. Mineralized contacts may act as low permeability barriers due to a reduction of pore space. The mineralized contact shows that the introduction of warm brines by modern injection may result in mineralization or chemical weathering, dynamically affecting permeability over time depending on the mineralogy of the host rock and chemical composition of the injected brine.

Isolation and purification of anthocyanins from black bean canning wastewater by column chromatography with macroporous resins were investigated in this study. Different adsorption materials and adsorption conditions were compared and the most effective material and adsorption conditions were selected to purify anthocyanins. Purified anthocyanins then were identified by high performance liquid chromatography electrospray tandem mass spectrometry. The most effective macroporous resin was selected by comparing the adsorption performance of five different types of macroporous resins (Diaion Hp20, Sepabeads Sp70, Sepabeads Sp207, Sepabeads Sp700, and Sepabeads Sp710). Equilibrium adsorption isotherms of five resins with wastewater were measured and analyzed using Langmuir and Freundlich isotherm models. Both Langmuir and Freundlich models could describe the adsorption process. The adsorption and desorption behaviors of anthocyanins were studied using a dynamic method on the five types of resins, and Sp700 presented the highest adsorption capacity as well as desorption capacity, indicating that Sp700 is a good candidate for purification of anthocyanins from black bean canning wastewater. The most effective adsorption conditions were tested using Sp700. Dynamic adsorption and desorption were performed in glass columns packed with Sepabead Sp700 to optimize the purification process. Temperature during adsorption and desorption (25°C and 35°C) did not significantly affect the adsorption and desorption ratio. Adsorption ratio was significantly reduced when the flow rate increased from 1.5 mL/min to 2.5 mL/min. However, desorption ratio was not affected by flow rate (from 1.5mL/min to 0.3mL/min). Ethanol concentration (from 30% to 60%) did not affect desorption ratio. Four kinds of anthocyanins were identified in black bean canning wastewater. The major anthocyanins were delphinidin 3-glucoside, petunidin 3-glucoside, and maldvidin 3-glucoside, with a small amount of petunidin 3, 5-diglucoside also in the final product.

Two computational models, including several calculation and analysis submodels, were developed to create a tool for assessing the impact of different treated wastewater reuse options on irrigated agriculture. The models consider various aspects of treated wastewater availability (past, present, and future), wastewater quality, agricultural water demand, and the economics of conveying wastewater from treatment plants to farms. The two models were implemented using Visual BASIC.NET in a GIS environment to facilitate visualization of some of the features of an area under study, and to provide a convenient interface for user application. One of the models is for treated wastewater availability calculations, and the other is for wastewater reuse. The water availability model has sub-models including urban population predictions, agricultural land use changes, residential water demand, agricultural water demand (evapotranspiration) for over 40 crop types, and treated wastewater analysis. The water reuse model is composed of three sub-models, including soil water and salt balance calculations, nutrient calculations, and pumping and conveyance costs calculations. The nutrient calculationssub-model is based on an existing model, but was completely rewritten and modified in some parts to accommodate the needs and features of the water reuse model presented herein. A sample application of the models is presented for Cache Valley, Utah. The results show a comparison of treated wastewater reuse schemes for the study area, highlighting how irrigated agriculture would best benefit from the total or partial use of treated wastewater. Two wastewater reuse scenarios were considered. The water availability model shows good agreement with other sources of information in terms of population forecast and calculation of future residential and agricultural water demand. However, according to the results from the model, the rate of increase of the urban area was much higher than the rate of decrease of the agricultural areas between the years 1992 and 2001.The future population growth and water demand increases for urban areas was calculated and validated for Logan City. Also, in the case study the model was shown to be a good tool for wastewater influent analysis for Logan City.

Alternative fuels are necessary to meet the increasing demands for fuels. Alternative fuels such as biodiesel are produced using vegetable oils, which are prominentt in the food industry. An alternate feedstock could be oil-producing microorganisms. These oleaginous microorganisms are defined as accumulating more than 20% of their weight in oil as lipids. Cultivating these microorganisms for oil production is not economical due to the high production costs from the sugars in the culture medium. Municipal wastewater could be a potential growth medium that has not previously been considered for cultivating oleaginous microorganisms. However, municipal wastewater contains a low concentration of carbon, which does not promote oil accumulation in the oleaginous microorganisms. To increase the carbon concentration in the wastewater, lignocellulosic sugars could be added to the municipal wastewater. These sugars are a potential alternative to sugars that are in the food industry. The goal of this research is to determine the efficacy of using municipal wastewater to cultivate a consortium of oleaginous microorganisms, thus, producing oil for biodiesel production. First, a consortium of oleaginous microorganisms was cultivated on autoclaved wastewater to determine if the wastewater contains any inhibiting substances for the microorganisms. In addition to the substances in the wastewater, indigenous microorganisms are possible inhibitors to the consortium. Therefore, to determine the effect these indigenous microorganisms have on the oleaginous microorganisms, the consortium was cultivated on raw municipal wastewater amended with varying amounts of sugar. Since the municipal wastewater can be used as a cultivation medium, the effect of the addition of lignocellulosic sugars was determined. During the production of lignocellulosic sugars, furfural and acetic acid, known microbial inhibitors, are formed. The effect of these inhibitors on the consortium’s growth and oil accumulation ability was ascertained, and inhibition models were developed to describe their impact. With these results, SuperPro Designer v6.0 was used to perform simulations and economic analyses to determine the efficacy of incorporating an oleaginous microorganism consortium in a wastewater treatment facility.

Acid mine drainage (AMD) is a major issue for the mining industry and a global environmental concern. It is facilitated heavily by microbially catalyzed sulfur oxidation/ disproportionation reactions involving reduced sulfide components in mine wastes that result in the release of harmful acidity and metals. The microbial processes catalyzing sulfide mineral leaching of mine waste rock resulting in AMD have been previously studied. However, the initiation of AMD processes in mining wastewaters has not been well studied. Post extraction, highly contaminated, mining wastewater is stored in retention (tailings) impoundments on site and treated to prevent impacts such as toxicity, contamination, and acidity prior to discharge to the receiving environment. Tailings, the waste stream generated through extraction of sulfide ores contains a variety of aqueous sulfur oxidation intermediate compounds (SOIs), such as polysulfides, elemental sulfur, and sulfur oxyanions, of the form SxOy2- termed “thiosalts” by the industry, which can also generate AMD. These wastewaters typically initially start off with circumneutral pH, but if thiosalts are present in high enough concentrations, microbial processes can cause net-acid generation leading to AMD. The microbial ecology and functionality of endemic tailings impoundment microbial assemblages in the “pre-net acid generating phase” (PNAG) of circumneutral mine wastewaters, as well as the associated sulfur species and reactions are not well understood. Thus, early-stage indicators that would offer mines proactive monitoring tools for improved tailings impoundment management are currently lacking. In collaboration with our mining industry partners, who provided tailings impoundment water samples, this dissertation tackles these limitations. This dissertation aimed to identify important microbes and the SOI important to the initiation of AMD in PNAG tailings impoundment wastewaters and to determine potential markers (microbial and/or geochemical) associated with these initiating AMD processes that would inform the development of monitoring tools in mine water management. The objectives of this doctoral research were to constrain the S biogeochemistry of the PNAG phase by characterizing both expression levels of sulfur oxidation genes and sulfur speciation under experimental conditions designed to assess the roles of microbial community, SOI geochemistry and pH. Specifically three well constrained laboratory experiments determined: (1) gene expression by a pure culture of Acidithiobacillus thiooxidans and sulfur speciation with either thiosulfate or elemental sulfur added as the starting SOI substrate (Chapter 3); (2) parent mining wastewater communities and associated sulfur oxidizing bacteria (SoxB) enrichments collected seasonally at two mines and grown at either pH 7-5 or pH 5-3 genetically (16S amplicon) (Chapter 4); and (3) geochemical sulfur pathways of three unique mine wastewater SoxB enrichment communities for six distinct simulated mine wastewater thiosulfate and/or tetrathionate treatments (Chapter 5). Results presented in Chapter 3 expand the understanding of the reactions and enzymes involved in S0 and S2O32- metabolism by a pure strain sulfur oxidizing bacteria A. thiooxidans ATCC 19377 by developing the first models integrating gene expression, solution sulfur speciation, electron microscopy and spectroscopy. These novel results reveal that A. thiooxidans S2O32- metabolism involves the conversion of S2O32- to SO42-, S0 and S4O62-, mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr) and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO32-, S2O32- and polythionates in the A. thiooxidans S0 metabolism model. The results of this chapter advance understanding by revealing (1) the important role specifically of TetH in S2O32- metabolism; (2) Hdr proteins, rather than Sdo proteins, are likely associated with S0 oxidation; (3) that formation of intracellular S2O32- is a critical step in S0 metabolism, and (4) that recycling of internally generated SO32- occurs, through comproportionating reactions that result in S2O32-. Results summarized in Chapter 4, identify that pH is the most important factor influencing which sulfur oxidizing bacteria (SoxB) occur irrespective of total S concentration of SOI substrate provided in enrichment experiments for two mines of different parent wastewater geochemistry. Mine 1 exhibited a lower total S and reactive soluble sulfur compounds (oxidation state < +VI) concentrations, and greater parent wastewater microbial community diversity with more unique sequences relative to Mine 2. All experimental SoxB enrichment experiment microbial communities evidenced a shift in dominance from primarily Alphaproteobacteria (28% - 77%) at circumneutral pH to Gammaproteobacteria (>80%) under moderately acidic pH values. A further pH dependent shift was observed at the genus level, from Halothiobacillus spp. dominating the circumneutral pH SoxB enrichments to Thiomonas spp. dominating the mildly acidic SoxB enrichments. These results provide some of the first putative biological indicators to improve prediction and management of sulfur processes and AMD onset within mining wastewaters. Chapter 5 results importantly assess the influences of SOI chemistry and SoxB consortia identity on SOI cycling. Results identify that SOI substrate, whether S2O32- , S4O62-, or S2O32- + S4O62-, was a more important determinant of microbial sulfur outcomes than the relative abundances of Halothiobacillus spp. and Thiomonas spp.. Further, three pH dependent phases of microbial sulfur processing were identified: Phase 1, pH > 5, dis/comproportionation reactions and acid consuming reactions were prevalent alongside oxidation, with S2O32- identified as an important indicator; Phase 2, pH 5 – 3, further dis/comproportionation occurred, with S0 emerging as an important indicator signalling the progression of the system towards net acid generation with S4O62- or S2O32- as the major S species present and; Phase 3, pH < 3, i.e. full AMD conditions, with dominant oxidation reactions resulting in SO42- and acid generation. Collectively, these results identify specific SOI species important at different pH stages in AMD initiation and development across different microbial SoxB, providing new indicators that may serve as signals associated with predictive tool development. The integration of the novel results of this thesis revealed some of the first biologically informed possible indicators (bacterial and geochemical) that will enable improved management through proactive monitoring tools for AMD initiation in mining wastewaters. / Dissertation / Doctor of Science (PhD)

Microplastic (MP) pollution is ubiquitous in the aquatic environment. Though their properties are known to vary considerably, these particles are typically 1–5,000 μm in size and irregular in shape. Research suggests that MPs pose a significant hazard to aquatic ecosystems, lead to negative economic consequences, and may cause adverse human health effects. The effluents of municipal wastewater treatment plants (WWTPs) comprise a significant source of MPs, containing < 1 MP/L to > 440 MPs/L. Pursuant to the large volumes of wastewater processed, estimated daily effluxes can exceed one billion MPs/day in some WWTPs. Membrane technologies, like those used in some tertiary wastewater treatment applications, appear well-positioned to mitigate releases of MPs. However, research directly characterizing the performance of membranes in these applications is lacking. The studies in this work address this knowledge gap. To this end, irregularly-shaped MPs were produced in a novel milling/sieving process. Ultrafiltration and microfiltration membranes were challenged to these MPs suspended in secondary effluent wastewater to elucidate their fouling behaviour under realistic solution conditions. Subsequently, MPs milled/sieved from a fluorescently-labelled plastic feedstock were utilized in microfiltration experiments. Bulk MP concentrations in samples were easily measured using a plate reader to quantify MP rejection. Improving upon this technique, a new protocol involving a flow cytometer was developed, enabling the identification of individual fluorescent MPs in filtration samples, even when complex solutions chemistries were used. A culminating investigation was performed to bridge a gap in the literature between studies considering small-scale laboratory filtration phenomena and observations of large-scale WWTPs. Thus, the performance of a hollow fiber crossflow microfiltration module was evaluated in the filtration of wastewater containing MPs. Overall findings suggest that incidences of fouling by MPs can be managed via periodic cleaning processes, and the well-informed selection/operation of membrane technologies can contribute to high MP removal efficiencies (> 99%) in tertiary wastewater applications. / Thesis / Doctor of Philosophy (PhD) / The term "microplastics" (MPs) is used to describe microscopic plastic particles. Recent investigations have reported these MPs in lakes, rivers, and oceans across the globe. These reports are concerning as other studies demonstrate that MP pollution can be hazardous to aquatic life, yet the potential effects of MPs on human health remain largely unknown. Many MPs originate from municipal wastewater treatment plants (WWTPs) which discharge large numbers of these particles into the aquatic environment. Researchers often recommend the use of membranes as a barrier to prevent MPs from leaving in the final treated wastewater. This work seeks to assess that recommendation. Assessments of the effectiveness of membranes at withholding MP particles in wastewater are performed over various conditions. The propensity of MPs to interfere with the desired output of treated wastewater is also measured. Overall, findings indicate that carefully designed and operated membranes processes can be well-suited to this application.