Ryan Ziels

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Urban development relies on biological wastewater treatment to metabolize organic material, nutrients, and transform pollutants before entering the receiving environment. Activated sludge is a commonly used biological treatment process and hosts a complex and largely unidentified microbial community. Wastewater treatment operators utilize various monitoring parameters to optimize removal efficiencies in the activated sludge system; yet a knowledge gap exists regarding the specific roles certain microbes play in water treatment, and how microbial community data can be generated and used for routine process monitoring. This thesis studied the benefits of using microbial community composition as an additional monitoring parameter for wastewater operators to utilize for making informed decisions. Activated sludge samples were taken twice a week at a regional wastewater treatment plant for over a year and analyzed using a full-length 16S rRNA amplicon sequencing method with the Nanopore platform. To discern which microorganisms were active in the wastewater treatment process, an ecological genomics-based mass balance model (EGMB) was established using full-length 16S rRNA zero-radius operational taxonomic units (zOTU’s). Previous EGMB models utilized short read data that have lower taxonomic resolution than long-read data, presenting an opportunity here to develop a more accurate model. The EGMB model was able to accurately distinguish process-critical species from inactive immigrating species, find their seasonal differences, and predict an incoming process disturbance. An additional analysis was conducted on the activated sludge microbial community to determine which operational parameters and influent characteristics had the greatest influence on community composition and found temperature and stirred sludge volume index to have the highest association with the active microbial community.

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Global plastic production has increased exponentially since the 1950s, ushering in an era of cheap synthetic polymers that have revolutionized human manufacturing and promoted socioeconomic development. However, the benefits of this plastics revolution are contrasted with human and environmental health problems resulting from its disposal. Not only has plastic waste contributed to burgeoning accumulation in landfills and the ocean, but additives and leachates from plastic polymers have been linked to negative health effects including endocrine disruption and cancer. Less well understood is the potential impact of microplastic particles and fibers (MPs) between 0.3mm and 5mm on human and environmental health. MPs frequently enter coastal marine waters through wastewater treatment where they are rapidly colonized by microbes. The drivers of community assembly on MPs remain unconstrained with implications for carbon cycling, antimicrobial resistance and mobilization of additives and leachates through marine food webs. Here, I investigated microbial community assembly on marine plastic fibers and the impact of MP pollution on planktonic microbial community composition. A time-resolved mesocosm experiment using different concentrations of MP fibers indicated that increased fiber concentrations did not have a significant effect on planktonic microbial community composition or chemical concentrations but did induce finer-scale taxonomic changes. A related in-situ textile degradation experiment indicated rapid microbial colonization coalescing into relatively stable community structures after one month. Plastic-attached communities varied significantly by polymer type but not by chemical additive or color. These results demonstrate that marine microbial community assembly varies by plastic type, but MP pollution may not significantly affect the surrounding planktonic microbial community.

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Anaerobic digestion (AD) is a well-established organic waste treatment biotechnology that enables resource recovery in the form of biomethane and nutrient-rich biosolids. AD systems treating municipal organic waste are prone to reduced biomethane yields and process upsets due to ammonia inhibition within their underlying microbial community. Microbial communities that are resilient to high ammonia conditions within AD are therefore of interest, given their ability to maintain stable and efficient AD reactor operation. Such microbial communities are generally characterized by the presence of a microbial guild performing syntrophic acetate oxidation (SAO) in partnership with hydrogenotrophic methanogens. So far, few members of the SAO guild have been identified and characterized. The objective of this study was to characterize the microbial community present within a high ammonia AD system and identify the taxa that were performing SAO. In this study, a commercial-scale biogas facility with a dry mesophilic AD system treating municipal organic wastes was monitored over the course of a year. Chemical monitoring of the AD system revealed high in situ ammonia concentrations, while a microbial community profile obtained via 16S rRNA gene amplicon sequencing suggested SAO as a dominant means of acetate utilization. In situ activity profiling was performed in batch microcosms with biomass sampled from the AD reactor using DNA stable isotope probing (SIP) amended with universally labelled ¹³C-acetate. DNA extracts recovered from the SIP incubations revealed a 5% shift in total community DNA ¹³C content. Following a consensus-based approach of high-resolution and quantitative SIP data analysis, 13 bacterial amplicon sequencing variants (ASVs) belonging to 8 genera were determined as isotopically enriched. Linking short-read 16S rRNA ASVs to long-read metagenomic assemblies allowed for accurate phylogenetic placement of acetate-incorporating taxa, as well as for an examination of potential metabolic pathways used for acetate oxidation by the active members. The results of this study provide new insights that can help guide process operations and optimization of the study AD system, as well as contribute to the emerging body of knowledge regarding SAO and microbial communities underpinning dry AD systems with high ammonia levels.

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Linking the genomic potential of uncultivated microbes to their in situ metabolic functions remains a critical challenge in the field of microbial ecology. This knowledge gap is especially relevant to our understanding of methanogenic microbial communities inhabiting engineered anaerobic digestion (AD) bioreactor environments. For instance, one of the major methane-yielding pathways in AD is mediated by the coupling of syntrophic acetate oxidation (SAO) and hydrogenotrophic methanogenesis. Our understanding of the stoichiometry and enzymes involved in this relationship continues to develop, yet we still lack data to support many relevant metabolic processes, or even a complete census of the microorganisms capable of carrying out SAO. So far, very few SAO bacteria have been isolated, and conventional multi-omic methods have been unable to elucidate their in situ physiologies due in part to their low abundance in AD environments. To bridge this knowledge gap, this thesis proposes a novel application of molecular techniques to quantitatively resolve microbial activity in uncultivated consortia. The combination of metabolic profiling via protein-based stable isotope probing (protein-SIP) and bio-orthogonal non-canonical amino acid tagging (BONCAT) with fluorescence-activated cell sorting (FACS) was used to study the flux of acetate through anaerobic digestion microcosms seeded with digestate from a full-scale biogas facility. As a result, the proteome of a novel SAO bacteria was highly enriched and yielded sufficient expression information to describe its metabolic pathway for acetate oxidation. This study marks the first application of microliter processing of trace samples in one pot (μPOTS) to microbial communities, and generated the first known proteomic profile of the ‘oxidative glycine pathway’ in a putative SAOB. This work demonstrates that BONCAT-FACS coupled with protein-SIP can be used to generate activity-targeted metaproteomes, and is extensible to other microbial communities where rare but active microorganisms underpin essential ecosystem functions or services.

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Over the past few years, microplastics (MPs) have emerged as contaminants of global environmental concern, but important questions remain regarding their source, transport and fate. Because of their complexity, quantification and identification of MPs remain a major analytical challenge. The goals of this study were to examine the importance of Combined Sewer Overflows (CSOs) as one of the pathways for MPs to urban watersheds, and the fate of urban MPs in the receiving waters. In addition, a new statistical protocol to guide efficient and accurate analysis of MPs was also developed. Focusing on the Vancouver metropolitan area, this study provides novel data on MPs in CSOs in Canada and in the Fraser River, BC – a major river in western North America. MPs were detected in all CSOs and the Fraser River samples, ranging in concentration from 1,833─13,673 MPs/m³ and 4─30 MPs/m³, respectively. A preliminary assessment of the annual emission rates of MPs by the two CSOs under study was carried out, with values ranging from 0.82 to 14.6 × 10⁹ MPs/year. This implies that CSO emissions of MPs require further research and should not be overlooked in management strategies. The much lower concentrations of MPs in the receiving waters of CSOs and significantly shorter fibers suggest a complex fate, which may involve mixing with other sources, sinking and/or rapid dilution upon entry to the environment. This research also suggests that the Fraser River is an important conduit for MPs to the Strait of Georgia, given the estimates of 0.67─3.35 trillion annual microparticles flux derived in this study. While the dimensions, polymer types, and shapes of MPs varied in space and time, synthetic fibers were most common (58─78% in CSO samples and 76─86% in river samples) and were dominated by polyester. Fibers emitted with CSOs had a median width of 24 µm, which is consistent with the average width of textile fibers. Fragments were also present and comprised mostly of polyethylene in both CSOs and river samples (0─70%). This study helps better understand the sources and transfer of MPs in urbanized watersheds, with management strategies and other solutions are also discussed.

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Partial nitritation/anammox (PN/A) is a novel pathway for nitrogen removal in wastewater treatment that offers advantages of low oxygen and organic carbon demands as well as high potential for energy recovery. However, the partial nitritation process remains a key hurdle for the widespread implementation of the PN-A process in mainstream treatment due to the difficulty in washing out nitrite-oxidizing bacteria (NOB) from active sludge. Exposing biomass to high concentrations of free ammonia (FA) has been reported as an effective strategy to achieve partial nitritation. This study examined the effectiveness of treating 20% of return activated sludge with synthetic centrate containing FA at 200 mg N/L for 24 hours to promote partial nitritation in mainstream wastewater treatment. Experimental and control bioreactors were operated under two different conditions, with or without FA treatment, respectively, after reaching similar nitrification performance. Biokinetic parameters of ammonium-oxidizing bacteria (AOB) and NOB were estimated by performing respirometric batch tests with activated sludge biomass from the two bioreactors under different operational conditions, and calibrating a process model based on oxygen mass balance. The bioreactor performance showed that the FA treatment strategy promoted the PN process, with a maximum nitrite accumulation ratio (NAR) of 41.9 ± 2.1% after treating return sludge with high FA solution for 37 days. However, this nitrite accumulation was not stable, and the NAR decreased to 10.9 ± 6.0% after 33 days, indicating that NOB were able to acclimate to the temporary exposure to a high FA concentration. The biomass yield coefficient (Y) of AOB increased during FA treatment, while the maximum specific growth rate (μmax) of AOB and NOB decreased under this condition. Microbial community analysis on activated sludge under FA treatment, and further investigations on the optimization of the FA treatment strategy combined with other NOB out-selection strategies are required to better facilitate the application of PN-A to full-scale mainstream wastewater treatment.

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Biofilm photobioreactors rely on cooperation between algae and bacteria within a single biofilm to treat wastewater. Algae growth produces oxygen, which can subsequently be utilized by aerobic bacteria to degrade organic matter and produce carbon dioxide, which is then utilized as a carbon source by algae. Due to their relatively low maintenance and energy inputs, biofilm photobioreactors could be amenable for decentralized wastewater treatment. However, the impact of biofilm photobioreactor design on treatment efficiency has received little attention. Here, it was hypothesized that open (i.e. unsealed) versus closed (i.e. sealed) photobioreactors could promote different nitrogen removal pathways by altering redox conditions throughout a diel cycle. This study explored the effect of open versus closed photobioreactor configurations on nitrogen removal and the microbial community structure in two parallel photobioreactors treating microscreened decentralized wastewater. The reactors were intermittently lit in a 16hr-8hr light-dark cycle, and operated at an HRT of 2 days and SRT of 9 days. The influent feed regime and alkalinity addition were varied over three successive 30-day experimental phases. Microscreening was an effective primary treatment step, removing 70 ± 6% (95% c.i.) of suspended solids and 39 ± 9% of COD. The photobioreactors removed 90 ± 6 % and 83 ± 3% of the remaining suspended solids and COD respectively, independent of operating conditions. Alternating oxic and anoxic conditions were observed in both reactors during the lit and unlit periods, respectively, resulting nitrification and denitrification. Optimal nitrogen removal conditions were observed under a sequencing batch feed regime with alkalinity addition. Under these conditions, TKN removal was significantly higher in the open reactor at 93 ± 5% compared to 78 ± 6% in the closed reactor due to higher rates of nitrification and N assimilation. TN removal was similar at 77 ± 9% and 76 ± 8% in the open and closed systems, respectively. The dominant bacterial genus in the reactors was Tychonema, a cyanobacteria which comprised up to 87% of 16S rRNA gene amplicon reads. Overall, this study demonstrated that nitrogen removal pathways differ significantly in open and closed photobioreactors when operated at the same COD loading rate.

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