Browsing by Author "Hug, Laura"

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Composition of the North American Wood Frog (Rana sylvatica) Bacterial Skin Microbiome and Seasonal Variation in Community Structure
(Springer Nature, 2020-07-01) Douglas, Alexander; Hug, Laura; Katzenback, Barbara
While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity, however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs’ respective pond water. Across seasons, diversity analyses suggest there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.
Exploring microbial diversity across a Southern Ontario landfill
(University of Waterloo, 2019-09-11) Sauk, Alexandra; Hug, Laura
Sanitary landfills are highly engineered environments that receive a heterogeneous mixture of organic waste, metals, and plastics. Global waste production continues to grow every year and waste management is an increasing environmental and financial concern for municipalities. Over the last 50 years, many municipalities have improved recycling efforts and hazardous waste disposal to limit inputs to landfills; however, landfills still contain and receive a number of hard to degrade and/or dangerous materials, including heavy metals and volatile compounds. Conventional landfills are designed to entomb municipal solid waste and prevent its degradation by microorganisms. Despite this engineering goal, waste degradation in landfills via aerobic and anaerobic decomposition by microorganisms actively reshapes the municipal solid waste over time and must be accounted for in landfill design. Our depth of knowledge on the diversity of landfill microorganisms and how this microbial diversity changes across and between landfills is limited. Much of the current research into landfill microbial diversity has investigated specific groups with known functions, such as cellulose degraders, methanotrophs, and methanogens. Recently, research groups have taken a community-based approach to studying landfill microbes, relating community composition to environmental and chemical parameters. Many of these previous studies rely solely on 16S rRNA gene amplicon sequencing for taxonomic identification, which limits functional predictions to what is already known about related groups. Understanding landfill microbial communities and their functions is important for informing waste management practices, and has the potential to reveal novel degradation metabolisms that can be used in waste remediation. This research combines 16S rRNA gene amplicon and metagenomic sequencing to examine the taxonomic and functional diversity of a Southern Ontario landfill and to relate this microbial diversity with site geochemistry. Eight samples were collected from a municipal landfill in 2016 via filtration of the liquid from three leachate wells, two temporal samples of a leachate-collecting cistern (one size-filtered in two fractions), and two groundwater wells from the adjacent aquifer. The DNA for all eight samples underwent 16S rRNA gene amplicon sequencing by the U.S. Department of Energy’s Joint Genome Institute. In parallel, total community DNA was extracted and then shotgun sequenced by the U.S. Department of Energy’s Joint Genome Institute for six samples, generating assembled and annotated metagenomes. Maximum likelihood phylogenies were inferred from the metagenome-derived 16S rRNA genes and a concatenated alignment of 16 syntenic ribosomal proteins co-located on single scaffolds. Based on a comparison between taxonomy derived from the two phylogenies, high quality metagenome assembled genomes (MAGs), and the 16S rRNA gene amplicon data, 22 bacterial and 2 archaeal phyla were present at >1% relative abundance within at least one landfill sample. The Patescibacteria, Bacteroidota, Firmicutes, and Proteobacteria had the highest relative abundances, whereas most other phyla were present at low abundance, with some fluctuations among sites. Below the phylum level, very few microorganisms were identified across multiple sites, with only 37 of 2989 populations (represented by 16S rRNA exact sequence variants) present in two or more sites. This indicates that, although phylum-level signatures are conserved, individual landfill populations vary widely both spatially and temporally. Three leachate and two groundwater sites had partial or complete volatile and non-volatile compound concentration data, allowing for limited correlation of geochemical conditions and contaminant concentrations to microbial diversity patterns across the landfill. Significant differences in geochemistry exist across the sites, with calcium, iron, magnesium, boron, meta and para xylenes, ortho xylenes, and ethylbenzene concentrations contributing most strongly to site differences. The genera Sulfurovum, Proteiniphilum, and Ferritrophicum are relatively abundant in the amplicon data, and have predicted roles in nutrient and contaminant cycling in the landfill related to benzene, proteins, and iron. Access to more sites with complete geochemistry data is required to allow direct connections to be drawn between microbial populations and site geochemistry. A phylogenetic and metabolic analysis was conducted examining the Tenericutes and Erysipelotrichia, which are related radiations dominated by parasitic and host-associated organisms. Landfill metagenomes allowed reconstruction of 12 Tenericute MAGs distributed within four orders, as well as five MAGs within the order RFN20 that are sister to the Erysipelotrichales. The landfill Tenericutes exhibited small genome sizes below 2 Mb, which is expected for this group, and encode a range of biosynthetic capabilities. Genome features suggest potential lifestyles: free-living, commensal, or parasitic, with the potentially parasitic bacteria showing a greater loss of amino acid pathways. The RFN20 MAGs’ genome sizes are below 3 Mb, closer to the Tenericute than the Erysipelotrichia reference genomes. One RFN20 MAG showed similar metabolic capacities to Dielma fastidious, an Erysipelotrichia species, whereas the other RFN20 MAGs are predicted to be metabolically more similar to the Tenericutes. The reconstruction and analysis of these Tenericute and RFN20 genomes expands our current knowledge of these abundant groups’ potential lifestyles in the landfill, and expands genomic representation of these understudied orders.
From genomes to metagenomes: Development of a rapid-aligner for genome assembly and application of macroecological models to microbiology
(University of Waterloo, 2019-09-26) Hilts, Angus; Hug, Laura
Since the development of the modern computer, many scientific fields have undergone paradigm shifts due to an increasing facility in data collection and analysis. Microbiology has been impacted by computational advances, especially in DNA sequencing applications, and this has led to an interesting problem: there is too much raw data for any person to understand. It is important to have tools that are able to process and analyze these vast amounts of data, so that microbiologists can robustly test hypotheses and predict patterns. Long-read sequencers are capable of sequencing entire genomes with very few reads, but exhibit much higher error rates compared to short-read sequencing platforms. Most current genome assemblers were developed for highly accurate short-read data, and so there is a need to build new tools that can handle these long, error-filled reads. Here, we developed an alignment algorithm in the C programming language for error-prone long reads, as part of a larger genome assembler. This alignment algorithm creates a profile of ordered kmers representing all of the reads, then clusters these kmers to generate a consensus sequence. We show that the alignment algorithm can handle long-read error rates and produce useful results. Using a low-coverage test data set, the algorithm was able to produce a consensus sequence with 85.3% identity to a reference sequence built with extremely high coverage data. Future work will aim to improve this accuracy by error correcting kmers and identifying close repeats of kmers. The field of metagenomics is entering a new state of maturation. Isolation of total community DNA, shotgun sequencing, and assembly of draft genomes for populations has become standard practice in many microbial ecology labs, and many pipelines for manipulating metagenomic sequence data exist. What is not as well understood, however, is how to analyze the growing databases of metagenomic datasets with statistical rigour. To examine the relationships and interactions of different groups of microorganisms across the planet requires strong statistical models that can be used to assess hypotheses. We borrowed occupancy modelling from the macroecological toolbox, and adapted it to microbial metagenomic datasets. Occupancy models are designed to assess the occupancy states of sample sites, while accounting for possible missed detections by re-sampling these sites. We emulate re-sampling by searching for multiple genes associated with functions of interest, where each gene is considered an independent sampling event. We use detection of these genes as proxies for presence of functional potential within environments, and can assess occurrence and, importantly, co-occurrence patterns. We applied this method to nearly 10,000 metagenomes to assess global occupancy patterns for methanogens and methanotrophs, key contributors to the methane cycle. To assess the occupancy patterns of methane cyclers, we looked for genes encoding the subunits for the methyl coenzyme M reductase complex (MCR) and the methane monooxygenases (MMO), biological markers of methanogenesis and methanotrophy, respectively. Our models predicted that occupancy probabilities for both functional groups changed with ecosystem type, latitude, and the date that the data were deposited to the database. The explanatory power of the models was relatively low, which is likely due to a lack of metadata that could be used to better inform models. Occupancy models have the potential to be powerful tools, but microbial ecologists will need to embrace better standards for metadata collection and reporting for metagenomes. This metadata could include the collection of data such as pH, temperature, and other key environmental factors. Future work should focus on establishing and enforcing these metadata requirements to enable statistical assessment of functionally important groups across environments.
A Metagenomic Examination of Virus Diversity, Host Interactions, and Genetic Potential Across Municipal Waste Sites
(University of Waterloo, 2023-09-18) George, Nikhil Anil; Hug, Laura
Viruses are the most abundant microbial guild on the planet, impacting microbial community structure and ecosystem services. Despite this, they have been underrepresented in reference databases compared to Bacteria and Archaea, which viruses are predicted to outnumber by at least an order of magnitude. The advent of metagenomics allowed the discovery of a multitude of viruses from a variety of environments. These metagenomic examinations illustrated that prokaryotic viruses are specifically understudied in engineered environments. In this thesis, I employ metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of prokaryotic viruses that were predicted from 27 in-house metagenomes from samples taken at three municipal landfills across North America. I identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~678 kbp), and note a cosmopolitan diversity of viruses in landfills that are distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and 11 viruses predicted to infect across multiple phyla, suggesting that some viruses are far less host- specific than is typically expected. Viruses in my study encode Auxiliary Metabolic Genes (AMGs) with the potential to augment their hosts’ methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes reported to contain these genetic features. Some virally encoded Cas effector proteins appear distinct relative to previously reported Cas systems, and are interesting targets for potential biotechnological tools. Virally encoded CRISPR arrays were predicted to target other viral elements, causing interviral conflicts; and CRISPR- encoding proviruses integrated into host chromosomes were latent examples of CRISPR- immunity-based superinfection exclusion. My observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics, meriting further study of these ecosystems.
Microbial Community Compositional Stability in Agricultural Soils During Freeze-Thaw and Fertilizer Stress
(University of Waterloo, 2024-01-22) Jensen, Grant; Hug, Laura
Microbial activity persists in cold region agricultural soils during the fall, winter, and spring (i.e., non-growing season) and frozen condition, with peak activity during thaw events. Climate change is expected to change the frequency of freeze-thaw cycles (FTC) and extreme temperature events (i.e, altered timing, extreme heat/cold events) in temperate cold regions, which may hasten microbial consumption of fall-amended fertilizers, decreasing potency come the growing season. In this thesis, I conducted a high-resolution temporal examination of the impacts of freeze-thaw and nutrient stress on microbial communities in agricultural soils across both soil depth and time. Four soil columns were incubated under a climate model of a non-growing season including precipitation, temperature, and thermal gradient with depth over 60 days. Two columns were amended with fertilizer, and two incubated as unamended soil. The impacts of repeated FTC and nutrient stress on bacterial, archaeal, and fungal soil community members were determined, providing a deeply sampled longitudinal view of soil microbial response to non-growing season conditions. Geochemical changes from flow-through leachate and amplicon sequencing of 16S and ITS rRNA genes were used to assess community response. Despite nitrification observed in fertilized columns, there were no significant microbial diversity, core community, or nitrogen cycling population trends in response to nutrient stress. FTC impacts were observable as an increase in alpha diversity during FTC. Community compositions shifted across a longer time frame than individual FTC, with bulk changes to the community in each phase of the experiment. My results demonstrate microbial community composition remains relatively stable for archaea, bacteria, and fungi through a non-growing season, independent of nutrient availability. This observation contrasts canonical thinking that FTC have significant and prolonged effects on microbial communities. In contrast to permafrost and other soils experiencing rare FTC, in temperate agricultural soils regularly experiencing such perturbations, the response to freeze-thaw and fertilizer stress may be muted by a more resilient community or be controlled at the level of gene expression rather than population turn-over. These results clarify the impacts of winter FTC on fertilizer consumption, with implications for agricultural best practices and modeling of biogeochemical cycling in agroecosystems.
Microbial Composition and Functional Diversity in Waste Environments
(University of Waterloo, 2023-01-09) Johnson, Lisa Anne; Hug, Laura
Landfilling remains the most common endpoint of discarded waste. At landfill sites, waste is transformed and degraded by microbially mediated processes conducted by different trophic groups, comprised of members of a heterogeneous and diverse microbial community. Resulting metabolic products from these processes commonly include methane (CH4) and carbon dioxide (CO2), potent greenhouse gases (GHGs), leading to concerns about the long-term efficacy of landfilling as a waste management practice. These concerns arise over the increasing amount of waste being generated worldwide that will place a strain on landfills and, therefore, impact the level of contamination from landfill sites to the environment, particularly regarding emissions of GHGs. These climate change challenges at landfill sites are exacerbated by the limited understanding of the microbial community composition and functional diversity that are present in these heterogeneous ecosystems. Investigating the functional potential of microbial communities with omics technologies, such as metagenomics and metaproteomics, can clarify microbial lineages and processes impacting waste decomposition, stability, and greenhouse gas management. This thesis examines microbial populations and their metabolic capacities using metagenomics, with a focus on waste management sites and anoxic environments. First, the microbial community within landfill-associated disruptive biofilms was investigated using a multi-omics approach (Chapter 2). Both 16S rRNA gene profiling and metagenomic datasets identified major biofilm phyla as the Proteobacteria, Firmicutes, Desulfobacterota, Campylobacterota, and Bacteroidota. However, the community composition across biofilm samples was very uneven, and varied at the genus-level across biofilm samples. Metaproteomics showed microbial lineages highly abundant in genomic datasets were frequently not the most active. Each biofilm sample had unique keystone species that were detected at low abundances in the genomic DNA, but comprised relatively large proportions of the metaproteomic dataset. The most abundant MAG in Biofilm 1 (BF1) from the protein data classified to genus Pseudodesulfuromonas (phylum Desulfobacterota), and the most abundant MAG in Biofilm 2 (BF2) was classified to genus Methanothrix (phylum Halobacteriota). In Biofilm 4 (BF4) the genus Thiothrix represented 0.2% of 16S rRNA gene sequences and the MAG Thiothrix BF4 Bin 143 accounted for 0.2% of total coverage in MAGs, but made up 33.7% of the total normalized abundance of proteins identified from BF4, highlighting this bacterium as highly active. Evaluation of the proteins at the highest abundance in each sample revealed methyl-coenzyme M reductase and adenylylsulfate reductase as proteins common to each biofilm. Methane and sulfur cycling were important processes occuring in each biofilm and are potential targets for biofilm disruption. As an enigmatic phylum with representatives found at landfill sites, the distribution and metabolic capacity of phylum Cloacimonadota was examined (Chapter 3) based on detection in the biofilm samples in Chapter 2. Cloacimonadota were abundant and diverse within metagenomic datasets from a municipal landfill, including biofilm metagenomes. A total of 24 new metagenome-assembled genomes (MAGs) were generated, expanding publicly available genomic datasets (30 genomes) by 80%. Cloacimonadota genomes were examined through phylogenetic relationships, metabolic capacity, and pangenome dynamics. Phylogenetic placement of genomes resulted in two distinct clades segregated by ecosystem. Clade I contained all genomes derived from engineered ecosystems, and Clade II contained the majority of genomes derived from environmental ecosystems. Metabolic reconstructions predict Cloacimonadota to be anaerobic and acetogenic, with mixed fermentative and anaerobic respiration lifestyles for the majority of Cloacimonadota surveyed. Genomes from engineered ecosystems encode a suite of genes not typically found in genomes from natural environments, including acetate kinase, genes for cysteine degradation to pyruvate, an increased diversity of carbon utilization enzymes, and different mechanisms for generating membrane potential and ATP synthesis. This phylum-level examination also clarifies the distribution of functions previously observed for members of the phylum, demonstrating that propionate oxidation and reverse TCA cycles are not common components of Cloacimonadota metabolism. Landfills contain heterogeneous and diverse habitats for microorganisms, and this includes both oxic and anoxic microenvironments. Therefore, microorganisms require mechanisms to manage damaging reactive oxygen species (ROS). To understand the distribution of enzymatic defenses against ROS, genomes spanning the domain Bacteria derived from diverse environments were surveyed for ROS defenses (Chapter 4). The diversity and distribution of enzymatic defenses were reviewed using peer-reviewed literature and publicly available genome databases. Distribution of bacterial genomic capacity for enzymatic ROS defenses did not identify gene complements that would allow for prediction of lifestyle (anaerobic or aerobic), or unique patterns of enzymes within and between bacterial phyla. Instead, the emerging trend was that many different strategies are employed. Further, specific strategies used by well-characterized organisms are described to highlight different oxidative stress responses by organisms using aerobic, facultatively anaerobic, and anaerobic lifestyles. The research in this thesis uses metagenomics as the methodological approach to describe microbial diversity and function. The use of metagenomics enabled the first description of landfill-associated biofilms, with a focus on microbial diversity and key metabolic processes, and additional examination of the enigmatic phylum Cloacimonadota and the distribution of ROS defenses in domain Bacteria.
Microbial diversity and cellulosic capacity in municipal waste sites
(University of Waterloo, 2019-01-25) Co, Rebecca; Hug, Laura
Cellulose is the most abundant organic compound found on earth. Cellulose’s recalcitrance to hydrolysis is a major limitation to improving the efficiency of industrial applications. The biofuel, pulp and paper, agriculture, and textile industries employ mechanical and chemical methods of breaking down cellulose. Enzymatic methods are attractive choices for industry due to their selectivity in their mode of action and high product yields. However, cellulases are not as economic as mechanical means of degrading cellulose, and few cellulases are optimized for large scales. Investigating the cellulolytic microbiome and functional potential of municipal waste sites, which house large amounts of paper waste, can identify novel cellulose degraders robust for industrial applications. The microbial diversity and metabolic potential in landfills have not been well studied. In this thesis, the cellulose degradation capacity was investigated at two municipal waste sites (MWS). First, the microbial composition and the cellulose degradation capacity of a leachate pond from a dump in Jamaica and the river adjacent to the dump were assessed using metagenomics. The diversity of metagenome-assembled metagenomes (MAGs) was greater in the leachate compared to the river, with thirteen high-quality MAGs identified across seven phyla, including Bacteroidetes, Proteobacteria and Firmicutes. In contrast, two high-quality MAGs, both members of the Proteobacteria, were reconstructed from the river metagenome. A MAG assigned to the candidate phylum CPR2 is the first candidate phylum radiation MAG to be reported from a landfill. The metagenomes were screened for genes belonging to glycosyl hydrolase (GH) families containing cellulases as a measure of cellulolytic potential at the sites. Beta-glucosidases were detected at both sites. In the metagenomes, the taxonomic affiliation of most potential cellulases in the leachate metagenome were to the Bacteroidetes, Firmicutes, Actinobacteria, Spirochaetes, and Tenericutes, whereas Bacteroidetes and Proteobacteria cellulases were most abundant in the river metagenome. The microbial composition of the leachate and river did not overlap based on read mapping, suggesting no contamination of the river by the leachate at the times and sites sampled. Secondly, the cellulolytic microbial diversity was also analyzed in six metagenomes from a landfill in Southern Ontario. The samples included a composite leachate cistern (CLC), three leachate wells, and one groundwater well. Twelve GH families containing cellulases were detected across the six metagenomes, with genes from GH3 and GH5 being the most prevalent. Beta-glucosidases and endocellulases were detected across all sites, but exocellulases were only detected in some of the leachate sites and the groundwater well. A large number of hypothetical proteins and non-specifically annotated proteins were also detected across all sites, which likely represent novel carbohydrate-modifying enzymes. The majority of the potential cellulase genes across the six sites were affiliated with the Bacteroidetes and Firmicutes. Thirdly, the potential cellulolytic capacity established from the metagenomes from the Ontario landfill was confirmed by enrichment cultivations of leachate biomass grown in synthetic leachate amended with cellulose. Several isolates from the enrichment cultures showed carboxymethylcellulose and cellobiose degradation capacities, signifying endocellulase and beta-glucosidase activities. Results from 16S rRNA gene amplicon sequencing of copy-paper, cardboard, newsprint, and filter paper-enriched cultures showed enrichment of exact sequence variants assigned to Paenibacillus, Cytophaga, and Proteiniphilum bacteria over time. The research in this thesis represents the first connections between the cellulolytic potential and relevant taxonomic groups in MWS to cellulose degradation by isolates enriched from landfill leachate.
Towards a Better Understanding of Activated Carbon-Based Amendments for In Situ Treatment of Petroleum Hydrocarbons in Anaerobic Groundwater Systems
(University of Waterloo, 2024-01-11) Marrocco, Andrea; Thomson, Neil; Hug, Laura
Injected activated carbon (AC) particulate amendments for the in situ treatment of groundwater impacted by petroleum hydrocarbons (PHCs) is relatively new, and relies on a combination of AC sorption and biodegradation. Currently the performance of this technology remains unclear, primarily related to the long-term interplay between sorption and biodegradation and whether the presence of AC enhances the anaerobic biodegradation of PHCs. To address these uncertainties, this research investigated the sorption and anaerobic biodegradation (sulfate reducing and methanogenic) behaviour in microcosm experiments amended with AC and column experiments designed to mimic an AC permeable reactive barrier (PRB) over a period of 1 to 2 years. The powdered AC (PAC) used in this research (WPC from Calgon Carbon Corporation) had a rough, irregular surface with potential macropore openings of 0.8 ± 0.3 µm, and variable particle sizes with an average diameter of 11.5 ± 4.4 µm. Sorption and desorption equilibrium experiments showed that the magnitude of single-solute (benzene [B], toluene [T], or o-xylene [X]) and multi-solute (BTX combined) sorption or desorption followed X > T > B and B > T > X, respectively. Due to competitive sorption, the magnitude of B, T, and X sorption in the multi-solute system was reduced relative to the single-solute systems. Sorption and desorption equilibrium conditions differed suggestive of hysteresis; however, this behaviour was not fully explored in this research. The best-fit single-solute Freundlich isotherm parameters for benzene, toluene and o-xylene were 36.1 ± 3.8, 0.484 ± 0.045, and 88.2 ± 7.7 for K_(f_i ) ([mg/g][L/mg]n) and 0.421 ± 0.044, 132 ± 20 and 0.371 ± 0.099 for n_(f_i ) (-), respectively. The improved simplified ideal adsorption solution (ISIAS) model was fit to the multi-solute sorption data and the competition factors (ai) were estimated for benzene, toluene and o-xylene as 1.42 ± 0.38, 1.43 ± 0.16 and 1.08 ± 0.08, respectively. Temporal sorption (up to 48 hours) and desorption (up to 720 hours) experiments showed that the time to reach sorption or desorption equilibrium for single-solute benzene and toluene was rapid (≤ 0.5 hours). Single-solute (toluene-only) and multi-solute (BTX) abiotic and bioactive (including sulfate-limited [10-20 mg/L SO42-]) or sulfate amended [138-275 mg/L SO42-]) microcosms with and without PAC were constructed (in addition to starved controls without toluene, BTX or PAC). Aqueous and solid phase toluene or BTX concentrations from the single- and multi-solute PAC amended microcosms, respectively, were compared to the single- and multi-solute Freundlich or ISIAS model predictions. In general, both the single- and multi-solute sorption isotherm models were found to overestimate the measured solid phase concentrations in the microcosms. This disparity is presumably due to differences in mixing conditions and solution matrix chemistry, or competitive sorption by metabolites and biofilm formation in the PAC amended microcosms. In the multi-solute microcosm systems, the observed o-xylene solid phase concentrations deviated the most from the ISIAS model predicted solid concentrations followed by toluene and then benzene. In the subset of single- and multi-solute sulfate-limited bioactive microcosms, evidence of methanogenesis coupled to a background substrate (other than toluene or BTX) was evident from the geochemical (i.e., CH4 production) and molecular (i.e., Methanomethylovorans, Methanosaeta and Methanobacterium enrichment) data. Methane production and methanogenic enrichment were consistently elevated in the bioactive microcosms with PAC, potentially supporting enhanced methanogenesis and archaeal growth in the presence of AC. Contrarily, in the subset of single- and multi-solute sulfate-amended bioactive microcosms, sulfate reduction was coupled to the oxidation of toluene or o-xylene (not benzene, which was recalcitrant) evidenced by the repetitive or preferential biodegradation of toluene followed by o-xylene in the multi-solute microcosm and supported by geochemical (i.e., SO42- reduction, and HS- and total inorganic carbon [TIC] formation) and molecular (i.e., enrichment of sulfate reducing bacteria, including Desulfosporosinus, Desulfoprunum and Desulfobacteraceae) data. In the single- and multi-solute sulfate-amended bioactive microcosms with PAC, the solid phase mass of toluene was repetitively reduced by ≥ 96% showing that PAC regeneration occurred during anaerobic biodegradation. Although anaerobic biodegradation of toluene and o-xylene were repetitively demonstrated, there was no substantial difference in the PHC, geochemical or molecular data collected between the single- or multi-solute sulfate amended bioactive microcosms with and without PAC indicating that the presence of PAC did not influence the anaerobic microbial activity. Additionally, the anaerobic biodegradation rate of toluene was not enhanced in the presence of PAC. Collectively, there were no discernible differences in the anaerobic biodegradation of toluene between the sulfate amended bioactive microcosms with and without PAC over the 1-year monitoring period. Three types of single-solute (toluene-only) and multi-solute (BTX) columns (37 cm long, 3.75 cm inner diameter) were constructed to represent PAC sorption alone, bioactivity alone, and PAC sorption with bioactivity. The columns were operated for approximately 2 years, with Year-1 serving as an acclimation period, and Year-2 used for high-resolution temporal monitoring. For the columns containing PAC, a 6-cm long PAC zone (0.5% wt/wt) was located near the middle of the column to mimic an AC PRB. During Year-2 for the multi-solute column with PAC sorption alone, the change in BTX concentration between the influent and effluent followed X > T > B given that o-xylene has the highest sorption capacity followed by toluene and then benzene. For the single-solute and multi-solute columns with PAC sorption alone the change in toluene concentration between the influent and effluent was greater for the single-solute column relative to the multi-solute column given that the sorption capacity of toluene was reduced in the multi-solute competitive system. For the multi-solute bioactive column without PAC sorption the change in concentration between the influent and effluent followed T > X > B due to the preferential biodegradation of toluene prior to o-xylene and the recalcitrance of benzene (as observed in the microcosm experiments). For the multi-solute bioactive column with PAC sorption the change in concentration between the influent and effluent followed T > X > B due to the preferential biodegradation of toluene and preferential sorption of o-xylene as toluene was biodegraded. The solid phase concentration of toluene within the PAC zone of the single- and multi-solute bioactive columns was highest at the leading edge followed by a gradual reduction towards the end of the PAC zone. The gradient in the solid phase toluene concentration shows that toluene desorbed more due to biodegradation towards the end of the PAC zone, furthest from the influent where toluene was continually replenished. In the bioactive multi-solute column, the magnitude of solid phase BTX concentrations within the PAC zone followed X > T > B at the leading edge; however, directly downgradient the solid phase BTX concentrations within the PAC zone followed X > B > T due to the biodegradation of toluene which resulted in higher sorption of o-xylene and benzene. Using the Year-2 data, an overall column mass balance was estimated by subtracting the cumulative effluent mass from the cumulative mass injected. The single- and multi-solute bioactive columns with PAC sorption yielded the largest removal of toluene mass compared to columns with either only PAC sorption or only bioactivity. For example, among the single-solute columns the mass removal of toluene was greatest for the bioactive column with PAC sorption (99.5% reduction), followed by the column with only PAC sorption (74.6% reduction) and the column with only bioactivity (44.4% reduction). The depletion in effluent toluene mass during Year-2 was consistent between the single- and multi-solute bioactive columns with PAC sorption; however, for the multi-solute column breakthrough of benzene and o-xylene occurred given the recalcitrance of benzene and the competitive inhibition of o-xylene during toluene biodegradation. Anaerobic biodegradation within the single- and multi-solute bioactive columns with or without PAC sorption was supported by changes in geochemical parameters that would be expected under sulfate reducing and methanogenic conditions (i.e., SO42- reduction, and HS-, TIC and CH4 formation). Like the microcosm systems, there was no difference in the magnitude of change of the geochemical parameters between columns with or without PAC, suggesting that the PAC zone in the bioactive columns did not influence the microbial activity. However, the PAC zone did influence the spatial distribution of anaerobic microbes. For the columns with only bioactivity the relative abundance of Methanosarcina and Methanomethylovorans were highest at the influent ends relative to the effluent ends. Whereas, for the bioactive columns with PAC sorption the relative abundance of Methanosaeta, Methanobacterium and Methanosarcina were highest within the PAC zone relative to outside of the PAC zone, in addition to being higher in abundance relative to all bacteria detected within the PAC zone (primarily Desulfosporosinus, Edwardsbacteria and Berkelbacteria). In the multi-solute bioactive column with PAC sorption, the abundance of Desulfosporosinus was also notably elevated at the leading edge of the PAC zones (coinciding with the location of the highest solid phase toluene concentration sorbed to the PAC). Compound specific isotope analysis (CSIA) revealed enrichment of 2H-T in the single- and multi-solute columns with only bioactivity or PAC sorption, although the average value of δ2H-T between the single- and multi-solute columns with only bioactivity was 36.1 ± 8‰ greater than the columns with only PAC sorption, as expected. The magnitude of hydrogen isotope fractionation of toluene associated with a combination of PAC sorption and biodegradation is unknown given toluene was depleted in the bioactive column with PAC sorption. Unlike toluene, a direct comparison of the hydrogen isotope fractionation of o-xylene between the multi-solute columns showed that the average value of δ2H-X was 54‰ and 58‰ greater in the bioactive column with PAC sorption relative to the columns with only PAC sorption or only bioactivity, respectively. 2H-X enrichment in the multi-solute bioactive column with PAC sorption was presumably due to a significant amount of o-xylene sorption to the PAC as toluene was biodegraded as opposed to substantial o-xylene biodegradation (supported by the solid phase data). Collectively the compiled data sets provide comprehensive insight into how AC particulate amendments behave in anaerobic systems in contact with PHCs, and the interplay between BTX sorption and anaerobic biodegradation under sulfate reducing and methanogenic conditions. These data provide direct evidence that the presence of PAC particulate amendments does not enhance the biodegradation of BTX relative to systems with no PAC under sulfate reducing conditions. Instead, the presence of the PAC provides rapid reductions in contaminant concentrations relative to systems without PAC and sustains reductions in the aqueous phase concentration of the most preferentially degraded solute under variable loading conditions as the PAC is regenerated. The PAC also influences microbial activity during PHC biodegradation by promoting microbial growth on the PAC, with notably high methanogenic enrichment. This research also provides evidence that ideal isotherms are not representative of the sorption behaviour in bioactive systems with AC and tend to overestimate sorption. Finally, PAC sorption, most notably for the most preferentially sorbed solute in the multi-solute system, generates substantial hydrogen isotope enrichment which may lead to overestimations in the fractionation presumed to be associated with biodegradation when integrating CSIA into monitoring approaches for bioactive systems with AC.
Using Metagenomic Approaches to Determine Antimicrobial Resistance Diversity and Prevalence within Four Landfills
(University of Waterloo, 2023-08-21) Ippolito, Isabella; Hug, Laura
Antibiotics are one of the many hazardous substances present in municipal solid waste (MSW) landfills. Antibiotics can contaminate the leachate generated in landfills, which poses a threat to human health as leachate can permeate and contaminate the surrounding ground water and surface water systems. The presence of antibiotics in landfills can create selection pressures on the microorganisms present in the landfill, selecting for antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB). This makes landfills important reservoirs of ARGs and ARB. This study used genome resolved metagenomic sequencing to observe ARG diversity and prevalence from four active municipal landfills, their adjacent ground or surface water systems, as well as other environments for comparison. The Resistance Gene Identifier (RGI) and DeepARG were used to predict resistance mechanisms, drug classes, and ARGs. The results from RGI and DeepARG highlighted landfill signatures that were distinct from other environments, and that landfill ARG profiles were more similar to each other than to other environments. PlasClass and Phage and Plasmid Recognizer for Metagenomes (PPR-Meta) were used to predict plasmids within the landfill metagenome datasets. Plasmids encoding ARGs were identified to assess the role of plasmids in ARG mobility in landfills, and to identify enriched ARG types on these mobile elements, such as multidrug resistance and antibiotic inactivation resistance mechanisms. The results of this study clarify target drug classes of interest, such as tetracyclines, aminoglycosides, macrolide-lincosamide-streptogramin (MLS) antibiotics, glycopeptides, and peptide antibiotics, as well as the impact that landfills along with their ground and surface water systems may have on surrounding environments.