Autism spectrum disorder (ASD) currently lacks a biological diagnostic test, ongoing research is being conducted to develop a urine biomarker test for autism. Researchers are investigating possible anions, such as sulfur-based anions, as a biomarker for autism. Although studies have not measured the quantification of sulfate-based anions within a biospecimen while using Ion Chromatography (IC) for a 24-hour period. Research studies on autism biomarker development could greatly benefit by investigating and quantifying sulfur-based anions such as sulfate, sulfide, sulfite, or thiosulfate. Our research investigated the quantifications of anions through the analysis of biospecimens across 24-hours in an IC. The results of our research indicate that sulfate fluctuates the least and was consistently read by the IC at each time point across 24 hours whereas the other anions of interest presented greater fluctuations and were not detected at each time point across the 24 hours under the conditions tested.

As the utilization of tyrosine is needed by both eukaryotes and prokaryotes, this versatile amino acid contributes towards a variety of operations including protein synthesis, pigment production, and host or habitat impacting metabolite creation. While there are numerous pathways which involve the degradation of tyrosine to create different products, the one that is central in this thesis is a pathway with homogentisate as an intermediate. This pathway brings an interest due to its association with metabolic disorders like Tyrosinemia (I, II, or III), and its impact within an agricultural environment. In other words, for humans and plant microbiomes to maintain their optimal metabolic homeostasis, tyrosine is required to participate in numerous demands. This necessity can ultimately create competition between organisms present in microbial communities, as there are a multitude of species that can metabolize tyrosine for the creation of diverse products. In this work, a primary objective is to characterize the breakdown of tyrosine within a competitive environment where there are multiple available pathways. There are many factors that could influence the catabolism of tyrosine like catalytic efficiency of enzymes, availability of breakdown routes, and pathway regulations. Here, the start will be creating a proof of concept developed by studying the competition for tyrosine utilization by environmental microbial enzymes; 4-hydroxyphenylpyruvate dioxygenase from Streptomyces avermitilis, 4-hydroxymandelate synthase from Amycolatopsis orientalis, and tyrosine ammonia lyase from Flavobacterium johnsoniae. Through phenotypic assays and by quantifying secreted metabolites, rerouting of this pathway is observed. This insight towards the ability of diverting the homogentisate pathway was then utilized for the analysis of contest between human enzyme, 4-hydroxyphenylpyruvate dioxygenase, and gut microbial enzyme, tyrosine ammonia lyase from Bacteroides ovatus. Within both aims it is seen that due to successful diversion of the pathway, there is a reduction in tyrosine with the formation of more favorable products. The strategy of redirecting this tyrosine catabolism pathway will provide baseline knowledge for future efforts to contribute towards alternative methods of intervention to alleviate the burdens from tyrosine metabolic dysfunction and disorders.

For my thesis I investigated an abnormal gut-derived metabolite of interest identified as 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) that may serve as a potential biomarker for autism, and help us get a better understanding of the underlying mechanisms of this disorder. Currently a laboratory test for autism does not exist, posing severe consequences on individuals with autism. In order to gather research on my metabolite of interest and its connection to autism as well as disorders correlated with autism, I analyzed different pieces of scientific literature investigating HPHPA and compiled this data into a literature review.

While most household surfactants are biodegradable in aerobic conditions, their presence in a microbiological treatment process can lead to the proliferation of antimicrobial-resistance genes (ARG) in bacteria, such as Pseudomonas aeruginosa. Surfactants can be cationic, anionic, or zwitterionic, and these different classes may have different effects on the proliferation of ARG. This study evaluated how the three classes of surfactants affected the microbial community’s structure and ARG in O2-based membrane biofilm reactors (O2-MBfRs) that provided at least 98% surfactant removal. Cationic cetrimonium bromide (CTAB) had by far the strongest impact with highest ARG abundance in the biofilm. In particular, Pseudomonas and Stenotrophomonas, the two main genera in the biofilm treating CTAB, were highly correlated to the abundance of ARG for efflux pumps and antibiotic inactivation. CTAB also promoted potential of horizontal gene transfer (HGT) of ARG. Combining results from the metabolome and metagenome identified four possible pathways for CTAB biodegradation. Of special important is a new pathway: β-carbon oxidation of CTAB to produce betaine. An insufficient nitrogen source could lead to irreversible ARB and ARG enrichment in the MBfR biofilm. Finally, a two-stage O2-MBfR successfully removed a high concentration (730 mg/L) of CTAB: Partial CTAB removal in the Lead reactor relieved inhibition in the Lag reactor. Metagenomic analysis also revealed that the Lag reactor was enriched in genes for CTAB and metabolite oxygenation.

This dissertation encompasses the interaction of antimicrobial chemicals and emerging contaminants with multi-drug resistant (MDR) bacteria and their implications in engineered systems. The aim is to investigate the effect of combination antimicrobials on MDR bacteria E. coli, evaluate the extent of synergism and antagonism of utilizing two distinct biocidal chemicals, and evaluate the influence of endocrine-disrupting chemicals (EDCs) on protein production in response to stressors. Resistance mechanisms of bacteria such as E. coli include the use of protein systems that efflux excess nutrients or toxic compounds. These efflux proteins activate in response to environmental stressors such as contaminants and antimicrobials to varying degrees and are major contributors to antibiotic resistance in pathogenic bacteria. As is the case with engineered microbial environments, large quantities of emerging contaminants interact with bacteria, influencing antibiotic resistance and attenuation of these chemicals to an unknown degree. Interactions of antimicrobials on MDR bacteria such as E. coli have been extensively studied for pathogens, including synergistic combinations. Despite these studies in this field, a fundamental understanding of how chemicals influence antibiotic resistance in biological processes typical of engineered microbial environments is still ongoing. The impacts of EDCs on antibiotic resistance in E. coli were investigated by the characterization of synergism for antimicrobial therapies and the extrapolation of these metrics to the cycling of EDCs in engineered systems to observe the extent of antibiotic resistance proteins to the EDCs. The impact of this work provides insight into the delicate biochemistry and ongoing resistance phenomena regarding engineered systems.

Environmentally harmful byproducts from solid waste’s decomposition, including methane (CH4) emissions, are managed through standardized landfill engineering and gas-capture mechanisms. Yet only a limited number of studies have analyzed the development and composition of Bacteria and Archaea involved in CH4 production from landfills. The objectives of this research were to compare microbiomes and bioactivity from CH4-producing communities in contrasting spatial areas of arid landfills and to tests a new technology to biostimulate CH4 production (methanogenesis) from solid waste under dynamic environmental conditions controlled in the laboratory. My hypothesis was that the diversity and abundance of methanogenic Archaea in municipal solid waste (MSW), or its leachate, play an important role on CH4 production partially attributed to the group’s wide hydrogen (H2) consumption capabilities. I tested this hypothesis by conducting complementary field observations and laboratory experiments. I describe niches of methanogenic Archaea in MSW leachate across defined areas within a single landfill, while demonstrating functional H2-dependent activity. To alleviate limited H2 bioavailability encountered in-situ, I present biostimulant feasibility and proof-of-concepts studies through the amendment of zero valent metals (ZVMs). My results demonstrate that older-aged MSW was minimally biostimulated for greater CH4 production relative to a control when exposed to iron (Fe0) or manganese (Mn0), due to highly discernable traits of soluble carbon, nitrogen, and unidentified fluorophores found in water extracts between young and old aged, starting MSW. Acetate and inhibitory H2 partial pressures accumulated in microcosms containing old-aged MSW. In a final experiment, repeated amendments of ZVMs to MSW in a 600 day mesocosm experiment mediated significantly higher CH4 concentrations and yields during the first of three ZVM injections. Fe0 and Mn0 experimental treatments at mesocosm-scale also highlighted accelerated development of seemingly important, but elusive Archaea including Methanobacteriaceae, a methane-producing family that is found in diverse environments. Also, prokaryotic classes including Candidatus Bathyarchaeota, an uncultured group commonly found in carbon-rich ecosystems, and Clostridia; All three taxa I identified as highly predictive in the time-dependent progression of MSW decomposition. Altogether, my experiments demonstrate the importance of H2 bioavailability on CH4 production and the consistent development of Methanobacteriaceae in productive MSW microbiomes.

Under current climate conditions northern peatlands mostly act as C sinks; however, changes in climate and environmental conditions, can change the soil carbon decomposition cascade, thus altering the sink status. Here I studied one of the most abundant northern peatland types, poor fen, situated along a climate gradient from tundra (Daring Lake, Canada) to boreal forest (Lutose, Canada) to temperate broadleaf and mixed forest (Bog Lake, MN and Chicago Bog, NY) biomes to assess patterns of microbial abundance across the climate gradient. Principal component regression analysis of the microbial community and environmental variables determined that mean annual temperature (MAT) (r2=0.85), mean annual precipitation (MAP) (r2=0.88), and soil temperature (r2=0.77), were the top significant drivers of microbial community composition (p < 0.001). Niche breadth analysis revealed the relative abundance of Intrasporangiaceae, Methanobacteriaceae and Candidatus Methanoflorentaceae fam. nov. to increase when MAT and MAP decrease. The same analysis showed Spirochaetaceae, Methanosaetaceae and Methanoregulaceae to increase in relative abundance when MAP, soil temperature and MAT increased, respectively. These findings indicated that climate variables were the strongest predictors of microbial community composition and that certain taxa, especially methanogenic families demonstrate distinct patterns across the climate gradient. To evaluate microbial production of methanogenic substrates, I carried out High Resolution-DNA-Stable Isotope Probing (HR-DNA-SIP) to evaluate the active portion of the community’s intermediary ecosystem metabolic processes. HR-DNA-SIP revealed several challenges in efficiency of labelling and statistical identification of responders, however families like Veillonellaceae, Magnetospirillaceae, Acidobacteriaceae 1, were found ubiquitously active in glucose amended incubations. Differences in metabolic byproducts from glucose amendments show distinct patterns in acetate and propionate accumulation across sites. Families like Spirochaetaceae and Sphingomonadaceae were only found to be active in select sites of propionate amended incubations. By-product analysis from propionate incubations indicate that the northernmost sites were acetate-accumulating communities. These results indicate that microbial communities found in poor fen northern peatlands are strongly influenced by climate variables predicted to change under current climate scenarios. I have identified patterns of relative abundance and activity of select microbial taxa, indicating the potential for climate variables to influence the metabolic pathway in which carbon moves through peatland systems.

This study investigated the difference in biofilm growth on pristine and aged polypropylene microplastics exposed to Tempe Town Lake water for 8 weeks. The research question here is, does the aging of microplastic (MPs) change the biofilm formation rate and composition of the biofilm in comparison with the pristine MPs. To answer this question, the biofilm formation was quantified using different methods over time for both pristine polypropylene and aged polypropylene using agar plate counts and crystal violet staining. Colony counts based on agar plating showed an increase in microbial growth over the 8 weeks of treatment, with the aged MPs accumulating higher microbial counts than the pristine MPs. The diversity of the biofilm decreased over time for both MPs and the aged MPs had overall less diversity in biofilm, based on phenotype enumeration, in comparison with the pristine MPs. Higher biofilm growth on aged MPs was confirmed using crystal violet staining, which stains the negatively charged biological compounds such as proteins and the extracellular polymeric substance matrix of the biofilm. Using this complementary approach to colony counting, the same trend of higher biofilm growth on aged MPs was found. Further studies will focus on confirming the phenotype findings using microbiome analysis following DNA extraction. This project created a methodology to quantify biofilm formation on MPs, which was used to show that MPs may accumulate more biofilms in the environment as they age under sunlight.

Electroactive bacteria connect biology to electricity, acting as livingelectrochemical catalysts. In nature, these bacteria can respire insoluble compounds like iron oxides, and in the laboratory, they are able to respire an electrode and produce an electrical current. This document investigates two of these electroactive bacteria: Geobacter sulfurreducens and Thermincola ferriacetica. G. sulfurreducens is a Gramnegative iron-reducing soil bacterium, and T. ferriacetica is a thermophilic, Grampositive bacterium that can reduce iron minerals and several other electron acceptors. Respiring insoluble electron acceptors like metal oxides presents challenges to a bacterium. The organism must extend its electron transport chain from the inner membrane outside the cell and across a significant distance to the surface of the electron acceptor. G. sulfurreducens is one of the most-studied electroactive bacteria, and despite this there are many gaps in knowledge about its mechanisms for transporting electrons extracellularly. Research in this area is complicated by the presence of multiple pathways that may be concurrently expressed. I used cyclic voltammetry to determine which pathways are present in electroactive biofilms of G. sulfurreducens grown under different conditions and correlated this information with gene expression data from the same conditions. This correlation presented several genes that may be components of specific pathways not just at the inner membrane but along the entire respiratory pathway, and I propose an updated model of the pathways in this organism. I also characterized the composition of G. sulfurreducens and found that it has high iron and lipid content independent of growth condition, and the high iron content is explained by the large abundance of multiheme cytochrome expression that I observed. I used multiple microscopy techniques to examine extracellular respiration in G. sulfurreducens, and in the process discovered a novel organelle: the intracytoplasmic membrane. I show 3D reconstructions of the organelle in G. sulfurreducens and discuss its implications for the cell’s metabolism. Finally, I discuss gene expression in T. ferriacetica in RNA samples collected from an anode-respiring culture and highlight the most abundantly expressed genes related to anode-respiring metabolism.