Matching Items (19)
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- All Subjects: Microbiology
- Creators: Wang, Xuan
Description
Methane (CH4) is very important in the environment as it is a greenhouse gas and important for the degradation of organic matter. During the last 200 years the atmospheric concentration of CH4 has tripled. Methanogens are methane-producing microbes from the Archaea domain that complete the final step in breaking down organic matter to generate methane through a process called methanogenesis. They contribute to about 74% of the CH4 present on the Earth's atmosphere, producing 1 billion tons of methane annually. The purpose of this work is to generate a preliminary metabolic reconstruction model of two methanogens: Methanoregula boonei 6A8 and Methanosphaerula palustris E1-9c. M. boonei and M. palustris are part of the Methanomicrobiales order and perform hydrogenotrophic methanogenesis, which means that they reduce CO2 to CH4 by using H2 as their major electron donor. Metabolic models are frameworks for understanding a cell as a system and they provide the means to assess the changes in gene regulation in response in various environmental and physiological constraints. The Pathway-Tools software v16 was used to generate these draft models. The models were manually curated using literature searches, the KEGG database and homology methods with the Methanosarcina acetivorans strain, the closest methanogen strain with a nearly complete metabolic reconstruction. These preliminary models attempt to complete the pathways required for amino acid biosynthesis, methanogenesis, and major cofactors related to methanogenesis. The M. boonei reconstruction currently includes 99 pathways and has 82% of its reactions completed, while the M. palustris reconstruction includes 102 pathways and has 89% of its reactions completed.
ContributorsMahendra, Divya (Author) / Cadillo-Quiroz, Hinsby (Thesis director) / Wang, Xuan (Committee member) / Stout, Valerie (Committee member) / Barrett, The Honors College (Contributor) / Computing and Informatics Program (Contributor) / School of Life Sciences (Contributor) / Biomedical Informatics Program (Contributor)
Created2014-05
Description
Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.
ContributorsDufault, Matthew Elijah (Co-author, Co-author) / Wang, Xuan (Thesis director) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Many bacteria actively import environmental DNA and incorporate it into their genomes. This behavior, referred to as transformation, has been described in many species from diverse taxonomic backgrounds. Transformation is expected to carry some selective advantages similar to those postulated for meiotic sex in eukaryotes. However, the accumulation of loss-of-function alleles at transformation loci and an increased mutational load from recombining with DNA from dead cells create additional costs to transformation. These costs have been shown to outweigh many of the benefits of recombination under a variety of likely parameters. We investigate an additional proposed benefit of sexual recombination, the Red Queen hypothesis, as it relates to bacterial transformation. Here we describe a computational model showing that host-pathogen coevolution may provide a large selective benefit to transformation and allow transforming cells to invade an environment dominated by otherwise equal non-transformers. Furthermore, we observe that host-pathogen dynamics cause the selection pressure on transformation to vary extensively in time, explaining the tight regulation and wide variety of rates observed in naturally competent bacteria. Host-pathogen dynamics may explain the evolution and maintenance of natural competence despite its associated costs.
ContributorsPalmer, Nathan David (Author) / Cartwright, Reed (Thesis director) / Wang, Xuan (Committee member) / Sievert, Chris (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The purpose behind this research was to identify unknown transport proteins involved in lactate export. Lactate bioproduction is an environmentally beneficial alternative to petroleum-based plastic production as it produces less toxic waste byproduct and can rely on microbial degradation of otherwise wasted biomass. Coupled with appropriate product refinement, industrial microbial producers can be genetically engineered to generate quantities of bioplastic approaching 400 million metric tons each year. However, this process is not entirely suitable for large investment, as the fermentative bottlenecks, including product export and homeostasis control, limit production metrics. Previous studies have based their efforts on enhancing cellular machinery, but there remain uncharacterized membrane proteins involved in product export yet to be determined. It has been seen that deletion of known lactate transporters in Escherichia coli resulted in a decrease in lactate production, unlike the expected inhibition of export. This indicates that there exist membrane proteins with the ability to export lactate which may have another similar substrate it primarily transports.To identify these proteins, I constructed a genomic library of all genes in an engineered lactate producing E. coli strain, with known transporter genes deleted, and systematically screened for potential lactate transporter proteins. Plasmids and their isolated proteins were compared utilizing anaerobic plating to identify genes through sanger sequencing. With this method, I identified two proteins, yiaN and ybhL-ybhM, which did not show any significant improvement in lactate production when tested. Attempts were made to improve library diversity, resulting in isopropyl-β-D-1-thiogalactopyranoside induction as a likely factor for increased expression of potential fermentation-associated proteins. A genomic library from Lactobacillus plantarum was constructed and screened for transport proteins which could improve lactate production. Results showed that isolated plasmids contained no notable inserts, indicating that the initial transformation limited diversity. Lastly, I compared the results from genomic screening with overexpression of target transporter genes by computational substrate similarity search. Induced expression of ttdT, citT and dcuA together significantly increased lactate export and thus production metrics as well as cell growth. These positive results indicate an effective means of determining substrate promiscuity in membrane proteins with similar organic acid transport capacity.
ContributorsLee-Kin, Jared (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of
chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis.
However, to create industrially competitive strains to efficiently convert lignocellulose to
high-value chemicals, current challenges must be addressed. Redox constraints, allosteric
regulation, and transport-related limitations are important bottlenecks limiting the
commercial production of renewable chemicals from lignocellulose. Advances in
metabolic engineering techniques have enabled researchers to engineer microbial strains
that overcome some of these challenges but new approaches that facilitate the
commercial viability of lignocellulose valorization are needed. Biological systems are
complex with a plethora of regulatory systems that must be carefully modulated to
efficiently produce and excrete the desired metabolites. In this work, I explore metabolic
engineering strategies to address some of the biological constraints limiting
bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective
lignocellulose bioconversion.
ContributorsOnyeabor, Moses Ekenedilichukwu (Author) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Nielsen, David R (Committee member) / Geiler-Samerotte, Kerry (Committee member) / Arizona State University (Publisher)
Created2023
Description
This dissertation focused on studying risks associated with emerging drinking water contaminants and tradeoffs related to water management interventions. The built environment impacts health, as humans on average spend ~90% of their time indoors. Federal regulations generally focus on drinking water at the water treatment plant and within the distribution system as opposed to when it enters buildings after crossing the property line. If drinking water is not properly managed in buildings, it can be a source or amplifier of microbial and chemical contaminants. Unlike regulations for chemical contaminants that are risk-based, for pathogens, regulations are either based on recommended treatment technologies or designated as zero, which is not achievable in practice. Practice-based judgments are typically made at the building level to maintain water quality. This research focuses on two drinking water opportunistic pathogens of public health concern, Legionella pneumophila and Mycobacterium avium complex (MAC). Multiple aspects of drinking water quality in two green buildings were monitored in tandem with water management interventions. Additionally, a quantitative microbial risk assessment framework was used to predict risk-based critical concentrations of MAC for drinking water-related exposures in the indoor environment corresponding to a 1 in 10,000 annual infection target risk benchmark. The overall goal of this work was to inform the development of water management plans and guidelines for buildings that will improve water quality in the built environment and promote better public health. It was determined that a whole building water softening system with ion exchange softening resin and expansion tanks were unexplored reservoirs for the colonization of L. pneumophila. Furthermore, it was observed that typical water management interventions such as flushing and thermal disinfection did not always mitigate water quality issues. Thus, there was a need to implement several atypical interventions such as equipment replacement to improve the building water quality. This work has contributed comprehensive field studies and models that have highlighted the need for additional niches, facility management challenges, and risk tradeoffs for focus in water safety plans. The work also informs additional risk-based water quality policy approaches for reducing drinking water risks.
ContributorsJoshi, Sayalee (Author) / Hamilton, Kerry A (Thesis advisor) / Abbaszadegan, Morteza (Committee member) / Conroy-Ben, Otakuye (Committee member) / Halden, Rolf (Committee member) / Arizona State University (Publisher)
Created2023
Characterization and Manipulation of Microbiomes From Arid Landfills for Improved Methane Production
Description
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.
ContributorsReynolds, Mark Christian (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Wang, Xuan (Committee member) / Kavazanjian, Edward (Committee member) / Arizona State University (Publisher)
Created2022
Description
Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Recent efforts have sought to supplant these sugar-intensive processes through the metabolic engineering of cyanobacteria, which instead can directly utilize atmospheric carbon dioxide (CO2) and sunlight. One of the most promising among recently discovered photoautotrophic strains is Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973), which has been reported to have doubling times as low as 1.5 hours. While encouraging, there are still major challenges preventing the widespread industrial acceptance of engineered cyanobacteria, chief among them is the scarcity of genetic tools and parts with which to engineer production strains. Here, UTEX 2973 was engineered to overproduce L-lysine through the heterologous expression of feedback-resistant copies of aspartokinase lysC and the L-lysine exporter ybjE from Escherichia coli, as aided by the characterization of novel combinations of genetic parts and expression sites. At maximum, using a plasmid-based expression system, a L-lysine titer of 556 ± 62.3 mg/L was attained after 120 hours, surpassing a prior report of photoautotrophic L-lysine bioproduction. Modular extension of the pathway then led to the novel photosynthetic production of the corresponding diamine cadaverine (55.3 ± 6.7 mg/L by 96 hours) and dicarboxylate glutarate (67.5 ± 2.2 mg/L by 96 hours). Lastly, mass transfer experiments were carried out to determine if the solubility of CO2 in and its rate of mass transfer to BG-11 media could be improved by supplementing it with various amines, including cadaverine. Ultimately, however, cyanobacteria grown in the presence of all tested amines was worse than in BG-11 alone, demonstrating the need for additional tolerance engineering to successfully implement this strategy.
ContributorsDookeran, Zachary Anthony (Author) / Nielsen, David R (Thesis advisor) / Wang, Xuan (Committee member) / Nannenga, Brent L (Committee member) / Varman, Arul M (Committee member) / Peebles, Christie AM (Committee member) / Arizona State University (Publisher)
Created2022
Description
Heterotrophs such as E. coli contain metabolic pathways with enzymes called carboxylases that are capable of fixing CO2 gas to form metabolites, which has implications for aiding with CO2’s role in climate change. The reductive branch of the tricarboxylic acid (TCA) cycle serves as an important pathway for NAD+ regeneration in enteric bacteria in anaerobic conditions and leads to the production of succinate, a useful industrial product. The enzyme phosphoenolpyruvate carboxykinase is responsible for fixing CO2 in the conversion of PEP to OAA within this pathway and has potential to be a significant carbon fixation module in heterotrophic organisms. This project explored pck genes from select organisms by transforming plasmids to test if these variants have improved kinetics compared to the native E. coli Pck and to investigate their ability to improve succinate bioproduction.
ContributorsModukuri, Shree (Author) / Wang, Xuan (Thesis director) / Godar, Amanda (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor)
Created2024-05
Description
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and hydroxyl radicals occur naturally as a byproduct of aerobic respiration. To mitigate damages caused by ROS, Escherichia coli employs defenses including two cytosolic superoxide dismutases (SODs), which convert superoxide to hydrogen peroxide. Deletion of both sodA and sodB, the genes coding for the cytosolic SOD enzymes, results in a strain that is unable to grow on minimal medium without amino acid supplementation. Additionally, deletion of both cytosolic SOD enzymes in a background containing the relA1 allele, an inactive version of the relA gene that contributes to activation of stringent response by amino acid starvation, results in a strain that is unable to grow aerobically, even on rich medium. These observations point to a relationship between the stringent response and oxidative stress. To gain insight into this relationship, suppressors were isolated by growing the ∆sodAB relA1 cells aerobically on rich medium, and seven suppressors were further examined to characterize distinct colony sizes and temperature sensitivity phenotypes. In three of these suppressor-containing strains, the relA1 allele was successfully replaced by the wild type relA allele to allow further study in aerobic conditions. None of those three suppressors were found to increase tolerance to exogenous superoxides produced by paraquat, which shows that these mutations only overcome the superoxide buildup that naturally occurs from deletion of SODs. Because each of these suppressors had unique phenotypes, it is likely that they confer tolerance to SOD-dependent superoxide buildup by different mechanisms. Two of these three suppressors have been sent for whole-genome sequencing to identify the location of the suppressor mutation and determine the mechanism by which they confer superoxide tolerance.
ContributorsFlake, Melissa (Author) / Misra, Rajeev (Thesis advisor) / Shah, Dhara (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2024