Matching Items (4)
Filtering by
- All Subjects: Biofilms
- All Subjects: Metabolism
- Genre: Academic theses
- Creators: Torres, Cesar
Description
To further the efforts producing energy from more renewable sources, microbial electrochemical cells (MXCs) can utilize anode respiring bacteria (ARB) to couple the oxidation of an organic substrate to the delivery of electrons to the anode. Although ARB such as Geobacter and Shewanella have been well-studied in terms of their microbiology and electrochemistry, much is still unknown about the mechanism of electron transfer to the anode. To this end, this thesis seeks to elucidate the complexities of electron transfer existing in Geobacter sulfurreducens biofilms by employing Electrochemical Impedance Spectroscopy (EIS) as the tool of choice. Experiments measuring EIS resistances as a function of growth were used to uncover the potential gradients that emerge in biofilms as they grow and become thicker. While a better understanding of this model ARB is sought, electrochemical characterization of a halophile, Geoalkalibacter subterraneus (Glk. subterraneus), revealed that this organism can function as an ARB and produce seemingly high current densities while consuming different organic substrates, including acetate, butyrate, and glycerol. The importance of identifying and studying novel ARB for broader MXC applications was stressed in this thesis as a potential avenue for tackling some of human energy problems.
ContributorsAjulo, Oluyomi (Author) / Torres, Cesar (Thesis advisor) / Nielsen, David (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Popat, Sudeep (Committee member) / Arizona State University (Publisher)
Created2013
Description
Metabolic engineering is an extremely useful tool enabling the biosynthetic production of commodity chemicals (typically derived from petroleum) from renewable resources. In this work, a pathway for the biosynthesis of styrene (a plastics monomer) has been engineered in Escherichia coli from glucose by utilizing the pathway for the naturally occurring amino acid phenylalanine, the precursor to styrene. Styrene production was accomplished using an E. coli phenylalanine overproducer, E. coli NST74, and over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae. The styrene pathway was then extended by just one enzyme to either (S)-styrene oxide (StyAB from Pseudomonas putida S12) or (R)-1,2-phenylethanediol (NahAaAbAcAd from Pseudomonas sp. NCIB 9816-4) which are both used in pharmaceutical production. Overall, these pathways suffered from limitations due to product toxicity as well as limited precursor availability. In an effort to overcome the toxicity threshold, the styrene pathway was transferred to a yeast host with a higher toxicity limit. First, Saccharomyces cerevisiae BY4741 was engineered to overproduce phenylalanine. Next, PAL2 (the only enzyme needed to complete the styrene pathway) was then expressed in the BY4741 phenylalanine overproducer. Further strain improvements included the deletion of the phenylpyruvate decarboxylase (ARO10) and expression of a feedback-resistant choristmate mutase (ARO4K229L). These works have successfully demonstrated the possibility of utilizing microorganisms as cellular factories for the production styrene, (S)-styrene oxide, and (R)-1,2-phenylethanediol.
ContributorsMcKenna, Rebekah (Author) / Nielsen, David R (Thesis advisor) / Torres, Cesar (Committee member) / Caplan, Michael (Committee member) / Jarboe, Laura (Committee member) / Haynes, Karmella (Committee member) / Arizona State University (Publisher)
Created2014
Description
This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. For the first time, the renewable production of benzaldehyde and benzyl alcohol has been achieved in recombinant E. coli with a maximum titer of 114 mg/L of benzyl alcohol. Further strain development to knockout endogenous alcohol dehydrogenase has reduced the in vivo degradation of benzaldehyde by 9-fold, representing an improved host for the future production of benzaldehyde as a sole product. In addition, a novel alternative pathway for the production of protocatechuate (PCA) and catechol from the endogenous metabolite chorismate is demonstrated. Titers for PCA and catechol were achieved at 454 mg/L and 630 mg/L, respectively. To explore potential routes for improved aromatic product yields, an in silico model using elementary mode analysis was developed. From the model, stoichiometric optimums maximizing both product-to-substrate and biomass-to-substrate yields were discovered in a co-fed model using glycerol and D-xylose as the carbon substrates for the biosynthetic production of catechol. Overall, the work presented in this dissertation highlights contributions to the field of metabolic engineering through novel pathway design for the biosynthesis of industrially relevant aromatic fine chemicals and the use of in silico modelling to identify novel approaches to increasing aromatic product yields.
ContributorsPugh, Shawn (Author) / Nielsen, David (Thesis advisor) / Dai, Lenore (Committee member) / Torres, Cesar (Committee member) / Lind, Mary Laura (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
Factors affecting biofilm development, specifically the materials of the pipe, were investigated. Two laboratory scale bioreactor systems were constructed to study biofilm formations: a pipe loop bioreactor with continuous flow at 10.1 liters per minute (LPM), and a tank bioreactor under stagnant conditions with a minimal flow of 0.0095 LPM. The continuous flow bioreactors were constructed using cross-linked polyethylene (PEX), copper, and galvanized steel pipes. The tank bioreactors consisted of glass chambers containing coupons made from the pipe materials, as well as glass microscope slides. Municipality tap water was used in the experimentation, with no nutrients added. Legionella pneumophila was spiked into all the pipe loop bioreactors, and only in one tank bioreactor. Detection of heterotrophic bacteria, coliforms and Legionella using tryptic soy agar (TSA), Brilliance, and buffered yeast charcoal extract (BYCE), respectively. Over ten weeks, biofilms were developed on PEX, copper, and steel, in the pipe loop bioreactors and the tank bioreactors. Heterotrophic bacteria were detected in all systems; however, no coliforms were detected, and Legionella pneumophila was only detected on a coupon in the copper pipe loop bioreactor, as measured by bacterial concentration on test materials. In the tank bioreactors, biofilms developed the most rapidly on PEX, followed by galvanized steel, and finally copper. Out of the four materials, copper had the lowest bacterial growth, which can be ascribed to the bactericidal impact of copper ions on the bacterial cells attaching to the copper surface. After biofilm aging, higher bacterial colonization on copper and accumulation of dead bacterial layer on the surface may act as a protective barrier against copper ions. Bacterial densities in the biofilm reached a high concentration of 1.40 x 105 CFU/cm2 on the PEX pipe loop bioreactor, and 1.05 x 104 CFU/cm2in the PEX coupon in the tank bioreactors. Comparing the turbulent conditions in the pipe loop bioreactors to the stagnant conditions in the tank bioreactor, showed that biofilms formed more rapidly under stagnant conditions, but in larger quantities under turbulent conditions.
ContributorsGreenberg, Samuel Gabe (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2021