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- All Subjects: Microbiology
- Creators: Garcia-Pichel, Ferran
- Member of: Theses and Dissertations
- Resource Type: Text
- Status: Published
Description
Synechocystis sp PCC 6803 is a photosynthetic cyanobacterium that can be easily transformed to produce molecules of interest; this has increased Synechocystis’ popularity as a clean energy platform. Synechocystis has been shown to produce and excrete molecules such as fatty acids, isoprene, etc. after appropriate genetic modification. Challenges faced for large–scale growth of modified Synechocystis include abiotic stress, microbial contamination and high processing costs of product and cell material. Research reported in this dissertation contributes to solutions to these challenges. First, abiotic stress was addressed by overexpression of the heat shock protein ClpB1. In contrast to the wild type, the ClpB1 overexpression mutant (Slr1641+) tolerated rapid temperature changes, but no difference was found between the strains when temperature shifts were slower. Combination of ClpB1 overexpression with DnaK2 overexpression (Slr1641+/Sll0170+) further increased thermotolerance. Next, we used a Synechocystis strain that carries an introduced isoprene synthase gene (IspS+) and that therefore produces isoprene. We attempted to increase isoprene yields by overexpression of key enzymes in the methyl erythritol phosphate (MEP) pathway that leads to synthesis of the isoprene precursor. Isoprene production was not increased greatly by MEP pathway induction, likely because of limitations in the affinity of the isoprene synthase for the substrate. Finally, two extraction principles, two–phase liquid extraction (e.g., with an organic and aqueous phase) and solid–liquid extraction (e.g., with a resin) were tested. Two–phase liquid extraction is suitable for separating isoprene but not fatty acids from the culture medium. Fatty acid removal required acidification or surfactant addition, which affected biocompatibility. Therefore, improvements of both the organism and product–harvesting methods can contribute to enhancing the potential of cyanobacteria as solar–powered biocatalysts for the production of petroleum substitutes.
ContributorsGonzalez Esquer, Cesar Raul (Author) / Vermaas, Willem (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2013
Description
Human activity has increased loading of reactive nitrogen (N) in the environment, with important and often deleterious impacts on biodiversity, climate, and human health. Since the fate of N in the ecosystem is mainly controlled by microorganisms, understanding the factors that shape microbial communities becomes relevant and urgent. In arid land soils, these microbial communities and factors are not well understood. I aimed to study the role of N cycling microbes, such as the ammonia-oxidizing bacteria (AOB), the recently discovered ammonia-oxidizing archaea (AOA), and various fungal groups, in soils of arid lands. I also tested if niche differentiation among microbial populations is a driver of differential biogeochemical outcomes. I found that N cycling microbial communities in arid lands are structured by environmental factors to a stronger degree than what is generally observed in mesic systems. For example, in biological soil crusts, temperature selected for AOA in warmer deserts and for AOB in colder deserts. Land-use change also affects niche differentiation, with fungi being the major agents of N2O production in natural arid lands, whereas emissions could be attributed to bacteria in mesic urban lawns. By contrast, NO3- production in the native desert and managed soils was mainly controlled by autotrophic microbes (i.e., AOB and AOA) rather than by heterotrophic fungi. I could also determine that AOA surprisingly responded positively to inorganic N availability in both short (one month) and long-term (seven years) experimental manipulations in an arid land soil, while environmental N enrichment in other ecosystem types is known to favor AOB over AOA. This work improves our predictions of ecosystem response to anthropogenic N increase and shows that paradigms derived from mesic systems are not always applicable to arid lands. My dissertation also highlights the unique ecology of ammonia oxidizers and draws attention to the importance of N cycling in desert soils.
ContributorsMarusenko, Yevgeniy (Author) / Hall, Sharon J (Thesis advisor) / Garcia-Pichel, Ferran (Thesis advisor) / Mclain, Jean E (Committee member) / Schwartz, Egbert (Committee member) / Arizona State University (Publisher)
Created2013
Description
Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.
ContributorsBadalamenti, Jonathan P (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce E. (Committee member) / Torres, César I (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2013
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
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological soil crusts (biocrusts) are topsoil communities of organisms that contribute to soil fertility and erosion resistance in drylands. Anthropogenic disturbances can quickly damage these communities and their natural recovery can take decades. With the development of accelerated restoration strategies in mind, I studied physiological mechanisms controlling the establishment of cyanobacteria in biocrusts, since these photoautotrophs are not just the biocrust pioneer organisms, but also largely responsible for improving key soil attributes such as physical stability, nutrient content, water retention and albedo. I started by determining the cyanobacterial community composition of a variety of biocrust types from deserts in the Southwestern US. I then isolated a large number of cyanobacterial strains from these locations, pedigreed them based on their 16SrRNA gene sequences, and selective representatives that matched the most abundant cyanobacterial field populations. I then developed methodologies for large-scale growth of the selected isolates to produce location-specific and genetically autochthonous inoculum for restoration. I also developed and tested viable methodologies to physiologically harden this inoculum and improve its survival under harsh field conditions. My tests proved that in most cases good viability of the inoculum could be attained under field-like conditions. In parallel, I used molecular ecology approaches to show that the biocrust pioneer, Microcoleus vaginatus, shapes its surrounding heterotrophic microbiome, enriching for a compositionally-differentiated “cyanosphere” that concentrates the nitrogen-fixing function. I proposed that a mutualism based on carbon for nitrogen exchange between M. vaginatus and its cyanosphere creates a consortium that constitutes the true pioneer community enabling the colonization of nitrogen-poor, bare soils. Using the right mixture of photosynthetic and diazotrophic cultures will thus likely help in soil restoration. Additionally, using physiological assays and molecular meta-analyses, I demonstrated that the largest contributors to N2-fixation in late successional biocrusts (three genera of heterocystous cyanobacteria) partition their niche along temperature gradients, and that this can explain their geographic patterns of dominance within biocrusts worldwide. This finding can improve restoration strategies by incorporating climate-matched physiological types in inoculum formulations. In all, this dissertation resulted in the establishment of a comprehensive "cyanobacterial biocrust nursery", that includes a culture collection containing 101 strains, isolation and cultivation methods, inoculum design strategies as well as field conditioning protocols. It constitutes a new interdisciplinary application of microbiology in restoration ecology.
ContributorsGiraldo Silva, Ana Maria (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Barger, Nichole N (Committee member) / Bowker, Mathew A (Committee member) / Sala, Osvaldo (Committee member) / Arizona State University (Publisher)
Created2019
Description
The mechanisms of extracellular respiration in Geobacter sulfurreducens, commonly considered to be a model organism for anode respiration, are yet to be completely understood. The interplay between electron and proton transport especially could be a key to gaining further insights. One way to investigate the mechanisms of extracellular respiration under varying environmental conditions is by analyzing the electrochemical response of the biofilm with respect to pH, buffer concentrations, and acetate concentrations. I seek to increase the understanding of the electrochemical response of the G. sulfurreducens biofilm through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques in concert with chronoamperometry. I used Geobacter sulfurreducens PCA biofilms in single-chamber electrochemical cells (approximately 100 mL volume) with a small gold working electrode (3.14 mm2). I observed limitations in the initial methods used for media replacement. I tracked changes in the CV data, such as EKA (midpoint potential), as a function of pH and buffer concentration. The media replacement method developed demonstrates success in pH experiments that will be transferrable to other environmental conditions to study electron transport. The experiments revealed that the clarity of data collected is dependent on the quality of the biofilm. A high quality biofilm is characterized by a high current density and normal growth behavior. The general trends seen in these experiments are that as pH increases the potential decreases, and as buffer concentration increases the potential decreases and pH increases. Acetate-free conditions in the reactor were unable to be achieved as characterized by non-zero current densities in the acetate-free experiments.
ContributorsHolzer, Denton Gene (Author) / Torres, Cesar (Thesis director) / Popat, Sudeep (Committee member) / Yoho, Rachel (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol. The normal process for butanol production is very intensive but there is a method to produce butanol from bacteria. This process is better because it is more environmentally safe than using oil. One problem however is that when the bacteria produce too much butanol it reaches the toxicity limit and stops the production of butanol. In order to keep butanol from reaching the toxicity limit an adsorbent is used to remove the butanol without harming the bacteria. The adsorbent is a mesoporous carbon powder that allows the butanol to be adsorbed on it. This thesis explores different designs for a magnetic separation process to extract the carbon powder from the culture.
ContributorsChabra, Rohin (Author) / Nielsen, David (Thesis director) / Torres, Cesar (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
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
p-Coumaric acid is used in the food, pharmaceutical, and cosmetic industries due to its versatile properties. While prevalent in nature, harvesting the compound from natural sources is inefficient, requiring large quantities of producing crops and numerous extraction and purification steps. Thus, the large-scale production of the compound is both difficult and costly. This research aims to produce p-coumarate directly from renewable and sustainable glucose using a co-culture of Yeast and E. Coli. Methods used in this study include: designing optimal media for mixed-species microbial growth, genetically engineering both strains to build the production pathway with maximum yield, and analyzing the presence of p-Coumarate and its pathway intermediates using High Performance Liquid Chromatography (HPLC). To date, the results of this project include successful integration of C4H activity into the yeast strain BY4741 ∆FDC1, yielding a strain that completely consumed trans-cinnamate (initial concentration of 50 mg/L) and produced ~56 mg/L p-coumarate, a resting cell assay of the co-culture that produced 0.23 mM p-coumarate from an initial L-Phenylalanine concentration of 1.14 mM, and toxicity tests that confirmed the toxicity of trans-cinnamate to yeast for concentrations above ~50 mg/L. The hope for this project is to create a feasible method for producing p-Coumarate sustainably.
ContributorsJohnson, Kaleigh Lynnae (Author) / Nielsen, David (Thesis director) / Thompson, Brian (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12