Browsing by Subject "Azotobacter"

Now showing 1 - 3 of 3
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    The genetic basis of nitrogen fixation and carbon metabolism in Azotobacter vinelandii
    (2022-06) Knutson, carolann
    Natural nitrogen fixation is done primarily by prokaryotes that reduce nitrogen gas(N2) to ammonia (NH3) using the enzyme nitrogenase in a process termed biological nitrogen fixation (BNF). BNF is an alternative to industrial fertilizers that could supply crops with the nitrogen they need, either through symbiotic or free-living associations. Engineering these associations requires an understanding of BNF that extends beyond nitrogenase, to the dynamic suite of proteins that support it. Azotobacter vinelandii is a model organism for studying BNF and contains more than 80 suspected BNF genes, many of which have an unknown function or lack experimental data showing direct BNF association. A. vinelandii is known for being a free-living and aerobic nitrogen fixer, making it both convenient for laboratory studies and biotechnologically relevant. Since most research in this organism has focused on nitrogen fixation, there have been few studies on how various carbon sources are metabolized. Characterizing this metabolism in A. vinelandii in the context of nitrogen fixation would help in engineering viable alternatives to Haber-Bosch. Consequently, this research builds on the contextual knowledge of BNF in A. vinelandii by using transposon mutagenesis to identify genes important to growth and/or nitrogen producing phenotypes. First, we used transposon sequencing (Tn-seq) to determine the genome-wide fitness of genes under diazotrophic, non-diazotrophic, and differing carbon sources. We then used the Tn-seq data from growth on the carbon source galactose to identify two galactose dehydrogenases predicted to complete the pathway of galactose metabolism missed by routine genomic annotation algorithms. Lastly, we explored the gene redundancy of NifA in nitrogenase regulation by characterizing two transposon mutagenesis mutants able to support the growth of algae in co-culture. Overall, this thesis expands on the knowledge of BNF in A. vinelandii by providing genome-wide fitness that quantify individual gene contributions to BNF, carbon metabolism and, which also explores gene redundancy in BNF regulation.
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    The Sequencing, Assembly and Annotation of Sugar Producing Green Algae and the Design of a Low Cost Turbidostat
    (2017-10) Arriola, Matthew
    The number of sequenced genomes continues to rise at an astounding rate, however, only thirty-one green algae genomes have been sequenced to date. In this work, two green algae, Micractinium conductrix SAG 241.80 (formerly named Chlorella sp. SAG 241.80) and Chlorella sorokiniana UTEX 1602, have had their genomes sequenced and assembled de novo and functionally annotated. These two new genomes were used to investigate the genes responsible for a sugar-secreting phenotype previously described in M. conductrix SAG 241.80. When grown at a lowered pH of approximately 5.7, M. conductrix SAG 241.80 releases maltose and glucose outside of the cell, while at pH 7.6, sugar production is minimal. An additional green alga with a high sugar-releasing phenotype was isolated from the environment (Scenedesmus sp. PABB004) and found to release a quantity of sugars five-fold higher than what was found for M. conductrix SAG 241.80. Additionally, this strain did not require the low pH of 5.7 for sugar release, and produced these sugars at a physiological pH of 7.0. This work, as well as additional efforts toward developing a low-cost turbidostat for performing evolutionary experiments in microbes will be presented.
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    SUSTAINABLE HYDROGEN, AMMONIUM, AND BIOFUELS PRODUCTION
    (2021-03) Knutson, Carolann
    Sustainable technologies are are often guided by our understanding of their natural equivalents. As such, our incomplete understanding of these natural processes limit our ability to design efficient and optimized synthetic schemes to address the challenges facing food and energy security. In particular, schemes using live organisms or purified proteins require that we either attempt to remove evolutionary features such as regulation or cope with unknown confounding factors resulting from the gaps in our knowledge. All three of the following chapters focus on furthering our understanding of the natural systems we wish to co-opt. The first chapter discusses producing biohydrogen via Azotobacter vinelandii, a soil bacterium that fixes nitrogen gas into ammonia aerobically using the enzyme, nitrogenase. Additionally, it shows how the produced biohydrogen can act as a proxy of nitrogenase activity such that we can quantify the in vivo inhibition that results from various nitrogenous compounds commonly found in the environment. The second chapter discusses growing green algae in turbidostat reactors as way to screen their compatibility with divergent growing technologies and commercial ventures. Lastly, the third chapter discusses how an oil-degrading marine bacterium, Marinobacter aquaeolei , produces wax esters via a partially defined wax ester biosynthesis pathway.