Browsing by Subject "Hydrogen Metabolism"

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    Microbial Interactions: From Microbes to Metals
    (2016-01) Kane, Aunica
    Microbial communities are the major drivers of biochemical cycling and nutrient flux on the planet, yet despite their importance, the factors that influence and shape behavior and function of microbial ecosystems remain largely undefined. The knowledge gap existing for microbial communities stems partly from a focus of microbiologists on monoculture but also because studies of multispecies systems are impeded by their complexity and dynamic nature. Synthetic ecology, the engineering of rationally designed communities in well-defined environments, provides an innovative and robust approach to reduce the complexity inherent in natural systems and mimic microbial interaction in a controlled framework. Synthetic ecology was used to engineer a co-culture using two previously non-interacting bacteria, Shewanella oneidensis and Geobacter sulfurreducens, both organisms important for multiple applications in biotechnology. The S. oneidensis and G. sulfurreducens co-culture provided a model laboratory co-culture to study microbial interactions and revealed that genetic mutations in metabolic pathways can provide the foundation to initiate cooperation and syntrophic relationships in multispecies ecosystems. Syntrophy between S. oneidensis and G. sulfurreducens was studied further using three-electrode bioreactors. Both S. oneidensis and G. sulfurreducens are capable of respiring insoluble terminal electron acceptors, a process termed extracellular respiration. During extracellular respiration, electrons produced during oxidative metabolism are transferred across both membranes to the outer surface of the bacterial cell where they reduce terminal electron acceptors such as metal oxides. Extracellular respiration can be monitored in real time as current produced in bioreactors with electrodes serving as a proxy for metal oxides. The ability of both S. oneidensis and G. sulfurreducens to transfer electrons to their outer surface enabled the study of a process central to many syntrophic communities known as interspecies electron transfer – the transfer of reducing equivalents between organisms. Mutants in various electron transfer pathways revealed that interspecies electron transfer in an obligate S. oneidensis/G. sulfurreducens co-culture was mediated by soluble redox-active flavins secreted by Shewanella serving as electron shuttles between species. The second half of this thesis focuses on S. oneidensis metabolism and interactions of microbes with metals. Microbial transfer of electrons to metals has a large impact on biogeochemical cycles and can also be harnessed for biotechnology applications in bioenergy and bioremediation. In order to effectively engineer S. oneidensis for these applications, it is imperative to understand how Shewanella gains energy from the oxidation of electron donors and the efficiency of electron transfer to metals and electrodes. Work in Chapter 4 revealed formate oxidation to be a central strategy under anaerobic conditions for energy conservation through the generation of proton motive force in S. oneidensis. Work in Chapter 5 quantified the effect of hydrogen metabolism on electron transfer reactions in Shewanella three-electrode bioreactors. Deletion of the hydrogenase large subunits, hyaB and hydA, in Shewanella resulted in higher current density and coulombic efficiency in single-chamber three-electrode bioreactors by diverting electron flux to the anode instead of to hydrogen production. The final chapter of this thesis focused on harnessing microbial transformation of metals for bioremediation purposes. An engineered Escherichia coli strain containing a mercury resistance plasmid was constructed to facilitate the remediation of organic and ionic forms of mercury pollution. The engineered strain was then encapsulated using silica sol gel technology generating a bio-filtration material for use in bioremediation platforms. Work in this thesis highlights the importance of microbial interactions, both with other organisms and with metals in the environment. Comprehensive knowledge on microbial interactions is important not only for a better understanding of ecosystem function but can also be harnessed for biotechnology applications. Microbial interactions and transformation of metals shape the world around us and have also facilitated use and further engineering of microorganisms for bioenergy and bioremediation technologies.