Myers, Benjamin
(2024)
Engineering the spark into bacteria: Development of electroactive bacterial chassis via gene engineering and bio- hybridisation with conductive nanomaterials.
PhD thesis, University of Nottingham.
Abstract
Climate change from anthropogenic hydrocarbon fuel use is causing rapid and unprecedented changes to global environmental systems. To prevent environmental collapse, it is vital to develop technologies for producing carbon- negative energy and fuels. This is the grand scientific, industrial, and economic challenge of our time and requires novel energy production and waste valorisation strategies at all levels of society, from global- scale producers to individual households. While there is no individual solution, the unique chemical <> electricity energy conversion possibilities of Microbial Electrochemical Technologies (MET) may offer innovative routes for capturing the residual energy in waste, alongside valorising spent carbon into energy rich fuels and chemicals. However, a range of process- limiting challenges results in MET applicability mostly languishing at the laboratory scale. This thesis aims to address two of these challenges, specifically: (1) MET being bound to the intrinsic physiological properties and growth rates of native electroactive microbes, and 2) the inefficiency of biological extracellular electron transport (EET) between cells and electrodes.
The work presented in this thesis employed two distinct strategies to combat these challenges. First, a gene modification strategy was used to increase the electroactive potential of the industrially relevant microorganism Cupriavidus necator. Second, a ‘biohybrid’ strategy was proposed, based on the implantation of functionalised carbon nanotubes within the outer membrane of Cupriavidus necator to augment electron exchange, in addition to the native biological electron transport mechanisms.
The first strategy involved structural and aromatic amino acid modifications of Cupriavidus necator’s native type- IV pili, to provide an additional electron transfer mechanism. Cupriavidus necator strains expressing modified pili were characterised using PeakForce TUNA Atomic Force Microscopy and Cyclic Voltammetry, where increased pili filament conductivity and oxidative currents to electrodes were observed, respectively. During bio- electrochemical system operation, increased current generation rates were observed in some of the strains expressing modified pili, as described in detail in Chapters 3 and 4 of this thesis.
The second, bio-hybridisation strategy involved the implantation of Carbon Nanotube Porins within the bacterial membrane to facilitate electron exchange between cells and electrodes. Functionalisation of Single Walled Carbon nanotubes with a membrane lipid analogue and shortening to an axial length of 5-20nm was found promote Cupriavidus necator uptake of the nanotubes, with no significant detriment to cellular viability. However, investigations to assess if nanotube uptake facilitated electrochemical communication between cells and electrodes were not conclusive. Continuation of this research is vital to determine the effectiveness of the system.
In summary, this thesis provides insights for the development of novel microbial chassis, capable of extracellular electron transport available for Microbial Electrochemical Technology system architects via two distinct approaches. Future work is centred around finding mechanisms to promote microbial adhesion to electrode surfaces, to increase the number of cells involved in electron exchange, alongside verifying the effectiveness of biohybrid electron transfer during bio- electrochemical system operation.
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