Bennett, Mechelle
(2021)
Hijacking Bacterial Electron Transfer for Iron - Mediated
Polymerisations.
PhD thesis, University of Nottingham.
Abstract
Polymer synthesis offers a plethora of methods and techniques to develop diverse and functional materials for a variety of applications. Reversible Deactivation Radical Polymerisation (RDRP) methods are the most versatile methods of which, Atom Transfer Radical Polymerisation (ATRP) and Reversible Addition Fragmentation chain-Transfer (RAFT) are highly promising for applications which require control over polymer structure, whilst sustaining mild reaction conditions. In the emerging areas of bioenergy harvesting and self-healing systems, methods to generate engineered living materials (ELM)s and hybrid synthetic/natural superstructures are being considered. These might have applications in microbial fuel cells (MFC), biological sensing, bioreactors, bioremediation, or as implantable synthetic microbiomes.
Cell mediated polymerisation reactions have been carried out to generate these hybrid superstructures, but most require conditions which affect cell metabolism, and the methods of biological redox initiation are not well understood. The research carried out in this thesis aims to utilise bacterial extracellular electron transfer (EET) systems to create novel hybrid bio-polymerisations, and to investigate the mechanisms by which they operate. The first chapter of this thesis examines ATRP and RAFT techniques that could be utilised for such experiments, and bacterial EET, quorum sensing (QS) and MFCs are discussed to provide context for the study. Prior literature surrounding biocatalysts and living materials are reviewed and a proposal for each results chapter in this thesis is given.
In the second chapter of this thesis a novel Fe ATRP polymerisation technique initiated by living bacteria is presented. The method can be carried out whilst maintaining bacterial viability under biological conditions (room temperature (RT), 37 °C, phosphate buffered saline (PBS)), to produce polymers, whereas chemically killed bacteria are unable to initiate the polymerisation. Different parameters including bacteria concentration, catalyst concentration, bacteria type, initiator type, degree of polymerisation (DP) and monomer type are explored. The ‘livingness’ of the bacterial initiated Fe ATRP methods are shown to be somewhat compromised, likely due to biological interference with the Fe catalyst which is essential for maintaining polymerisation control. Although difficulty in molecular weight (Mn) control of the resulting polymers is displayed, the findings point towards a future platform technology for the manipulation of cells via a synthetic extracellular matrix (ECM) environment.
The third chapter of this thesis offers an alternative novel Fenton Glucose Oxidase - RAFT (FG-RAFT) technique that could be carried out in the presence of air without the need for prior degassing. This bacterial initiated polymerisation method introduces a less time-consuming and more economically viable technique than that of bacterial initiated Fe ATRP. Furthermore, the resulting polymers of FG-RAFT display lower dispersities (Đ ~ 1.12) with somewhat predictable Mns. The initial radical flux in these reactions is explored by altering component concentrations (Glucose, GOx, FeCl3), revealing that the catalyst concentration (FeCl3) can be tailored to generate polymers with lower Đs in the case of N-acryloylmorpholine (NAM), but in turn the Mn control is slightly compromised. The polymerisations can be carried out maintaining bacterial viability, whilst heat killed bacteria is shown to be unable to initiate the polymerisations, indicating the necessity of bacterial metabolism to the redox initiation. The quality of the resulting polymers are shown to differ depending on monomer type, with dimethylacrylamide (DMA) producing the most well-defined polymers, even with Fe concentrations as low as 7 µM.
The fourth chapter of this thesis investigates the Cytochrome C (C-Cyt) protein NapC of E. coli as an EET component in the reduction of Fe3+ by bacteria. Cloning techniques are used to upregulate the protein in E. coli of which the inducible promoter PBAD is successful. Sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) reveals greater NapC production for higher induced cultures. These are subsequently utilised in Fe ATRP polymerisations, showing higher reaction rates than those initiated by wild type cultures. Linear sweep voltammetry (LSV) is finally used to probe the Fe reduction capabilities of the clones, and although further research is necessary, the results suggest that Fe reduction is upregulated by the bacteria in times of environmental stress.
Overall, the development of 2 new synthetic polymerisation methods utilising bacterial redox chemistry as an initiation stimulus are presented, and cloning methods are used to investigate the involvement of NapC in bacterial EET. The methods presented contribute to advances in the fields of hybrid biosynthetic technologies, improved EET knowledge and innovations to sustainable polymerisation chemistry.
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