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Role of biological electron mediators in microbial extracellular electron transfer

Hassan, Md Mahamudul (2018) Role of biological electron mediators in microbial extracellular electron transfer. PhD thesis, Murdoch University.

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Electron mediators are redox active compounds capable of mediating electron transfer from a donor to acceptor. In microbial systems, electron mediators play a key role in extracellular electron transfer processes to assist the bacteria to thrive under unusual environmental conditions. Electron mediators are known to facilitate electron transfer from the bacterial cells to their electron acceptors which are insoluble (e.g. Fe3+, Mn4+) or toxic (e.g. oxygen for anaerobes). Interspecies electron transfer between different microbial species is also known to be driven by electron mediators. In this case, one species uses the oxidized mediator as electron acceptor and reduces it while the other species uses the reduced mediator as electron donor. The involvement of electron mediators in these electron transfer processes has led to extensive investigation to elucidate their contribution in microbial ecosystems.

The aim of this thesis is to investigate the role of microbially produced electron mediators in facilitating microorganisms to thrive in selected environments that are of human concern. In this study, a novel electrochemical tool was developed that allows characterization of the electron mediators more effectively than the conventional techniques. The proposed method offered much better sensitivity and resolution compared to the conventional technique in detecting electron mediators.

Conventional electrochemical studies use the three-electrode electrochemical cell which is equipped with only one controllable working electrode (WE). The other two electrodes serve as counter and reference electrodes. The traditional one-WE setup is based on the oxidation or reduction of the target molecule at different time interval as for example used in cyclic voltammetry. Having only one WE does not allow mimicking redox condition of the microbial systems where oxidation and reduction occur simultaneously.

In order to test for the presence of redox active mediators, a new apparatus and technique was developed that consists of two independently controllable WEs which enable the generation of redox gradient between the WEs to allow simultaneous oxidation and reduction of the target redox active mediator. By using this redox gradient generating property, a new method was developed that characterizes electron mediators within a thin layer microscale (250 μm) system without the need of a bulk solution and associated mass transfer.

Electrochemical properties of electron mediators were characterized by stepwise shifting a “voltage window” (maintaining 0.05 V potential difference between two WEs) within a range of potentials (between –1 V and +0.5 V vs. Ag/AgCl) and monitoring the establishment of steady equilibrium current in both WEs. The resulting current difference between two WEs was recorded for each voltage step of the “voltage window”. Results indicated that this technique enabled identification (by the distinct peak locations at the potential scale) and quantification (by the peak of current) of individual mediators as well as several mediators in an aqueous mixture. This technique enabled the precise determination of the mid-potential of hexacyanoferrate (HCF), riboflavin (RF) and two mediators from the pyocyanin-producing P. aeruginosa (WACC 91) culture. The capability of Twin-WE approach in detecting unknown electron mediators from a microbial culture confirms its suitability in studying microbial extracellular electron transfer (EET) processes.

The Twin-WE electrochemical cell was used to investigate the role of the bacterial mediator PYO in electron transfer processes accomplished by its producer P. aeruginosa (PA), a high impact bacterium from human health perspective. Pyocyanin (PYO) is a redox active compound present in the biofilm of P. aeruginosa and believed to mediate an electron transfer from PA cells to oxygen for assisting PA to respire under oxygen limited condition. In contrast to widely held belief, this study shows that reduced PYO v

(RedPYO) is not readily oxidized by oxygen unless catalyzed by living cells. The results are supportive to a scenario in which PYO can extract electrons from other living cells by oxidizing their NADH. The resulting RedPYO can be utilized as electron donor for oxygen or nitrate respiring PA cells. While this PYO mediated electron transfer resembles syntrophic interspecies electron transfer, it suggests, in this case, the existence of a not yet described form of energy parasitism. The discovery of this parasitic life style puts a new perspective on the role of PYO in biofilms, its natural soil environment and host infections.

The existence of a similar electron extracting mechanism of PYO was also investigated in microbially influenced corrosion (MIC). MIC is a complex bio-electrochemical process where the exposure of the metal to microorganisms and their metabolic products causes dissolution of metal ions. Corrosion of steel occurs due to the existence of simultaneous anodic and cathodic reactions on the steel surface. At the cathodic site, steel loses electrons which consequently causes the dissolution of ferrous ions at the anodic site. Under aerobic condition, steel loses electrons from the cathodic site to oxygen. MIC has been described as bacteria rely on mediators to use electrons from the cathode under anaerobic conditions. The potential role of bacterial mediators to influence corrosion in the presence of oxygen has not been investigated yet.

The capability of PYO to extract electrons from living cells was translated to electron extraction from corroding steel. Results showed that PYO can more efficiently harness electrons from the steel than oxygen alone. The reduced PYO thus generated (RedPYO) subsequently can transfer electrons to oxygen. The corrosion rate as determined by the release of dissolved iron was increased by two-fold when carbon steel was exposed to PYO compared to the exposure to a PYO free electrolyte under oxygen saturated environment. This increase in corrosion rate can be explained by the existence of a PYO mediated electron flow from the steel to the oxygen which accelerated the cathodic half reaction. PA cells can also benefit from this electron flow to generate cellular energy (ATP) using RedPYO as the electron donor for oxidative phosphorylation. Hence, PA and PYO containing biofilms could be described as catalyst of the cathodic reaction of corroding iron. To our knowledge, this is the first study to demonstrate the role of a biological electron mediator in influencing aerobic corrosion by cathodic stimulation.

Overall, this thesis has contributed towards improving the understanding of microbial mediators, their detection and possible role in microbial consortia and in interaction of microbes with reducing surfaces such as steel constructions.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Engineering and Information Technology
Supervisor: Cord-Ruwisch, Ralf
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