Photosynthetic microorganisms, including algae and cyanobacteria, are able to export electrons derived from the photosynthetic electPhotosynthetic microorganisms, including algae and cyanobacteria, are able to export electrons derived from the photosynthetic electron transport chain to the cell exterior, in a phenomenon known as exoelectrogenesis. This phenomenon has implications for an emerging field of biotechnology which seeks to employ photosynthetic biocatalysts to produce solar electricity, chemicals and fuels (bioelectrochemical systems). However, both the underlying mechanism by which exoelectrogenesis occurs and the function that exoelectrogenesis plays in photomicrobial communities are poorly understood. This gap in understanding represents a barrier to the intelligent design of biophotoelectrochemical systems, hindering their development into a practical technology. At a fundamental level this also represents a gap in the understanding of the biochemical mechanisms of photosynthesis, one of the most important and widespread biochemical pathways in life. The cyanobacteria Synechocystis sp. PCC 6803 is used throughout this work as a model organism for the study of photosynthesis and exoelectrogenesis. The research presented in this thesis aims to deepen understanding of the mechanism and function of exoelectrogenesis in cyanobacteria by generating and employing a workflow for identifying the endogenous redox mediator, developing a platform for in operando electrochemistry and microscopy, and using it to study the dynamic spatial variations in biofilm behaviour during exoelectrogenesis.

First, the question of the mechanism of exoelectrogenesis is examined. Exoelectrogenesis is known to occur in cyanobacteria through the secretion of a small, soluble redox mediator, with the identity of this mediator having remained unknown for many years. Recently, a proposal for the identity of this mediator was put forward: this work begins by critically assessing the veracity of this proposal and testing whether it holds true under standard conditions. Finding that it does not, a workflow is developed for the identification of the mediator. Through this workflow, spectroscopic and electrochemical assays are applied to mediator-enriched samples, identifying unique features that may be attributable to mediator-like species. Chemical species composing these samples are separated out by high performance liquid chromatography, and putative features annotated by liquid chromatography-mass spectrometry with post-processing metabolomics analysis. The workflow identifies several plausible candidates in the cyanobacterial exometabolome which are increased in concentration under light exposure, providing solid movement towards elucidating the identity of the endogenous mediator of exoelectrogenesis.

Next, insights are sought into the mechanism and function of exoelectrogenesis at a multicellular level. The current toolkit used in cyanobacterial exoelectrogenesis research predominantly employs bulk techniques to examine the process; what these bulk techniques gain in signal intensity, they sacrifice in dynamic, spatial information and understanding of diversity and heterogeneity within the multicellular community (the biofilm). Exoelectrogenesis is a single-cell behaviour that has a function within a multi-cellular community, so insights into both mechanism and function are to be gained by the study of single cells within the population. To this end, new tools are needed. A platform for in operando electrochemical microscopy is developed, to facilitate microscopic imaging during (photo)electrochemical measurements of the cyanobacterial biofilm. Similar techniques are increasingly being used to study other microbial community behaviours, however they have yet to be rigorously applied to the study of cyanobacterial exoelectrogenesis. This platform is optimised for both electrochemical and microscopic measurements, and further developed to apply spatially-patterned stimuli (voltage and light). A series of screening experiments are conducted to identify suitable fluorescence probes for biochemical parameters, as well as the most suitable chassis organism for microscopic studies.

During this screening, the Nernstian membrane potential reporter Thioflavin T (ThT) is identified as a compatible and favourable probe for use in this system. In recent years, membrane potential reporters (including ThT) have been used to inform on many aspects of microbial community behaviour, including exoelectrogenesis in heterotrophic bacteria. The connection between membrane potential (V_m) and exoelectrogenesis in cyanobacteria has so far not been studied, but it is hypothesised that exoelectrogenesis would influence V_m and so the application of a probe for membrane potential could act as a useful proxy for exoelectrogenic activity. By conducting photoelectrochemistry of cyanobacterial biofilms on the microscope, with ThT as a V_m reporter, a robust, reproducible V_m response is observed during exoelectrogenesis. This response is tested under a series of relevant conditions, including inhibiting and increasing exoelectrogenesis. Methods for investigating the heterogeneity across the biofilm in three dimensions are developed, and a diversity of V_m responses demonstrated. Finally, by application of precision light stimulation, it is shown that V_m changes propagate from cells experiencing high light to those in darkness, presenting a route toward understanding why cyanobacteria export photo-excited electrons.

Overall, this work presents movements towards identifying the mediator of exoelectrogenesis: a recent proposal is ruled out, several assays for the mediator identified and a system for isolating and identifying the mediator developed, which, with further work, can reveal its identity. A robust and flexible platform for in operando electrochemistry and microscopy is developed, optimised and successfully screened for use with appropriate fluorescence probes, and is shown to be well suited for further work, perhaps with other microorganisms or microscopes. Finally, membrane potential across the cyanobacterial biofilm is studied during exoelectrogenesis, showing that membrane potential changes occur and can be monitored during electron export, at the single- and multi-cellular level, and that changes in membrane potential are propagated across the biofilm in response to precise illumination. ron transport chain to the cell exterior, in a phenomenon known as exoelectrogenesis. This phenomenon has implications for an emerging field of biotechnology which seeks to employ photosynthetic biocatalysts to produce solar electricity, chemicals and fuels (bioelectrochemical systems). However, both the underlying mechanism by which exoelectrogenesis occurs and the function that exoelectrogenesis plays in photomicrobial communities are poorly understood. This gap in understanding represents a barrier to the intelligent design of biophotoelectrochemical systems, hindering their development into a practical technology. At a fundamental level this also represents a gap in the understanding of the biochemical mechanisms of photosynthesis, one of the most important and widespread biochemical pathways in life. The cyanobacteria Synechocystis sp. PCC 6803 is used throughout this work as a model organism for the study of photosynthesis and exoelectrogenesis. The research presented in this thesis aims to deepen understanding of the mechanism and function of exoelectrogenesis in cyanobacteria by generating and employing a workflow for identifying the endogenous redox mediator, developing a platform for in operando electrochemistry and microscopy, and using it to study the dynamic spatial variations in biofilm behaviour during exoelectrogenesis.

First, the question of the mechanism of exoelectrogenesis is examined. Exoelectrogenesis is known to occur in cyanobacteria through the secretion of a small, soluble redox mediator, with the identity of this mediator having remained unknown for many years. Recently, a proposal for the identity of this mediator was put forward: this work begins by critically assessing the veracity of this proposal and testing whether it holds true under standard conditions. Finding that it does not, a workflow is developed for the identification of the mediator. Through this workflow, spectroscopic and electrochemical assays are applied to mediator-enriched samples, identifying unique features that may be attributable to mediator-like species. Chemical species composing these samples are separated out by high performance liquid chromatography, and putative features annotated by liquid chromatography-mass spectrometry with post-processing metabolomics analysis. The workflow identifies several plausible candidates in the cyanobacterial exometabolome which are increased in concentration under light exposure, providing solid movement towards elucidating the identity of the endogenous mediator of exoelectrogenesis.

Next, insights are sought into the mechanism and function of exoelectrogenesis at a multicellular level. The current toolkit used in cyanobacterial exoelectrogenesis research predominantly employs bulk techniques to examine the process; what these bulk techniques gain in signal intensity, they sacrifice in dynamic, spatial information and understanding of diversity and heterogeneity within the multicellular community (the biofilm). Exoelectrogenesis is a single-cell behaviour that has a function within a multi-cellular community, so insights into both mechanism and function are to be gained by the study of single cells within the population. To this end, new tools are needed. A platform for in operando electrochemical microscopy is developed, to facilitate microscopic imaging during (photo)electrochemical measurements of the cyanobacterial biofilm. Similar techniques are increasingly being used to study other microbial community behaviours, however they have yet to be rigorously applied to the study of cyanobacterial exoelectrogenesis. This platform is optimised for both electrochemical and microscopic measurements, and further developed to apply spatially-patterned stimuli (voltage and light). A series of screening experiments are conducted to identify suitable fluorescence probes for biochemical parameters, as well as the most suitable chassis organism for microscopic studies.

During this screening, the Nernstian membrane potential reporter Thioflavin T (ThT) is identified as a compatible and favourable probe for use in this system. In recent years, membrane potential reporters (including ThT) have been used to inform on many aspects of microbial community behaviour, including exoelectrogenesis in heterotrophic bacteria. The connection between membrane potential (Vm) and exoelectrogenesis in cyanobacteria has so far not been studied, but it is hypothesised that exoelectrogenesis would influence Vm and so the application of a probe for membrane potential could act as a useful proxy for exoelectrogenic activity. By conducting photoelectrochemistry of cyanobacterial biofilms on the microscope, with ThT as a Vm reporter, a robust, reproducible Vm response is observed during exoelectrogenesis. This response is tested under a series of relevant conditions, including inhibiting and increasing exoelectrogenesis. Methods for investigating the heterogeneity across the biofilm in three dimensions are developed, and a diversity of Vm responses demonstrated. Finally, by application of precision light stimulation, it is shown that Vm changes propagate from cells experiencing high light to those in darkness, presenting a route toward understanding why cyanobacteria export photo-excited electrons.

Overall, this work presents movements towards identifying the mediator of exoelectrogenesis: a recent proposal is ruled out, several assays for the mediator identified and a system for isolating and identifying the mediator developed, which, with further work, can reveal its identity. A robust and flexible platform for in operando electrochemistry and microscopy is developed, optimised and successfully screened for use with appropriate fluorescence probes, and is shown to be well suited for further work, perhaps with other microorganisms or microscopes. Finally, membrane potential across the cyanobacterial biofilm is studied during exoelectrogenesis, showing that membrane potential changes occur and can be monitored during electron export, at the single- and multi-cellular level, and that changes in membrane potential are propagated across the biofilm in response to precise illumination.