The mechanisms of extracellular electron transfer

Abstract

In bioelectrochemical systems (BESs) microbial activity facilitates electricity generation and product synthesis. Using the microbial process of extracellular electron transfer (EET) Shewanella and Geobacter species can respire using a solid terminal electron acceptor, such as an anode in BES. Study of these microorganisms and how they behave at the molecular level is important for shining light on geomicrobial processes and development of BES. Through the use of molecular and electrochemical techniques, this PhD thesis will focus on the molecular mechanisms employed by bacterial biofilms on the anode of a BES, specifically Shewanella oneidensis MR-1 and Geobacter sulfurreducens DL-1. The physiology of these microorganisms appears to be directly associated with the operational conditions of the BES. The application of electrochemical and molecular studies enables the comparison and understanding of the cellular response to the BES operation, in particular on how they respond to changes in the anode potential. Quantitative proteomics from low biomass, biofilm samples is not well documented. The first objective of this thesis was to show the successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm of Shewanella oneidensis MR-1. After growth at different potentials (+0.5 V, 0.0 V & -0.4 V vs. Ag/AgCl), biofilm proteins were extracted from anodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicated an increase in metabolic activity. This objective demonstrated SWATH-MS as a suitable method for studying differences between biomass limited biofilm samples. Subsequently, SWATH-MS and electrochemical methods were used to characterize anodic biofilms of S. oneidensis MR-1 and G. sulfurreducens DL-1. Experiments were conducted at the different electrode potentials of +0.5 V, 0.0 V and -0.4 V or at +0.1 V and +0.6 V for S. oneidensis and G. sulfurreducens respectively. SWATH-MS analysis revealed different strategies of adaption to changes in potential for both microorganisms, with S. oneidensis showing an increase in the relative abundance of its EET cytochromes with increased potential. In addition, these findings support the model that S. oneidensis nanowires are extensions of the outer membrane. Conversely, the majority of EET cytochromes quantified for G. sulfurreducens showed little or no significant change in relative abundance in response to electrode 3 potential. These results suggest S. oneidensis has greater adaptability in its regulation of EET cytochromes compared to G. sulfurreducens. Proteomic, bioelectrochemical and UV-HPLC methods, confirm the involvement and dominance of mediated electron transfer in biofilms of S. oneidensis respiring with an electrode. Furthermore, the relative higher abundance of a riboflavin biosynthesis protein, suggested the involvement of flavins in the EET of biofilms of G. sulfurreducens. Biofilms of G. sulfurreducens grown at the highly oxidative potential of +0.6 V, showed indications of oxidative stress, with lower current production, lower bioelectrochemical signals, and the presence of certain cellular protection mechanisms. Furthermore, similarities between the species were detected, with an increase in the relative abundance of TCA cycle proteins observed with higher rates of EET. In summary, this PhD thesis provides evidence that SWATH-MS is a reliable method to observe changes in the relative abundance of proteins in anodic biofilms of S. oneidensis and G. sulfurreducens. Furthermore, it provides insight into regulation of EET proteins, the physiological response to electrode potential, and how these differ between species of dissimilatory metal respiring bacteria.