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Bioelectrochemistry and, more specifically, microbial electrochemistry, are technologies based on the connection between microbes (named as exoelectrogens or, focusing only on bacteria, electrochemically active bacteria) and electrodes. The exchange of electrons to and from the electrode has been studied primarily in mixed cultures but also with pure strains, mostly using model species such as Geobacter and Shewanella; however, more efforts are needed to elucidate the interaction between microbes and electrode and to find new interesting niches of application for these microorganisms. A field of application is bioelectrochemical remediation, an effective strategy in environments where the absence of suitable electron acceptors limits classic bioremediation, and in which bioelectrochemical systems are used for the removal of pollutants from environmental matrices. Bioelectrochemical remediation of hydrocarbons with pure strains and microbial communities has been reported; however, only few exoelectrogenic hydrocarbonoclastic bacteria have been characterized, so far. The degradative potential of several hydrocarbon-degrading strains has been extensively studied, in terms of pollutants removal and mechanism of contaminant mineralization, but not much is known about their exoelectrogenic capacity and possible application for bioelectrochemical remediation. Bioelectrochemistry and its application for bioremediation purposes, has primarily focused on testing the hydrocarbonoclastic capacities of already known exoelectrogenic strains. In this study we took a different approach, and we aimed at studying the exoelectrogenic activity of three strains that showed great potential for bioremediation applications: Cupriavidus metallidurans CH34, and Pseudomonas sp. strains DN34 and DN36. C. metallidurans CH34 is a model metal-resistant strain, whose hydrocarbonoclastic capacities have recently been individuated, and Pseudomonas sp. strains DN34 and DN36 that are two hydrocarbon-degrading strains isolated from an oil-polluted site in central Chile. By analyzing current production, bacterial growth and substrate consumption in bioelectrochemical systems (BES), we determined that the three strains possess exoelectrogenic activity. Moreover, C. metallidurans CH34 showed the most promising results with a non-recalcitrant substrate and was selected to assess bioremediation experiments with toluene as model hydrocarbon. We demonstrated for the first time that strain CH34 is able to degrade toluene under denitrifying conditions. Further experiments in Microbial Fuel Cells (MFC) linked toluene degradation to current production by strain CH34, showing current peaks after toluene respike (maximum current density 0.24 mA/m2). Moreover, a Microbial Electrolysis Cell (MEC) was operated by applying an external voltage (800 mV) between anode and cathode to stimulate microbial metabolism of strain CH34 and to observe the behavior of the strain in terms of toluene removal and current generation. Current outputs increased by two orders of magnitude in comparison with MFC (up to 47 mA/m2), and coulombic efficiency raised up to 77%, demonstrating that the bacterial cells adjusted progressively to the system conditions and that electrochemical losses were, at least partially, overcome. In order to evaluate the effect of an electron carrier on current production, Neutral Red (NR) was selected as external transporter and amended in a MEC containing toluene and inoculated with strain CH34, but no relevant effect was observed on current production nor coulombic efficiency. Hence, we concluded that NR had no influence on current generation in our system and that a mediated mechanism with this electron carrier is not probable. The mechanism of extracellular electron transport (EET) is a key feature in BESs and the efficiency of the microorganism to exchange electrons with an electrode and to connect the EET to the cellular carbon metabolism, significantly influences the overall process performance. We demonstrated that the first step of the denitrification pathway is activated by nitrate reductases when NO3- was the only electron acceptor, but we also aimed at studying whether the pathway of denitrification is still active in absence of nitrate, if a solid the anode is potentiostetically-polarized at the same redox potential of nitrate reductase. Our results indicate that nitrate reductase is not involved in the transport of electrons in BES and that strain CH34 follows a different pathway of electron transport to the anode. However, current production and cells viability demonstrated that strain CH34 was actively performing oxidative phosphorylation, thus that, in a mechanism that has not been elucidated yet, an extracellular electron transfer takes place, either in a direct or indirect way.
PhD program in Chemical, Geological and Environmental Sciences Curriculum in Environmental Sciences Cycle XXXI Università degli Studi di Milano-Bicocca, Italy