BIOELECTROCHEMICAL SYSTEMS TO INVESTIGATE THE EXOELECTROGENIC ACTIVITY OF HYDROCARBONDEGRADING BACTERIA

Abstract

Bioelectrochemistry and, more specifically, microbial electrochemistry, aretechnologies based on the connection between microbes (named asexoelectrogens or, focusing only on bacteria, electrochemically active bacteria)and electrodes. The exchange of electrons to and from the electrode has beenstudied primarily in mixed cultures but also with pure strains, mostly using modelspecies such as Geobacter and Shewanella; however, more efforts are neededto elucidate the interaction between microbes and electrode and to find newinteresting niches of application for these microorganisms. A field of application isbioelectrochemical remediation, an effective strategy in environments where theabsence of suitable electron acceptors limits classic bioremediation, and in whichbioelectrochemical systems are used for the removal of pollutants fromenvironmental matrices. Bioelectrochemical remediation of hydrocarbons withpure strains and microbial communities has been reported; however, only fewexoelectrogenic hydrocarbonoclastic bacteria have been characterized, so far.The degradative potential of several hydrocarbon-degrading strains has beenextensively studied, in terms of pollutants removal and mechanism of contaminantmineralization, but not much is known about their exoelectrogenic capacity andpossible application for bioelectrochemical remediation. Bioelectrochemistry andits application for bioremediation purposes, has primarily focused on testing thehydrocarbonoclastic capacities of already known exoelectrogenic strains. In thisstudy we took a different approach, and we aimed at studying the exoelectrogenicactivity of three strains that showed great potential for bioremediationapplications: Cupriavidus metallidurans CH34, and Pseudomonas sp. strainsDN34 and DN36. C. metallidurans CH34 is a model metal-resistant strain, whosehydrocarbonoclastic capacities have recently been individuated, andPseudomonas sp. strains DN34 and DN36 that are two hydrocarbon-degradingstrains isolated from an oil-polluted site in central Chile. By analyzing currentproduction, bacterial growth and substrate consumption in bioelectrochemicalsystems (BES), we determined that the three strains possess exoelectrogenic activity. Moreover, C. metallidurans CH34 showed the most promising results witha non-recalcitrant substrate and was selected to assess bioremediationexperiments with toluene as model hydrocarbon. We demonstrated for the firsttime that strain CH34 is able to degrade toluene under denitrifying conditions.Further experiments in Microbial Fuel Cells (MFC) linked toluene degradation tocurrent 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 anodeand cathode to stimulate microbial metabolism of strain CH34 and to observe thebehavior of the strain in terms of toluene removal and current generation. Currentoutputs increased by two orders of magnitude in comparison with MFC (up to 47mA/m2), and coulombic efficiency raised up to 77%, demonstrating that thebacterial cells adjusted progressively to the system conditions and thatelectrochemical losses were, at least partially, overcome. In order to evaluate theeffect of an electron carrier on current production, Neutral Red (NR) was selectedas external transporter and amended in a MEC containing toluene and inoculatedwith strain CH34, but no relevant effect was observed on current production norcoulombic efficiency. Hence, we concluded that NR had no influence on currentgeneration in our system and that a mediated mechanism with this electron carrieris not probable. The mechanism of extracellular electron transport (EET) is a keyfeature in BESs and the efficiency of the microorganism to exchange electronswith an electrode and to connect the EET to the cellular carbon metabolism,significantly influences the overall process performance. We demonstrated thatthe first step of the denitrification pathway is activated by nitrate reductases whenNO3- was the only electron acceptor, but we also aimed at studying whether thepathway of denitrification is still active in absence of nitrate, if a solid the anode ispotentiostetically-polarized at the same redox potential of nitrate reductase. Ourresults indicate that nitrate reductase is not involved in the transport of electronsin BES and that strain CH34 follows a different pathway of electron transport tothe anode. However, current production and cells viability demonstrated thatstrain CH34 was actively performing oxidative phosphorylation, thus that, in a mechanism that has not been elucidated yet, an extracellular electron transfertakes place, either in a direct or indirect way.