Non-invasive characterization of electrochemically active microbial biofilms using confocal Raman microscopy

Electrochemically active biofilms rely on microorganisms capable of extracellular electron transfer. Such biofilms are involved in the dissimilatory reduction of metal oxides in natural environments as well as electricity driving and driven processes at the electrodes of microbial bioelectrochemical systems. In this work we present the application of confocal Raman microscopy (CRM) as a non-invasive, label-free, and in vivo characterization method of acetate oxidizing anodic biofilms, grown from primary wastewater inoculum and dominated by Geobacter species (>85% of sequences analysed using pyrotag sequencing). Using the resonance Raman effect of the heme protein cytochrome c (Cyt c)—an ubiquitous component of extracellular electron transfer reactions—it was possible to collect characteristic spectral information of electrochemically active biofilms at pixel integration times of 0.2 s and an excitation wavelength of 532 nm. This allowed monitoring of biofilm development at different growth stages, without impacting its structural or metabolic activity. Furthermore, we demonstrate the possibility of non-invasive investigation of the spatial redox electrochemistry (up to a compositional level) of electrochemically active biofilms, as we observed significant changes in the vibrational properties of Cyt c resulting from shifts in the anodic potential between different redox conditions. Compared to conventional methods requiring destructive sample manipulation and fixation, the proposed approach based on CRM allows the non-invasive analysis of microbial aggregates with minimal sample preparation or prior knowledge of the sample.