Several techniques have been proposed to characterize the
microbial anode performance, such as: cyclic voltammetry [17],
impedance spectroscopy [19], anode microbial identification [20],
surface-enhanced resonance Raman spectroscopy [21] and confocal
Raman microscopy [22]. However, information on the
physiological characteristics of bacteria is needed, in order to
comprehend how bacteria colonize and maintain viability at the
electrode surface and/or in the anode/cathode compartments.
Multi-parameter flow cytometry is a powerful tool to provide
important physiological information at the individual cell level and
near-real time scale. It has been used to study bacterial population
dynamics [23–25] and evaluate the physiological heterogeneity
of complex mixed microbial consortia, such as activated sludge
[26–28]. The important advantage of using flow cytometry is the
possibility of analyzing a vast amount of cells in a very short period
of time (about 1000 bacteria/s), which allows the gathering of larger
sets of data with more precise information when compared with
conventional microbiology methods (such as staining, followed by
microscope cell counting) used to monitor microbial populations.
Multi-parameter flow cytometry is therefore a robust technique for
bioprocess characterization and optimization, since it allows nearreal
time analysis of cell responses to different process parameters.
Harnisch et al. [29] and Patil et al. [30] reported the use of flow cytometry for the characterization of anode biofilm enrichments of
a bioelectrochemical system and the influence of pH in the anode
biofilm composition.
Bacteria maintain their integrity through the cytoplasmic membrane,
which allows for their communication with its immediate
environment. The existing passive and active transport systems
across the cytoplasmic membrane generate an electrochemical gradient
(cell polarization), which is essential for a fully functional
healthy cell [31]. However, membrane polarization may change due
to an external stimulus. Several fluorescent dyes (such as Propidium
iodide, Thiazole orange or 3,3-dihexyloxacarbocyanine iodide) are
used in combination with flow cytometry analysis for cell detection
and determination of both intact and polarized cell membranes.