research plan. Finally, we discuss the implications of microscale gradients in electrochemically active biofilms. In this chapter, a proposed mechanism for longdistance electron transport that occurs over distances that can exceed 20 μm within
electrochemically active biofilms comprised of Geobacter sulfurreducens wild-type
strain DL-1 is described. According to this mechanism, referred to as redox conduction, long-distance electron transport results from sequential short-distance electron
transfer reactions (“electron hops”) between adjacent redox cofactors distributed
throughout the biofilm that act as electron transport conduits. The general approach
to investigate the mechanism of electron transport through a material is to place the
material between two electrodes and measure the dependency of the rate of electron
transport through the material from one electrode to the other, in the form of
electrical current, on the potentials applied to the electrodes. Materials utilizing
different mechanisms of electron transport exhibit different current–potential
dependencies (Dominguez-Benetton et al. 2012). Derivation of the idealized
current–potential dependency for a G. sulfurreducens biofilm based on redox conduction is described here, which has been successfully applied to fit experimental
results. General methodology is also described for performing biofilm electron
transport rate measurements in the laboratory. The goal of this chapter is to describe
redox conduction and experimental methods to enable researchers to perform electron transport rate measurements for their own types of biofilms. Although biofilms
of most microorganisms are electronic insulators, biofilms of pure culture Geobacter
sulfurreducens as well as mixed species derived from wastewater, when grown on
the anodes of microbial fuel cell, are electronically conductive. Remarkably, the
electronic conductivity of biofilms is comparable to synthetic conducting polymers
(Dominguez-Benetton et al. 2012). Direct conductivity measurements have
demonstrated that the biofilm conductivity can be attributed to the network of pili
filaments known as microbial nanowires. Surprisingly, the conductivity of pili is
metallic-like rather than previously known methods of electron flow in proteins
using redox-active cofactors such as c-type cytochromes (Ghach et al. 2014).
Electronic conductivity enables microorganisms to access electron acceptors that
are many cell lengths away. In addition, biologically produced conductive films,
which can be synthesized from inexpensive feedstocks and when alive, can selfrepair and replicate, introduce new concepts and materials for bioelectronics. This
chapter summarizes the methods used to directly measure the newly discovered
conductive properties of biofilms. Firstly, we describe the physical meaning of
electronic conductivity (Logan et al. 2006). Then, we discuss three different
mechanisms of conductivity in materials—tunneling, hopping, and delocalization.
Later, we discuss in detail the experimental methods applied to directly measure
conductivity in living biofilms and the results obtained using these methods. We start
with four-probe method widely used to measure DC electronic conductivity of
materials. Then, we describe a complementary and independent method using
two-probe AC impedance spectroscopy to confirm biofilm conductivity and to
further distinguish between electronic and ionic conductivity. We summarize the
results from different organisms that reveal that biofilm conductivity depends on cell
type and physiology. Finally, we discuss two key physical probes—temperature and
110
S. Sevda et al.
electrochemically active biofilms comprised of Geobacter sulfurreducens wild-type
strain DL-1 is described. According to this mechanism, referred to as redox conduction, long-distance electron transport results from sequential short-distance electron
transfer reactions (“electron hops”) between adjacent redox cofactors distributed
throughout the biofilm that act as electron transport conduits. The general approach
to investigate the mechanism of electron transport through a material is to place the
material between two electrodes and measure the dependency of the rate of electron
transport through the material from one electrode to the other, in the form of
electrical current, on the potentials applied to the electrodes. Materials utilizing
different mechanisms of electron transport exhibit different current–potential
dependencies (Dominguez-Benetton et al. 2012). Derivation of the idealized
current–potential dependency for a G. sulfurreducens biofilm based on redox conduction is described here, which has been successfully applied to fit experimental
results. General methodology is also described for performing biofilm electron
transport rate measurements in the laboratory. The goal of this chapter is to describe
redox conduction and experimental methods to enable researchers to perform electron transport rate measurements for their own types of biofilms. Although biofilms
of most microorganisms are electronic insulators, biofilms of pure culture Geobacter
sulfurreducens as well as mixed species derived from wastewater, when grown on
the anodes of microbial fuel cell, are electronically conductive. Remarkably, the
electronic conductivity of biofilms is comparable to synthetic conducting polymers
(Dominguez-Benetton et al. 2012). Direct conductivity measurements have
demonstrated that the biofilm conductivity can be attributed to the network of pili
filaments known as microbial nanowires. Surprisingly, the conductivity of pili is
metallic-like rather than previously known methods of electron flow in proteins
using redox-active cofactors such as c-type cytochromes (Ghach et al. 2014).
Electronic conductivity enables microorganisms to access electron acceptors that
are many cell lengths away. In addition, biologically produced conductive films,
which can be synthesized from inexpensive feedstocks and when alive, can selfrepair and replicate, introduce new concepts and materials for bioelectronics. This
chapter summarizes the methods used to directly measure the newly discovered
conductive properties of biofilms. Firstly, we describe the physical meaning of
electronic conductivity (Logan et al. 2006). Then, we discuss three different
mechanisms of conductivity in materials—tunneling, hopping, and delocalization.
Later, we discuss in detail the experimental methods applied to directly measure
conductivity in living biofilms and the results obtained using these methods. We start
with four-probe method widely used to measure DC electronic conductivity of
materials. Then, we describe a complementary and independent method using
two-probe AC impedance spectroscopy to confirm biofilm conductivity and to
further distinguish between electronic and ionic conductivity. We summarize the
results from different organisms that reveal that biofilm conductivity depends on cell
type and physiology. Finally, we discuss two key physical probes—temperature and
110
S. Sevda et al.