Electrical Conductivity in a Mixed-Species Biofilm
Nikhil S. Malvankar,a,bJoanne Lau,bKelly P. Nevin,bAshley E. Franks,b* Mark T. Tuominen,aand Derek R. Lovleyb
Department of Physicsaand Department of Microbiology,bUniversity of Massachusetts, Amherst, Massachusetts, USA
Geobacter sulfurreducens can form electrically conductive biofilms, but the potential for conductivity through mixed-species
withlowchargetransferresistanceeventhoughmicroorganismsotherthan Geobacteraceae accountedfornearlyhalfthemicro-
crobe-electrode interactions and bioelectronics (11, 12, 17, 22)
and has revealed the potential for microorganisms to make direct
Most biofilms that have been studied are insulating (1, 2, 17, 20).
The possibility of electrically conductive biofilms was first sug-
duced thick (40 to 50 ?m) biofilms when growing on anode sur-
faces; (ii) biofilm cells not in contact with the anode contributed
to current production as much as cells in direct contact; and (iii)
the production of thick current-producing biofilms was depen-
dent on the presence of conductive pili (25). Subsequent studies
modeling current production in biofilms in which Geobacter spe-
cies predominated found that it was necessary to include an em-
pirically fitted conductivity value in the model in order to accu-
rately predict observed current densities (6, 29).
Direct measurements of conductivity in current-producing
biofilms of Geobacter sulfurreducens revealed high conductivities,
rivaling those of synthetic conducting polymers (17). Multiple
ductivity could be attributed to a network of pili (17). Surpris-
ingly, the pili have metal-like conductivity (17). Metal-like con-
ductivity is a new paradigm for long-range electron transport in
hopping between c-type cytochromes in biofilms, a more tradi-
experimental findings refute the electron-hopping hypothesis
Conductivity through biofilms is essential for high current
organisms not in direct contact with the anode to contribute to
current production (11, 15, 25, 28). Conductive networks may
also make it possible for microorganisms to directly exchange
electrons in syntrophic partnerships (19, 27), which may be a
more efficient mode of syntrophic interaction than interspecies
hydrogen transfer (9).
Electrical conductivity of mixed-species current-producing
biofilms. The anodes of microbial fuel cells generating current
from wastewater or organic matter in aquatic sediments can be
colonized by a diversity of microorganisms (7, 10). In order to
evaluate the conductivity of a mixed-species current-producing
biofilm, an inoculum of anaerobic digester sludge from the Pitts-
field, MA, wastewater treatment plant was prepared as described
he discovery of long-range electron transport through elec-
tronically conductive biofilms offers new possibilities in mi-
(17) “ministack” microbial fuel cells that contained two gold an-
odes (6.45-cm2total geometric area) separated by a 50-?m non-
conducting gap. Anodes were connected by a 560-? load to a
carbon-cloth cathode which was immersed in a 50 mM FeCN
solution. External potential was not applied to the anode for the
MFC operation, ensuring true fuel cell mode. Acetate (10 mM)
37°C. All results were confirmed by repeated measurements on
The production of current in the microbial fuel cells (Fig. 1a)
was associated with the growth of a biofilm that covered the two
anodes and converged, bridging the nonconducting gap (Fig. 1b;
see also Fig. 3a). When electrical conductance across the gap was
measured as previously described (17), there was significant bio-
film conductance (Fig. 1c). Biofilm conductivity (Fig. 1d), calcu-
lated with conformal mapping as previously described (17), was
comparable to that previously reported for current-producing
biofilms of strain KN400 (17) (see supplemental material for de-
tails). As previously described, the effluent from the anode cham-
ber was passed to another chamber which was identical with the
exception that the two gold electrodes were not connected to the
cathode (17). No biofilm grew in the control chamber, and con-
ductance between the two electrodes was low (Fig. 1c). The dem-
onstrated high electrical conductivity of mixed-species-de-
rived biofilms provides an explanation for their capacity for
high current densities (0.9 ? 0.45 A/m2) comparable to those
obtained with G. sulfurreducens biofilms grown in the same
type of ministack microbial fuel cells (0.7 A/m2) under similar
conditions (17, 21).
Charge transfer resistance. Charge transfer resistance repre-
sents an energy barrier at the electrode interface (15, 28). In addi-
tion to promoting long-range electron transport through bio-
films, high biofilm conductivity can lower the charge transfer
resistance (Rct) at the biofilm/anode interface because electrons
Received 3 June 2012 Accepted 4 June 2012
Published ahead of print 15 June 2012
Address correspondence to Nikhil S. Malvankar, firstname.lastname@example.org.
*Present address: Ashley E. Franks, Department of Microbiology, La Trobe
University, Bundoora, Victoria, Australia.
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 78 Number 16 Applied and Environmental Microbiologyp. 5967–5971aem.asm.org
film with higher conductivity have greater energy than electrons
transported through biofilms of lower conductivity (15, 28). This
higher energy reduces the energy barrier at the biofilm/anode in-
terface that lowers the charge transfer resistance. This possibility
was evaluated by measuring the charge transfer resistance using
electrochemical impedance spectroscopy (15, 23). In this config-
uration, the two sides of the split anode were connected and used
as the working electrode. The reference electrode was Ag/AgCl,
laser scanning microscopy image (three-dimensional [3D] reconstruction) of mixed-species biofilm showing that the biofilm bridged the nonconductive gap. Gap is
charge transfer resistance of mixed-species biofilm and that of corresponding control. Error bars represent standard deviations.
Malvankar et al.
aem.asm.org Applied and Environmental Microbiology
FIG 3 (a) Schematic of mixed-species biofilm formation. (b) Image of outer, top-layered brownish biofilm that is loosely attached to the anode. Bar, 1 cm. (c)
Image of inner, bottom-layered pinkish biofilm that is strongly attached to the anode. Bar, 1 cm. (d) Community analysis of wastewater inoculum as well as of
the inner and outer biofilms. Left: charts show the division based on phyla. Right: charts show the division based on species.
Mixed-Species Conductive Biofilms
August 2012 Volume 78 Number 16aem.asm.org 5969
placed in the anode chamber, and the counter-electrode was a
carbon cloth, placed in the cathode chamber. The anode was dis-
connected from the cathode, and all of the impedance measure-
ments were performed at the open-circuit potential of the anode
mixed-species and G. sulfurreducens biofilms, the amplitude exci-
tation was 0.1 V of alternating current (ac) (4, 5). Linearity of the
range of 0.001 V to 0.1 V at the open-circuit potential (see Fig. S2
in the supplemental material). The charge transfer resistance was
evaluated from the measured impedance spectra by fitting (see
Table S1 in the supplemental material) the previously described
tal material). As expected, the charge transfer resistance of the
mixed-species biofilms was much lower than that seen in unin-
oculated controls (Fig. 2a; see also Fig. S4 in the supplemental
measurements of charge transfer resistance made under similar
of the mature mixed-species biofilms grown on gold electrodes
trodes (24) but comparable with that seen with biofilms of G.
sulfurreducens strain KN400 (1.1 k?·cm2) and much lower than
the 204 k?·cm2reported for biofilms of Shewanella oneidensis
Community analysis. Current-producing mixed-species bio-
films had two distinct layers (Fig. 3a)—a top, outer brown layer
that was loosely attached to the anode (Fig. 3b) and a bottom,
inner pink layer that was strongly attached to the anode (Fig. 3c).
In order to identify the microorganisms which were conferring
conductivity to mixed-species biofilms, clone libraries of 16S
for the inner pinkish biofilm, which was closely attached to the
electrode, and for the outer brownish biofilm, which was loosely
attached to the electrode. At day 54, the outer and inner layers of
the biofilm were individually sampled with a micropipette for
community analysis. As previously described (3, 19), genomic
DNA was extracted and 16S rRNA gene sequences were amplified
cedure for community analysis is provided in the supplemental
material, and the results are presented in Fig. 3c. The proportion
of Geobacteraceae in the initial inoculum was 8%, whereas the
proportions of Geobacteraceae in the inner and the outer biofilm
zones were ca. 50% and 10%, respectively.
Implications. The finding that mixed-species biofilms can
possess electrical conductivity comparable to that of pure culture
biofilms of G. sulfurreducens with low charge transfer resistance
to produce the thick biofilms necessary for high current densities.
Modeling studies have previously demonstrated that invoking a
highly conductive biofilm could explain the effective function of
high-current-density multispecies biofilms in which Geobacter
species predominated (6, 29, 30). The conductivity of the mixed-
species biofilms was an order of magnitude higher than that of
multispecies methanogenic aggregates derived from wastewater
for higher conductivity in current-producing biofilms than in the
previously described conductive aggregates, because electrons re-
leased from microorganisms near the outer surface of current-
producing biofilms need to be transported much farther than in
cell aggregates, in which electrons need to be transported only to
It is not possible to determine from the data available whether
microorganisms other than Geobacter species contributed to the
coli and Pseudomonas aeruginosa grown on the two-electrode de-
biofilms were also found to have poor conductivity (1, 2, 20).
Other current-producing microorganisms such as Shewanella
oneidensis (8) and Thermincola strain JR (31) do not form thick
ble of forming highly conductive biofilms. Thus, these results in-
dicate that biofilms containing high proportions of organisms
other than Geobacter species may be conductive, but whether the
other organisms contribute to biofilm conductivity warrants fur-
This research was supported by the Office of Naval Research (grant no.
N00014-10-1-0084 and N00014-12-1-0229), the Office of Science (BER),
and the U.S. Department of Energy (award no. DE-SC0004114 and Co-
for Hierarchical Manufacturing (grant no. CMMI-1025020).
We thank Trevor Woodard for assistance with wastewater sludge and
Pravin Shrestha for help with community analysis.
1. Dheilly A, et al. 2008. Monitoring of microbial adhesion and biofilm
growth using electrochemical impedancemetry. Appl. Microbiol. Bio-
2. Herbert-Guillou D, Tribollet B, Festy D, Kiéné L. 1999. In situ detection
and characterization of biofilm in waters by electrochemical methods.
Electrochim. Acta 45:1067–1075.
3. Holmes DE, Nevin KP, Woodard TL, Peacock AD, Lovley DR. 2007.
Prolixibacter bellariivorans gen. nov., sp. nov., a sugar-fermenting, psy-
chrotolerant anaerobe of the phylum Bacteroidetes, isolated from a ma-
rine-sediment fuel cell. Int. J. Syst. Evol. Microbiol. 57(Pt 4):701–707.
4. Hong J, et al. 2005. AC frequency characteristics of coplanar impedance
sensors as design parameters. Lab Chip 5:270–279.
5. Hong J, et al. 2004. A dielectric biosensor using the capacitance change
with AC frequency integarated on glass substrates. Jpn. J. Appl. Phys.
6. Kato Marcus A, Torres CI, Rittmann BE. 2007. Conduction-based
7. Kiely PD, Regan JM, Logan BE. 2011. The electric picnic: synergistic
requirements for exoelectrogenic microbial communities. Curr. Opin.
8. Lanthier M, Gregory KB, Lovley DR. 2008. Growth with high planktonic
biomass in Shewanella oneidensis fuel cells. FEMS Microbiology Lett. 278:
9. Lovley D. 2011. Reach out and touch someone: potential impact of DIET
mediation, and bioenergy. Rev. Environ. Sci. Biotechnol. 10:101–105.
10. Lovley DR. 2006. Bug juice: harvesting electricity with microorganisms.
Nat. Rev. Microbiol. 4:497–508.
11. Lovley DR. 2011. Live wires: direct extracellular electron exchange for
bioenergy and the bioremediation of energy-related contamination. En-
ergy Environ. Sci. 4:4896–4906.
12. Lovley DR. 2011. Powering microbes with electricity: direct electron
transfer from electrodes to microbes. Environ. Microbiol. Rep. 3:27–35.
13. Malvankar NS, Lovley DR. 2012. Microbial nanowires: a new paradigm
Malvankar et al.
aem.asm.org Applied and Environmental Microbiology
for biological electron transfer and bioelectronics. ChemSusChem Download full-text
14. Malvankar NS, Mester T, Tuominen MT, Lovley DR. 2012. Superca-
pacitors based on c-type cytochromes using conductive nanostructured
networks of living bacteria. ChemPhysChem 13:463–468.
15. Malvankar NS, Tuominen MT, Lovley DR. 2012. Biofilm conductivity is
a decisive variable for high-current-density microbial fuel cells. Energy
Environ. Sci. 5:5790–5797.
16. Malvankar NS, Tuominen MT, Lovley DR. 2012. Comment on “On
electrical conductivity of microbial nanowires and biofilms” by S. M.
Strycharz-Glaven, R. M. Snider, A. Guiseppi-Elie, L. M. Tender, Energy
Environ. Sci. 2011, 4, 4366. Energy Environ. Sci. 5:6247–6249.
17. Malvankar NS, et al. 2011. Tunable metallic-like conductivity in micro-
bial nanowire networks. Nat. Nanotechnol. 6:573–579.
18. Manohar AK, Bretschger O, Nealson KH, Mansfeld F. 2008. The use of
electrochemical impedance spectroscopy (EIS) in the evaluation of the
electrochemical properties of a microbial fuel cell. Bioelectrochemistry
19. Morita M, et al. 2011. Potential for direct interspecies electron transfer in
methanogenic wastewater digester aggregates. mBio 2:e00159–11. doi:
20. Muñoz-Berbel X, Muñoz FJ, Vigués N, Mas J. 2006. On-chip impedance
measurements to monitor biofilm formation in the drinking water distri-
bution network. Sens. Actuators B Chem. 118:129–134.
21. Nevin KP, et al. 2008. Power output and coulombic efficiencies from
crobial fuel cells. Environ. Microbiol. 10:2505–2514.
22. Qian F, Li Y. 2011. Biomaterials: a natural source of nanowires. Nat.
23. Rabaey K, Angenent L, Schröder U, Keller J (ed). 2009. Bioelectro-
chemical systems: from extracellular electron transfer to biotechnological
application. International Water Association, London, United Kingdom.
24. Ramasamy RP, Ren Z, Mench MM, Regan JM. 2008. Impact of initial
biofilm growth on the anode impedance of microbial fuel cells. Biotech-
nol. Bioeng. 101:101–108.
25. Reguera G, et al. 2006. Biofilm and nanowire production leads to in-
creased current in Geobacter sulfurreducens fuel cells. Appl. Environ. Mi-
26. Strycharz-Glaven SM, Snider RM, Guiseppi-Elie A, Tender LM. 2011.
On the electrical conductivity of microbial nanowires and biofilms. En-
ergy Environ. Sci. 4:4366–4379.
27. Summers ZM, et al. 2010. Direct exchange of electrons within aggregates
of an evolved syntrophic coculture of anaerobic bacteria. Science 330:
28. Torres CI, et al. 2010. A kinetic perspective on extracellular electron
transfer by anode-respiring bacteria. FEMS Microbiol. Rev. 34:3–17.
29. Torres CI, Marcus AK, Parameswaran P, Rittmann BE. 2008. Kinetic
experiments for evaluating the Nernst-Monod model for anode-
respiring bacteria (ARB) in a biofilm anode. Environ. Sci. Technol.
30. Torres CI, et al. 2009. Selecting anode-respiring bacteria based on anode
potential: phylogenetic, electrochemical, and microscopic characteriza-
tion. Environ. Sci. Technol. 43:9519–9524.
31. Wrighton KC, et al. 2008. A novel ecological role of the Firmicutes
identified in thermophilic microbial fuel cells. ISME J. 2:1146–1156.
Mixed-Species Conductive Biofilms
August 2012 Volume 78 Number 16 aem.asm.org 5971