Thesis submitted for the degree of Doctor of Philosophy (Ph.D.)
Dublin City University
School of Biotechnology
Shewanella loihica PV-4 electroactive biofilms (EABs) grown in
potentiostat-controlled electrochemical cells
by
Monica Epifanio (M.Sc. in Industrial Biotechnology)
Under the supervision of
Dr. Brendan O'Connor
Dr. Enrico Marsili
Dr. Ciaran Fegan
3th September 2015
Shewanella loihica PV-4
Polar flagella
S.loihica PV-4 biofilm
• Facultative anaerobes
• Gram negative
• Rod like bacterium
• Motile by polar flagella
• Incredible respiratory versatility
in anoxic condition
(e- acceptors: Fe (III) , Mn (IV)…)
• Biofilm forming bacterium
Flexible use of varied number of
electron acceptors
Bio-energy production
Bioremediation
Shewanella loihica PV-4
Extracellular Electron Transfer (EET)
MEDIATED EET
Phospholipid bilayer is not a barrier!
Extracellular Electron Transfer
(EET)
Shi et al.,2012
Porine
C-type cytochrome
Porine
Extracellular Electron Transfer
(EET)
MEDIATED EET S.loihica PV-4 nanowires
Reduction can occur at a distance up to 50 µm
Electron shuttles:
Quinones
Flavins
SHEWANELLA SP. GENUS
SHEWANELLA SP. GENUS
Although all members of the genus have the presence of c-type
cytochromes, the number and order of their genes varies and this might
affect their ability to reduce external insoluble substrates
∆omcA= current density in in both S.loihica and
S.oneidensis
∆MtrC= current density in MR-1
current density in PV-4
(MAIN PATH OF THE e- TOWARD THE ANODE)
S. loihica PV-4 S. oneidensis MR-1
Newton et. al 2009
RATIONALE OF THE STUDY
TO ELECTROCHEMICALLY CHARACTERIZE S. LOIHICA
PV-4 EXTERNAL ELECTRON TRANSFER
System design
parameters
Electrode material
Electrode surface area
Electrode surface hydrophilicity
Electrode Porosity
Biological parameters
Type of microorganisms
Capability to perform DET
Growth condition (presence or absence of
oxygen, nutrient concentration…)
Operating parameters
Aerobic vs anaerobic
Batch vs continuous flow
EET MECHANISM
BIOMASS GROWTH
RATE
BIOFILM THICKNESS
What does influence the performance of EAB in an
Electrochemical System?
INVESTIGATION OF BIOLOGICAL PARAMETERS:
I. Effect of lactate concentration on S. loihica PV-4 electroactivity
II. Effect of different electron acceptors during S. loihica PV-4 growth
on its electroactivity
OBJECTIVES OF THE STUDY
INVESTIGATION OF SYSTEM DESIGN PARAMETERS:
I. Effect of electrode surface abrasion and functionalization on S.
loihica PV-4 electroactivity
II. Effect of electrode surface coating on S. loihica PV-4 electroactivity
INVESTIGATION OF BIOLOGICAL PARAMETERS
Effect of lactate concentration on S. loihica PV-4
electroactivity
STATE OF THE ART
- Current generation is proportional with lactate concentration in S.
oneidensis (Kim et al.,1999);
- Multiple lactate feedings, generates higher current in S. oneidensis
(Biffinger et al.,2009).
Does S. loihica PV4 behave similarly?
Effect of lactate concentration on S.loihica PV-4
electroactivity
Effect of lactate concentration on S.loihica PV-4
electroactivity
Riboflavin 106 ± 5 µAcm-2
Stable biofilm formed at the
electrode
MET
Current generation is proportional with lactate concentration in
S.lohica PV4
However further increase in [lactate] from 20 mM to 40 mM probably
leads to a plateau of RF production
20 mM yielded higher current density and improved reaction
reversibility
CONCLUSIONS
INVESTIGATION OF SYSTEM DESIGN PARAMETERS:
Related publication
Monica Epifanio, Saikumar Inguva, Michael Kitching, Jean-Paul Mosnier, Enrico Marsili (2015)
Effects of atmospheric air plasma treatment of graphite and carbon felt electrodes on the anodic
current from Shewanella attached cells; Bioelectrochemistry (in press)
Effect of electrode surface abrasion and
functionalization on S. loihica PV-4 electroactivity
STATE OF THE ART
Surface chemistry and topography of the electrode material
determines the EET mechanism in EABs (Brutinel et al., 2012)
Atmospheric plasma treatment increases surface roughness and
bacterial adhesion due to the more hydrophilic surface generated
(Zaldivar et al., 2010)
In G.sulfureducens a higher electrode surface area resulted in
greater current generation, by the formation of a thicker biofilm, thus
enhancing the DET(Brutinel et al., 2007)
Would a rougher surface bring a greater current
density ? Would it be achieved either through thicker biofilm
formation (DET) or through higher electron shuttle
release/absorption (MET) at the interface
biofilm/electrode?
P240 P400 P600
CARBON
FELT GRAPHITE ATMOSPHERIC AIR
PLASMA DM medium
WASHED VS
UNWASHED
Connolly et al., 2012
Effect of electrode surface abrasion on S.loihica PV-4
electroactivity
P240
plasma
P20 P400
P600
Surface abrasion effects:
Higher current density in P240 by
higher biofilm coverage
Lag phase 600>400>240
104 ± 9 μA cm−2 P600 P600
P400
P600
P400
P240 P240
P400
P600
Functionalization effects:
Increasing of lag-phase
Overall current density wasn’t really
affected
Current slope increased by ~45%
P600
Effect of electrode surface functionalization on
S.loihica PV-4 electroactivity
Untreated Treated
Washed
- P240U
- P240W
- P240U plasma
- P240W plasma
- CFU
- CFW
- CFU plasma
- CFW plasma
Washed
Washed Washed
Unwashed Unwashed
Unwashed Unwashed
1) 2) 3) CF
CFP
VERY HYDROPHOBIC
VERY HYDROPHILIC
Presence of e- shuttles Air plasma treatment
(1) Current density
Coulombic efficency
+
+
+/-
-
(2-3) Biofilm attachment + +
~ 450%
4)
Nude electrodes
Presence of e- shuttles Air plasma treatment
(4) RF absorption + +
(5) Resistance
(EIS) - +/-
5)
Plasma pre-treatment is a feasible option to increase power output in
bioelectrochemical systems
However the effects of plasma pre-treatment are not all beneficial and the interplay
between DET and MET must be considered when designing optimal electrode pre-
treatment.
CONCLUSIONS
Effect of electrode surface coating on S.loihica PV-4
electroactivity
INVESTIGATION OF SYSTEM DESIGN PARAMETERS:
Related publication
Zhang Xiaoming, Epifanio Monica, Marsili Enrico (2013)
Electrochemical characteristics of Shewanella loihica on carbon nanotubes-modified graphite
surfaces. Electrochimica Acta 102, 252-258.
STATE OF THE ART
CNTs coating on graphite fibers gave 54% higher current
density than the unmodified GF electrodes (Y.Shen et
al.,2014)
CNTs coating on glassy carbon, gave 82 times higher current
density in S.oneidensis biofilms, than unmodified electrodes
(Peng et al. 2010)
To manipulate CNTs individually or collectively
To disperse CNTs homogeneously
Main challenge
Effect of electrode surface coating on
S.loihica PV-4 electroactivity
high deposition rate
excellent uniformity
controlled thickness
Electrophoresis deposition (EPD)
Height of oxidation and
reduction peaks increased
proportionally with the
number of CNT deposition
steps (0-8)
The peak current increased (0-
8) and the peak separation
decreased (0-8)indicating that
the redox reaction is more
reversible (Fe(CN)6 3−/4− )
Effect of electrode surface coating on S.
loihica PV-4 electroactivity
0 2 4 6 8 10 120.16
0.18
0.20
0.22
0.24
0.26
Ep
(vs.
SC
E)/
V
numbers of Layers
Epa
Epc
0 2 4 6 8 10 12-1200
-600
0
600
1200
Ip/
A
numbers of layers
Ipa
Ipc
|Ipc|
K3[Fe(CN)6]3−
CNT-modified electrode
Shorter lag phase
Much higher current density
However, lower e- shuttle absorption (current
drop after medium replacement)
Higher biofilm attachment
EET facilitated by CNTs coating
10 μm
D CNT8
PLAIN
Plain
CNT- coated
electrode
DET - 0.15 V - 0.10 V
MET -0.38 V - 0.38 V
MET -0.6 V - 0.6 V
Effect of electrode surface coating on
S.loihica PV-4 electroactivity
S. loihica PV-4 grown on CNT8-graphite electrodes developed
faster and showed a higher EET rate than on plain electrodes
CNT layers facilitate both DET and MET
CONCLUSIONS
The work undertaken in this research project demonstrates that
the performance of electroactive bacteria such as S. loihica can
be enhanced by optimizing culture conditions and working
electrode characteristics.
SUMMARY
Acknowledgment
Dr. Ciaran Fegan Dr. Brendan
O'Connor Dr. Enrico Marsili
Dr. Emanuele Barborini Dr. Marc Bauman Saikuma Inguva Michael Kitching Triona O’Connell