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