4ISMET, Melanie PIERRA

A L I M E N T A T I O N

A G R I C U L T U R E

E N V I R O N N E M E N T

Successful enrichment procedure for enhancing electron transfer in

electroactive biofilms

PIERRA Mélanie, TRABLY Eric, GODON Jean-Jacques, BERNET Nicolas.

4th International Microbial Fuel Cell Conference 1st - 4th September 2013 - Cairns, Queensland, Australia

.02

Electroactive biofilm

Rozendal et al., 2006 Int J Hydrog Energy, 31(12), pp.1632–1640. Rabaey & Verstraete, 2005. Trends Biotechnol, 23(6), pp.291–298. Liu et al., 2010. Biofuels, 1(1), pp.129–142.

Electro-active bacteria

are able to transfer

electrons to an insoluble

and external electron

acceptor.

MFC

MEC

etc…

MXC’s

Bioelectrochemical

Systems

(BES)

Anode

CxHyOz

CO2

e-

e-

H2

CH4

…

.03

Food Industry

Fish and seafood

Slaughterhouses,

salting

Dairy industry

Brined

vegetables

Petroleum Industry

Raffinerie

Chemical and

pharmaceutical industry

Saline pollutions in Industry

Lefebvre, O. et al, Water Res. 2006. 40: p. 3671-3682; Xiao, Y. et al, Environ. Technol. 2010. 31 (8-9): p. 1025-1043 3

Leather

Industry

Textile

Industry

.04

Food Industry

Fish and seafood

Slaughterhouses,

salting

Dairy industry

Brined

vegetables

Petroleum Industry

Raffinerie

Chemical and



Lefebvre, O. et al, Water Res. 2006. 40: p. 3671-3682; Xiao, Y. et al, Environ. Technol. 2010. 31 (8-9): p. 1025-1043 4

Leather

Industry

Textile

Industry

Industries generating saline effluents:

5% of worldwide effluents

Lefebvre et al., 2012 Bioresource technology, 112, pp.336–40

.05

Food Industry

Fish and seafood

Slaughterhouses,

salting

Dairy industry

Brined

vegetables

Petroleum Industry

Raffinerie

Chemical and



5

Leather

Industry

Textile

Industry

saline conditions => good conductivity in the anodic chamber => good charge transport

Lefebvre, O. et al, Water Res. 2006. 40: p. 3671-3682; Xiao, Y. et al, Environ. Technol. 2010. 31 (8-9): p. 1025-1043

Lefebvre et al., 2012 Bioresource technology, 112, pp.336–40 Rousseau et al., 2013. Electrochemistry Communications, 33, pp.1–4.

.06

Sources of Electroactive bacteria

Lefebvre et al, 2010. Applied microbiology and biotechnology. Chae et al., 2009. 100(14), pp.3518–3525. Harnisch et al., 2011. Energy & Environmental Science, 4(4), p.1265 Miceli et al., 2012. Environmental science & technology, 46(18), pp.10349–55.

Various sources of

electroactive bacteria

High variability in the

performances of

biofilm communities

[µA/m²-15 A/m²]

• Freshwater and marine sediments

• Salt marsh

• Anaerobic Sludge

• Wastewater treatment plants

• Mangrove swamp sediments

Mix of vinasse,

compost and soil :

0,2 A/m²

Soil :

3,92 A/m²

Salt marsh

sediments :

15,27 ± 1,76 A/m² Marine sediments :

7,19 ± 3,33 A/m²

Need to use a reliable

enrichment technique

.07

Enrichment to enhance biofilm formation and performance

Wang et al., 2010. Bioresource technology, 101(14), pp.5733–5735 Lovley, 2006. Nat Rev Microbiol, 4(7), pp.497–508. Nevin et al., 2008. Environ Microbiol, 10(10), pp.2505–14. Miceli et al., 2012. Environmental science & technology, 46(18), pp.10349–55. Kim et al., 1999. Microbiology and Biotechnology, 9(2), pp.127–131.

• Most of the known electroactive bacteria are dissimilatory metal

reducing bacteria (Shewanella putrefaciens, Geobacter spp,

Desulfuromonas spp)

• Most of the inoculating strategies consist in the re-use of electroactive

biofilm to inoculate new electrode in a BES system

• This study aims to develop an enrichment method to select

microorganisms which can use solid iron oxides as electron acceptor to

inoculate BES systems

Anode

CxHyOz

CO2

e-

Fe(III) oxides

CxHyOz

CO2

e-

.08

Experimental Design

Wang et al., 2010. Bioresource technology, 101(14), pp.5733–5735 Lovley & Phillips 1986. Applied and environmental microbiology, 51(4), pp.683–689.

Work

ing

ele

ctr

ode

Refe

rence e

lectr

ode

Counte

r ele

ctr

ode

U

I

0.2V vs SCE

Anode (Working-electrode) : graphite

Cathode (Counter-electrode) : platinium

Reference electrode : Hg/Hg2Cl2/Cl- SCE

3 electrodes system (Half cell MEC)

Inoculum : Salt marsh sediments

Substrate : Acetate (10 mM)

Initial pH : 7

Temperature : 37°C

Salinity : 35gNaCl/L

Enrichment culture

Electron acceptor :

Fe(III) oxides

Electron acceptor :

Graphite electrode

U

I I= f(t)

.09

Experimental Design

Wang et al., 2010. Bioresource technology, 101(14), pp.5733–5735 Lovley & Phillips 1986. Applied and environmental microbiology, 51(4), pp.683–689.

Work

ing

ele

ctr

ode

Refe

rence e

lectr

ode

Counte

r ele

ctr

ode

U

I

0.2V vs SCE

3 electrodes system (Half cell MEC)

Inoculum : Salt marsh sediments

Substrate : Acetate (10 mM)

Initial pH : 7

Temperature : 37°C

Salinity : 35gNaCl/L

Enrichment culture

Electron acceptor :

Fe(III) oxides

Electron acceptor :

Graphite electrode

U

I I= f(t)

.010

Materials & Methods

𝑄𝑚𝑎𝑥 𝐶 = 𝑖 𝑡 𝑑𝑡

Anode

CxHyOz

CO2

e-

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40

J(A

:m²)

time (days)

0

500

1 000

1 500

2 000

2 500

3 000

3 500

4 000

4 500

0 10 20 30 40

Q(C

)

time (days)

𝐶𝐸 = 𝑛𝑒−𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑒𝑑

𝑛𝑒−𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑓𝑟𝑜𝑚 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 Lag Phase

Qmax Charge transmitted :

Coulombic efficiency :

.011

Materials & Methods

4x

4x

Sediments

4x

4x

Effect of the enrichment culture stages on :

• bioelectrochemical performance

• electroactive biofilm community structure

E1

E2

E3

B0

B1

B2

B3

.012

Materials & Methods Genomic DNA, PCR-SSCP and pyrosequencing

•SSCP = Fingerprinting technique

•1 species => 1 peak

•Area under the peak => abundance of the species in the

microbial community

Elution time

Species 1

Flu

ore

scen

ce

inte

nsity

Species 2

CE-SSCP profile

Microbial

fingerprinting Removal of

Biofilm

Centrifugation of

liquid culture Pyrosequencing

0

5

10

15

20

25

30

35

40

Bacterial communities Re

lative

ab

un

da

nce (

%)

1 2 3 4



E N V I R O N N E M E N T .013

1 enrichment step :

Increase of the

coulombic efficiency

from 30,4±4% to

99±7% was shown

Electron transfer efficiency

Increase of Lag phase

Efficient electroactive

biofilm growth:

From 1,6 to 4,5 A/m²

obtained

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

B0 B1 B2 B3

Jmax

(A

/m²)

Enrichment biofilm step

Jmax (A/m²)

Lag Phase (d)

Lag

Ph

ase

(d

ays)

0%

20%

40%

60%

80%

100%

120%

140%

B0 B1 B2 B3

Co

ulo

mb

ic e

ffic

ien

cy (

%)




Microbial communities: structure

4x

4x Sediments

4x

4x E1

E2

E3

B0

B1

B2

B3




Microbial communities: structure

Sediments

Similar microbial structure

(1 or 2 most abundant

species as electroactive

bacteria)

High simplification of

microbial diversity

SSCP patterns

E1

E2

E3

B0

B1

B2

B3




Increase of Lag

Phase concurs with

the emergence of

Marinobacterium sp

0

5

10

15

20

25

30

35

0

1

2

3

4

5

6

B0 B1 B2 B3

Jmax

(A

/m²)

Enrichment biofilm step

Jmax (A/m²)

Lag Phase (d)

Lag

Ph

ase

(d

ays)

0%

20%

40%

60%

80%

100%

120%

140%

0

20

40

60

80

100

B0 B1 B2 B3

Re

lati

ve a

bu

nd

ance

(%

)

Co

ulo

mb

ic e

ffic

ien

cy (

%)

Microbial communities: structure Most abundant species

vs electroactive performance

Electroactive activity

of biofilm is

enhanced from the

first enrichment

culture due to the

selection of

Geoalkalibacter

subterraneus




Biofilms Enrichments

Microbial communities: structure • Liquid enrichment cultures : Geobacteraceae

• Biofilms : Geobacteraceae and Marinobacterium sp.

• Liquid enrichment procedure permits the selection of efficient

electroactive strain (of Geobacteraceae) from the first enrichment step




PCA on Enrichment and Biofilm Microbial Community profiles

BF3

-0.2 -0.1 0.0 0.1 0.2

-0.2

-0

.1

0.0

0

.1

Axis 1 - 37.1%

Ax

is 2

- 2

4.0

%

BF1

BF2

Sediment

Sediment BF

E1

E2

E3

Optimal performance is

obtained from

enrichment and biofilm

converging community

profiles

Lag Phase increases

from enrichment and

biofilm divergent

community profiles

Principal Component Analysis

Easier adhesion of

electroactive bacteria




Conclusions

• A successful enrichment strategy

• With only 1 step required

• Enrichment of Geoalkalibacter subterraneus

• After 3rd enrichment step

o Divergence of species selected

o Decrease of electroactive performance

.020

Acknowledgments

Nicolas BERNET Eric TRABLY Jean Jaques GODON Anais BONNAFOUS Alessandro CARMONA Mohanakrishna GUNDA

Documents

4ISMET, Melanie PIERRA