PIERRA M., TRABLY E., GODON J.J., BERNET N. Successful enrichment procedure for electroactive biofilm formation from environmental sample. MFC4 / 4th International Microbial Fuel Cell Conference, September 1st - 4th, 2013, Cairns, Queensland, AUSTRALIA

1. 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

2. .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
…

3. .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

4. .04
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
4
Leather Industry
Textile Industry
Industries generating saline effluents:
5% of worldwide effluents
Lefebvre et al., 2012 Bioresource technology, 112, pp.336–40

5. .05
Food Industry
Fish and seafood
Slaughterhouses, salting
Dairy industry
Brined vegetables
Petroleum Industry
Raffinerie
Chemical and pharmaceutical industry
Saline pollutions in Industry
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.

6. .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

7. .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-

8. .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.
Working electrode
Reference electrode
Counter electrode
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)
U
I I= f(t)

9. .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.
Working electrode
Reference electrode
Counter electrode
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
U
I I= f(t)

10. .010
Materials & Methods
푄푚푎푥 퐶 = 푖 푡 푑푡
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)
퐶퐸 =
푛푒−푡푟푎푛푠푓푒푟푒푑
푛푒−푡ℎ푒표푟푒푡푖푐푎푙 푓푟표푚 푠푢푏푠푡푟푎푡푒
Lag Phase
Charge transmitted : Qmax
Coulombic efficiency :

11. .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

12. .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
Fluorescence
intensity
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
Relative abundance (%)
1 2 3 4

13. 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
.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 Phase (days)
0%
20%
40%
60%
80%
100%
120%
140%
B0
B1
B2
B3
Coulombic efficiency (%)

14. 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
.014
Microbial communities: structure
4x
4x
Sediments
4x
4x
E1
E2
E3
B0
B1
B2
B3

15. 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
.015
Microbial communities: structure
Sediment
s
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

16. 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
.016
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 Phase (days)
0%
20%
40%
60%
80%
100%
120%
140%
0
20
40
60
80
100
B0
B1
B2
B3
Relative abundance (%)
Coulombic efficiency (%)
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

17. 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
.017
Enrichments Biofilms
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

18. 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
.018
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%
Axis 2 - 24.0%
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

19. 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
.019
Conclusions
•A successful enrichment strategy
•With only 1 step required
•Enrichment of Geoalkalibacter subterraneus
•After 3rd enrichment step
oDivergence of species selected
oDecrease of electroactive performance

20. .020
Acknowledgments
Nicolas BERNET Eric TRABLY Jean Jaques GODON Anais BONNAFOUS Alessandro CARMONA Mohanakrishna GUNDA