4ISMET, Melanie PIERRA
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Transcript of 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
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
.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.
.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
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
Ph
ase
(d
ays)
0%
20%
40%
60%
80%
100%
120%
140%
B0 B1 B2 B3
Co
ulo
mb
ic e
ffic
ien
cy (
%)
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
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
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
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
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
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
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
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%
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
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
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