Melanie King U.S. Environmental Protection Agency January 26, 2012.
ICABHPA-2012, Melanie Pierra
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Transcript of ICABHPA-2012, Melanie Pierra
Fermentative hydrogen production under moderate halophilic conditions
PIERRA Mélanie, TRABLY Eric, GODON Jean-Jacques, BERNET Nicolas
INRA, UR 50, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, 11100 Narbonne, France.
ICABHPA-2012 Hyderabad International conference on advances in biological hydrogen production and applications
H2 H2
2 Wrana et al, 2010; Clauwaert et al, 2008; Tommasi et al, 2012; Wang et al, 2011
Coupling dark fermentation and Microbial
Electrolysis Cells
Dark
fermentation
Microbial
electrolysis Any substrate Organic acids
(acetate, butyrate)
Outlet
Dark fermentation +
Microbial Electrolysis Any substrate Outlet
H2
Saline media
pH [7-8]
Food Industry
Fish and seafood
Slaughterhouses,
salting
Dairy industry
Brined
vegetables
Petroleum Industry
Reffineries
Chemical and
pharmaceutical industry
Saline wastewaters in Industry
Lefebvre et Moletta, 2006; Xiao et Roberts, 2010 3
Saline wastewaters in Industry
Lefebvre et Moletta, 2006; Xiao et Roberts, 2010
Leather Industry Textile Industry
4
• Halotolerant :
able to survive in a
salty environment
• Halophilic :
Growth (marine) and
requires a salty
environment
• Mecanisms :
Regulation of osmotic
pressure
Life in saline environment
Larsen, 1967; Lefebvre & Moletta, 2006 5
Gro
wth
rate
(arb
itra
iry
un
its)
NaCl concentration (g/L)
Extrem
halophilic
bacteria
Moderate
halophilic
bacteria
Halotolerant
bacteria
Non
halophilic
bacteria
35 0 >
6 Hawkes et al, 2007, Guo et al, 2010 , , Trably et al, 2011
Dark fermentation principles
Lactate
Acetone,
Butanol,
Ethanol,
Propionate
…
Acetate CO2 + H2
Organic matter
(biomass, solid waste, wastewaters)
Amino acids Single sugars Fatty acids
Volatile fatty acids
(acetate, butyrate)
CO2 + CH4 H2S
SO42-
hydrolytic bacteria
Lactic bacteria
Homoacetogenic
bacteria
Methanogenic
Archaea
Sulfate
reducing
bacteria Specific operating
conditions
(pH, T°, [S])
Materials & Methods
Wrana et al, 2010;
Inoculum : saline sediment
7 salinities from 9 to 75 gNaCl/L
Substrat : glucose (5g/L)
Initial pH : 8
Triplicates
Génomic DNA and PCR-SSCP
Single stranded DNA
fragment conformation
G
C
A
T T
A
C
G
PCR
Genomic DNA
Species 1
Denaturation
Double stranded DNA
fragments
Fluorescent
labeled primers
for DNA
detection
Elution time
Species 1
Species 2
Flu
ore
scen
ce
inte
nsi
ty
PCR products
sharing the same
length
Capillary
electrophoresis Species 2
H2 GC
VFAs : GC-FID
Metabolites : HPLC
H2 & Metabolites
7
Biological Hydrogen Potential tests
Materials & Methods
Wrana et al, 2010;
Time (days)
Vmax
H2max
Lag time
Rc
Cu
mu
lati
ve H
2 p
rod
uct
ion
(m
ol H
2/m
ol gl
uco
se)
Gompertz
model
8
Quéméneur et al, 2011; Quéméneur et al, 2011; Oren, 2001
H2 production performances
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
9 19 29 38 48 58 75
Vm
ax (
mo
lH2
mo
lGlc
d-1
)
salinity (gNaClL-1)0,0
0,2
0,4
0,6
0,8
1,0
9 19 29 38 48 58 75
H2
max
(m
olH
2 m
olG
LC-1
)
salinity (gNaClL-1)
0,0
1,0
2,0
3,0
4,0
5,0
9 19 29 38 48 58 75
Lag
tim
e(d
)
salinity (gNaClL-1)
-0,2
-0,1
0,0
9 19 29 38 48 58 75
Spé
cifi
c ra
te o
f H
2
con
sum
pti
on
(d
-1)
salinity (gNaClL-1)• First H2max decrease
• Constat increase to
0.90 (±0.02) molH2
molGlc-1 at 75 gNaClL
-1
• Highest hydrogen
production yields at
the highest NaCl
concentrations
• Specific impact on
H2 consumers !
• Homoacetogenesis
more sensitive
• Sharp decrease and
consistency of H2
production rate
• Increase of Lag
phase
9
Hawkes et al, 2007, Guo et al, 2010 , , Trably et al, 2011
Fermentative metabolic products
• Homoacetogenic consumption pathway.
• 9 gNaClL-1 = Clostridium spp as dominant bacteria
• Increase of lactate and ethanol concurrent routes for H2 production
• Inhibition of Propionate H2 consumption route
• Formate accumulation
10
0
4
8
12
16
20
24
28
32
0
2
4
6
8
10
12
14
16
9 19 29 38 48 58 75
H2
(m
mo
l)
met
ab
oli
c en
d-p
rod
uct
s
(mm
ol)
lactate
ethanol
propionate
formate
acetate
butyrate
H2
Salinity (gNaClL-1)
Quéméneur, 2011; Quéméneur, 2012
Bacterial community composition
• Only one or two dominant species
and few subdominants
• Clear community shift in bacterial
communities from 19 gNaClL-1
9 gNaClL-1
19 gNaClL-1
29 gNaClL-1
38 gNaClL-1
48 gNaClL-1
58 gNaClL-1
75 gNaClL-1
11
Bacterial community composition
• High reproductibility of
experiments
• Sample clustered
according to the
dominant species and
according to salinity
• Genetic differences
between bacterial
communities can be
correlated to their
metabolic activity
Salinity
H 2max
Lag phase
-0.2 -0.1 0.0 0.1 0.2
-0.1
0
.0
0.1
0
.2
Axis 1 - 38.3%
Axis
2 -
31.3
%
19gNaClL-1
29gNaClL-1
38gNaClL-
1
48gNaClL-1
9gNaClL-1
58gNaClL-1
75gNaClL-1
12
PCA statistical analysis
0
10
20
30
40
50
60
70
80
90
100
9 19 29 38 48 58 75
Others
VIBRIONALES
FUSOBACTERIALES
ENTEROBACTERIALES
CLOSTRIDIALES
BACTEROIDALES
ALTEROMONADALES
NaCl concentration (in gNaCl L-1)
Guo et al, 2010 , , Trably et al, 2011; Quéméneur, 2011; Quéméneur, 2012
Bacterial community composition
13
• 9gNaClL-1 : Clostridium, Enterobacter and Escherichia spp.
• % Clostridium, Enterobacter and Escherichia spp decreased as the salinity increased
• 58 & 75 gNaClL-1 : Vibrionales proportion reachs up to 79 & 92% !
Bacteria orders
Oh et al, 2003 14
0
10
20
30
40
50
60
70
80
90
100
9 19 29 38 48 58 75
Others
VIBRIONALES
Vibrio sp
Vibrionaceae
Vibrio ssp
Vibrio parahaemolyticus
Vibrio nereis
FUSOBACTERIALES
ENTEROBACTERIALES
CLOSTRIDIALES
BACTEROIDALES
ALTEROMONADALES
NaCl concentration (in gNaCl L-1)
Bacterial community composition
species or closest known phylogenetical level
• 58 gNaClL-1 and 75 gNaClL
-1 : a new Vibrionaceae spp
15
Vibrio spp.
Vibrio
Strains isolated from sewage sludge
Oh et al, 2003, Isolation of Hydrogen-producing Bacteria from Granular Sludge of an Upflow Anaerobic Sludge Blanket Reactor
16
• NaCl : an important parameter influencing process
performances as well as bacterial community structure.
• NaCl concentration : strong selective pressure on
bacterial communities, emergence of new species affiliated
to the family of Vibrionaceae.
• Vibrio spp : able to produce efficiently hydrogen in
moderate halophilic conditions
• Vibrio spp : higher hydrogen production yields at the
highest NaCl concentrations (0.90 ±0.02 molH2/molGlc at
75 gNaCl L-1, compared to 0.65 ±0.01 molH2 molGlc
-1 at 9 gNaCl
L-1)
• New strain belonging to Vibrionaceae in mixed cultures =
new perspectives for biotechnological purposes
Conclusions
0,0
0,2
0,4
0,6
0,8
1,0
9 19 29 38 48 58 75
H2
max
(m
olH
2 m
olG
LC-1
)
salinity (gNaClL-1)
0
10
20
30
40
50
60
70
80
90
100
9 19 29 38 48 58 75
Others
VIBRIONALES
Vibrio sp
Vibrionaceae
Vibrio ssp
Vibrio parahaemolyticus
Vibrio nereis
FUSOBACTERIALES
ENTEROBACTERIALES
CLOSTRIDIALES
BACTEROIDALES
ALTEROMONADALES
NaCl concentration (in gNaCl L-1)
0
10
20
30
40
50
60
70
80
90
100
9 19 29 38 48 58 75
Others
VIBRIONALES
FUSOBACTERIALES
ENTEROBACTERIALES
CLOSTRIDIALES
BACTEROIDALES
ALTEROMONADALES
NaCl concentration (in gNaCl L-1)
Salinity
H2max
Lag phase
-0.2 -0.1 0.0 0.1 0.2
-0.1
0.0
0.1
0.2
Axis 1 - 38.3%
Axis
2 -
31.3
%
19gNaClL-1
29gNaClL-1
38gNaClL-
1
48gNaClL-1
9gNaClL-1
58gNaClL-1
75gNaClL-1
Thank you for your attention
17
http://www.montpellier.inra.fr/narbonne
Laboratory of Environmental Biotechnology, INRA, Narbonne, France
18
Halanaerobaculum tunisienne
Hedi et al, 2008
Growth at NaCl concentrations between 14% and 30% (opt 20%-22%)
pH between 5.9 et 8.4 (opt 7.2-7.4)
Strict anaerobic bacteria
Substrates: glucose, galactose, cellobiose, mannose, maltose,
saccharose, pyruvate, amidon
End-Products de la fermentation du glucose: acetate,
butyrate, lactate, H2, CO2
From hypersaline sediments Tunisia, chott El-Djerid
Halanaerobaculum tunisienne
Biohydrogen production under halophilic
conditions
Only in pure culture: