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Dark fermentative hydrogen production is an intermediate microbial process occurring along anaerobic microbial degradation of organic matter. One direct application of this fermentative bioprocess consists in the production of renewable H2 and simultaneous treatment of organic pollutants. Nowadays, high amounts of saline effluents are generated by fish, seafood, petroleum and leather industries. Such saline effluents are rarely treated by biological anaerobic processes that are strongly inhibited by high salt concentrations. Alternative biological processes, such as dark fermentation, still remain to be investigated with the aim of removing organic pollution from such saline effluents. Moreover, more knowledge about the effect of saline conditions on fermentative microbial mixed cultures would provide new insights on the bacterial inhibition resulting from their exposition to saline conditions. This study deals with the characterization of hydrogen-producing microbial communities after increasing salt concentrations in a range compatible with a marine environment. A series of batch experiments was performed under anaerobic conditions favorable to hydrogen production, with a NaCl concentration ranging from 9 to 75 gNaCl/L. Marine sediments were used as inoculum. Biogas and bacterial metabolites were monitored over experimental time. The bacterial community structure dynamics were characterized using molecular tools based on the analysis of genomic 16S rDNA (CE-SSCP), and individual bacterial species were further identified by pyrosequencing. As a result, the significant and highest biohydrogen production yield (0.9±0.04 molH2.molGlucose-1) was observed at the highest NaCl concentration of 75 g.L-1. However, by increasing the NaCl concentration, the bioH2 production rates slowed down gradually, and longer lag phases were observed. A clear and gradual metabolic shift was also observed suggesting a substantial impact of the saline environment on anaerobic bacterial metabolism, as well as a high selection pressure on acidogenic bacteria. As expected, the composition of the bacterial community at 9gNaCl/L (control) was consistent with literature data, with Clostridium sp. and Enterobacter sp as main dominant species. Interestingly, a gradual shift of the bacterial community structure, concomitant to metabolic changes, was observed by increasing NaCl concentration, with Vibrio sp. as new dominant bacteria (87% in abundance) at the highest salinities. This is the first report on the presence of Vibrio sp. as main hydrogen-producing bacteria in such acidogenic mixed-cultures. Thus, this study provides new insights on anaerobic metabolism occurring in saline conditions with new possibilities of biotechnological applications from such saline effluents.
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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:
