Download pdf - 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


• 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


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



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


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


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: