La bioremédiation,
un théatre de l’innovation technologique
à l’interface de la biologie et de la chimie
Adaptations et interactions microbiennes dans l’environnement
S. Vuilleumier, GMGM, UMR 7156 UdS - CNRS
CNRS, 10 juillet 2014
http://basol.environnement.gouv.fr
>5400 polluted sites
in need of remediation
http://basias.brgm.fr
>500’000 installations
Polluted sites in France
The bioremediation market
«The market for the overall remediation market is estimated to be $23 billion»
ADEME (chiffres 2008) : 626 millions d’euros (estimation)
Pandey et al. (2009)
Vapor extraction
Air sparging
Multiphase extraction
in situ chemical degradation
Excavation
Phytoremediation
ex situ bioremediation
in situ bioremediation
Strategies for microbial bioremediation
PollutionNutrientinjection
Bioaugmentation
Biostimulation
"Engineered
bioremediation":
Active approaches
Decontamination without direct intervention :
«intrinsic bioremediation», «monitored natural attenuation»
Microbe injection
Microbial bioremediation:
Questions and experimental approaches
• Are the target pollutants (bio)degradable ?
• Who are the microbial players involved
(identification, isolation) ?
• How do they do it (metabolic pathways) ?
• Is there a contaminant-specific response
of the microbial flora in situ?
• Analytical chemistry, isotopic approaches
• Classical microbiology, genomics
• Genomics, isotopic approaches
• Bioindication (genomics)
Genomics and the environment
(mainly microbial)
sequenced genomes:
www.genomesonline.org
~20%
Microbial metabolism of C1 compounds
• From CH4 to CO2
halogenated pollutants
• Aerobic and anaerobic
• Across all three domains of life
(Bacteria, Archaea and Eukarya)
CH4
CH3OH / CH3Cl
CH2O / CH2Cl2
HCOOH / CHCl3
CO2 / CCl4
Microbial adaptation to pollutants:
chlorinated methanes
catabolic
genomic islands
H+ Cl-
central
metabolismBiomass
Energy
Dehalogenase expression
R-Cl
Dehalo-
genase
Biomass
Energy
Core genome
Solventeffects
Cell
changesDNA repair
Stress response1
GenotoxicityAcid
production
Metabolic tuning
3 Regulation
4H+ Cl- efflux2
3330800 337400 3346200
b3187 b3188
b3183 through b3186
b3196 through b3206
b3189 through b3195 b3208 b3209 b3210
b3207
3330800 337400 3346200
b3187 b3188
b3183 through b3186
b3196 through b3206
b3189 through b3195 b3208 b3209 b3210
b3207
Vuilleumier et al. 2009
Muller et al. 2011a,b
Torgonskaya et al., 2011
Vuilleumier et al., 2011
Marx et al. 2012
Roselli et al. 2013
Michener et al. 2014
Molecular biological investigations of polluted sites:
Analysis of metagenomic DNA
Environmental
sampleTotal DNA extraction
Identification
of microbial
populations
Follow-up
of key microbial functions
PCR, sequencing
Microbial diversity at polluted sites:
CCl4-contaminated groundwater
Method development:
anaerobic cultivation,
D-HPLC and T-RFLP
…and sequencing!
Penny et al., AEM 2010
Profiling, selection and isolation of CCl4-degrading consortia and strains
C4
C4
C2 C2C3C2
C1 C1C1 C3
C1: Sporotalea propionica TM1
Penny et al., in revision
Penny et al., FEMS Microbiol Ecol 2010
Microbial bioremediation
Interdisciplinarity at the biology – chemistry interface
• Are the target pollutants biodegradable ?
• Who are the microbial players involved
(identification, isolation) ?
• How do they do it (metabolic pathways) ?
• Is there a contaminant-specific response
of the microbial flora in situ?
• Analytical chemistry, isotopic approaches
• Classical microbiology, genomics
• Genomics, isotopic approaches
• Bioindication (genomics) BioGeoLink platform G. Imfeld, CR1 CNRS, UMR 7517
Compound specific isotope analysis (CSIA)
• Biological transformations : preferential use of light isotopes
Substrate residual fraction : 13C > 1%
Substrate: 13C:12C ~1%
C13C
13
C13
C12
C12
C12
C12
C12
C12 C
12
C12 C
12
C12
C12
C12
C12
C12
C12
C12
C12
C12
C13
C13
C13
C12
AfterBefore
C12
C12C
12
C12
C12
C12
C12
C12C
12
C12
C12
C12
C12
C12
C12
GC-IRMS
Gas chromatography
(-GC-) Isotope Ratio Mass Spectrometry
(-IRMS-)
He
Oven
CO2
CO2
CO2
Combustion
Ion source
Probe
valve
Magnet
separation
C: PDB
(Fossil Peedee-Belminite-Formation)
Standard
m/z 44
(12C16O2)
m/z 45
(13C16O2)
m/z 46
(12C18O16O)
Detector
BioGeoLink platform (LHyGeS, UMR 7517 CNRS,
Gwenaël Imfeld, CNRS)
CSIA: Compound-specific isotope fractionation
of chloroacetanilide pesticides in lab-scale wetlands
Elsayed et al. (2014)
chloroacetanilides
R3 R4
NR2
O
Cl
R1> Pollutant dissipation
occurs by biodegradation!
Nadalig et al., MicrobiologyOpen (2013)
Frontiers in Microbiology (in preparation)
Three chloromethane-degrading strains
of known genome sequence
Methylobacterium extorquens CM4
Hyphomicrobium sp. MC1
Leisingera methylohalidovorans
> Strains CM4 and MC1
possess the known cmu pathway
> Leisingera methylohalovorans
unknown degradation pathway
(no cmu genes)
-120
-100
-80
-60
-40
-20
0
-20 0 20 40 60 80 100
Bacterial chloromethane degradation:
CSIA evidence for different degradation pathways
δδδδ13C (‰)
δδ δδ2H
(‰
)
CH3Cl
CmuA
CmuB
CoI
CH3Cl
CH3-H
4F
+ HCl
H4F
CmuA
CmuB
CoI
CH3Cl
CH3-H
4F
+ HCl
H4F
Microbiology and bioremediation:
space for more interdisciplinarity!
• Are the target pollutants biodegradable ?
• Who are the microbial players involved
(identification, isolation) ?
• How do they do it (metabolic pathways) ?
• Is there a contaminant-specific response
of the microbial flora in situ?
• Analytical chemistry, isotopic approaches
• Classical microbiology, genomics
• Genomics, isotopic approaches
• Bioindication (genomics)
single cell biology,
microfluidics
metabolomics
microbial
ecotoxicology
Université de Strasbourg
Gwenaël Imfeld (LHyGeS)
Dimitri Heintz (IBMP)
Barbara Ernst (IPHC)
Sylvie Massemin (IPHC)
Maurice Millet (IPCEES)
Cuong Pham-Hu (IPCEES)
Mireille del Nero (IPHC)
Hubert Schaller (IBMP)
Philippe Hammann (IBMC)
Réseau REALISE
Génoscope
BRGM Orléans
Nicole Baran/Catherine Joulian
Université Lyon I
Guy Perrière
INRA Dijon
Fabrice Martin
IMBE Marseille
Isabelle Lafont-Schwob
Harvard University > Idaho
Chris Marx / Joshua Michener
MPI Mainz
Frank Keppler
University of Bayreuth
Steffen Kolb
UFZ Leipzig-Halle
Ivonne Nijenhuis
University of Washington
Marina Kalyuzhnaya
University of East Anglia/Warwick
Colin Murrell/Hendrik Schäfer
University of Michigan
Jeremy Semrau
Iowa State University
Alan DiSpirito
University of Luxembourg
Emilie Muller/Paul Wilmes
Adaptations et interactions
microbiennes dans l’environnement
(AIME) UMR 7156 UdS – CNRS
Stéphane Vuilleumier (PR)
Françoise Bringel (CNRS)
Thierry Nadalig (MC)
Christelle Gruffaz (Technician)
Yousra Louhichi (Tech. assistant)
PhDs
Omniea Azzam Elsayed
Pauline Chaignaud
N.N. (ADEME-BRGM 2014)
Internships 2014
Pan-Yu Kuang (M2)
Theo Schwab (M1)
Thomas Sonntag (BTS)
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