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)