Implication des microorganismes dans les ...horizon.documentation.ird.fr/exl-doc/pleins_textes/... · Laboratoire Biochimie et Physiologie Moléculaire des Plantes (BPMP, Patrick

UNIVERSITE MONTPELLIER

Habilitation à Diriger des Recherches

Spécialité : Sciences de la Terre et de l’Environnement

Implication des microorganismes dans les biotransformations et processus de transfert des métaux et métalloïdes dans les

environnements contaminés par les mines

par

Odile BRUNEEL

Chargée de Recherches à l’IRD

HydroSciences Montpellier UMR 5569

Soutenue le 29 mars 2016 devant le jury composé de

Bernard OLLIVIER Directeur de Recherche IRD, UMR 7294, Marseille Rapporteur

Philippe NORMAND Directeur de Recherche CNRS UMR 5557, Lyon Rapporteur

Pascale BAUDA Professeur Université de Lorraine, UMR 7360, Metz Rapporteure

Pascal SIMONET Directeur de Recherche CNRS, UMR 5005, Lyon Examinateur

Michel LEBRUN Professeur Université de Montpellier, UMR LSTM Examinateur

Ecole Doctorale : Systèmes Intégrés en Biologie, Agronomie, Géosciences, HydroSciences, Environnement

2

SOMMAIRE

I CURRICULUM VITAE....................................................................................................... 3

Diplômes et formation......................................................................................................... 3

Parcours professionnel........................................................................................................ 3

Responsabilités récentes, animations scientifiques, comités............................................ 4

Collaborations récentes....................................................................................................... 4

Evaluation de la recherche.................................................................................................. 5

II CONTRATS DE RECHERCHE ET FINANCEMENTS ............................................... 6

III ENCADREMENT D’ETUDIANTS ET ENSEIGNEMENT…...................................... 8

Encadrement d’étudiants.................................................................................................... 8

Activité d’enseignement....................................................................................................... 9

IV PUBLICATIONS ET COMMUNICATIONS............................................................... 10

Synthèse de la production scientifique............................................................................. 10

Publications........................................................................................................................ 11

Communications, conférences et poster........................................................................... 14

V ACTIVITE DE RECHERCHE…......................................................................................15

Préambule ........................................................................................................................... 15

Travaux antérieurs ............................................................................................................ 19

Travaux actuels................................................................................................................... 47

VI PROJET DE RECHERCHE .......................................................................................... 55

VII REFERENCES BIBLIOGRAPHIQUES……….…..................................................... 60

VIII ANNEXES : SELECTION DE 5 PUBLICATIONS……….……............................. 67

3

I CURRICULUM VITAE

Odile BRUNEEL

IRD-CR1 Née le 1er

avril 1973

[email protected] Mariée, un enfant

Affectation actuelle :

En expatriation depuis février 2012 au Laboratoire de Microbiologie et Biologie Moléculaire

Université Mohammed V, Faculté des Sciences, Av Ibn Batouta BP1014

Rabat, Maroc

Laboratoire HydroSciences Montpellier, UMR5569 (CNRS/IRD/UM)

Université de Montpellier, CC0057 (MSE), 163 rue Auguste Broussonet

34090 Montpellier, France

Domaine de recherche :

Implication des microorganismes dans les biotransformations et processus de transfert des

métaux et métalloïdes dans les environnements contaminés par les mines

DIPLOMES ET FORMATIONS

• 2004 : Doctorat en Sciences de l’eau dans l’Environnement Continental, Ecole Doctorale

Sciences de la Terre et de l’Eau. Laboratoire HydroSciences Montpellier. Université

Montpellier II

• 2001 : DESS Diagnostic, Prévention et Traitements en Environnement, Faculté Libre des

Sciences de Lille, Mention Bien

• 1997 : DEA de Biologie, option Biologie des Protistes de Clermont-Ferrand I et II

PARCOURS PROFESSIONNEL

Recherche

• Février 2012 - aujourd’hui : en affectation au sein du Laboratoire de Microbiologie et

Biologie Moléculaire, Faculté des Sciences, Université Mohammed V, Rabat, Maroc

• Depuis Octobre 2008: Chargée de Recherches 1ère

classe à l’IRD

• Octobre 2004 - aujourd’hui : Chargée de Recherches à l’IRD au sein du Laboratoire

HydroSciences Montpellier (UMR 5569, CNRS-Université Montpellier-IRD)

• 2001-2004 : Recherche en géomicrobiologie à l’Université Montpellier II dans le cadre de

ma thèse. Laboratoire HydroSciences Montpellier, UMR 5569

4

Activités salariées

• 1999-2000 : Professeur des écoles en CE2 à Djibouti (Afrique de l’Est) dans le cadre d’une

coopération civile d’aide au développement

RESPONSABILITES RECENTES, ANIMATIONS SCIENTIFIQUES, COMITES

• Représentante par Intérim de l’IRD au Maroc (août 2014-aujourd’hui)

• Membre du comité de pilotage du réseau SICMED Mistrals « Activités minières dans le

bassin méditerranéen – Interactions contaminants métalliques / écosystèmes − Interfaces avec

la santé, l’environnement »

• Membre élue depuis 2012 de la commission scientifique sectorielle n°1 (CSS1, Sciences

physiques et chimiques de l’environnement planétaire) de l’IRD

COLLABORATIONS RECENTES

Instituto de Biologıa Molecular y Biotecnologıa (Volga Iñiguez), Facultad de Ciencias

Puras, Universidad Mayor de San Andres, C. 27 Campus Universitario Cota Cota, La Paz,

Bolivie (laboratoire soutenus par le DSF de l'IRD dans le cadre du programme "jeunes

équipes")

Laboratoire de Microbiologie et Biologie Moléculaire (LMBM, L. Sbabou, J. Aurag et A.

Filali-Maltouf), Faculté des Sciences, Université Mohamed V, Rabat, Maroc

Laboratoire de Physiologie et Biotechnologie Végétale (LPBV, A. Smouni, M. Fahr),

Faculté des Sciences, Université Mohamed V, Rabat, Maroc

Equipe de recherche E2G, (R. Hakkou) Département des Sciences de la terre, Faculté des

Sciences et Techniques de Guéliz, Université de Cadi Ayyad, Avenue Abdelkarim Elkhattabi,

Gueliz, P.O. Box 549, Marrakech, Maroc

Laboratoire Géoexplorations et Géotechniques (A. Ddekayir), Département de Géologie,

Faculté des Sciences, BP. 11201, Zitoune, Meknès, Maroc

Institut de Minéralogie et de Physique des milieux Condensés (IMPMC, G. Morin), UMR

CNRS 7590, UPMC, 4 Place Jussieu, 75252 Paris, France

Laboratoire AMPERE (E. Navarro), UMR CNRS 5005, Ecole Centrale de Lyon, Université

de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France et Laboratoire des Symbioses

Tropicales et Méditerranéennes, LSTM, UMR 113, TA A-82/J Campus de Baillarguet, 34398

Montpellier, France

Laboratoire Biochimie et Physiologie Moléculaire des Plantes (BPMP, Patrick Doumas), 2,

place Pierre Viala, 34060 Montpellier, France

Equipe Environnement et Microbiologie (EEM, R. Duran, B. Lauga), UMR 5254 IPREM-

EEM, Pau, France

Laboratoire de Génétique Moléculaire, Génomique et Microbiologie (GMGM, P. Bertin, F.

Ploetze), UMR 7156, Univ Louis Pasteur–CNRS, Strasbourg, France

5

EVALUATION DE LA RECHERCHE

Participaption à un jury de thèse

L. Giloteaux en décembre 2009

Participation à différents jurys de Licence, M1 et M2 tous les ans depuis 2005

Evaluations pour les journaux:

FEMS Microbiology Ecology, Microbial Ecology, Extremophiles, Environmental Science and

Pollution Research, Geomicrobioloy Journal, Journal of Applied Microbiology, PLOS ONE

Evaluations de projets :

ANR (Blanc, JC), Ec2co (Microbiologie environnementale), FRB (Fondation pour la recherche

sur la biodiversité

6

II CONTRATS DE RECHERCHE ET FINANCEMENT

Contrats de recherches nationaux et internationaux

• 2003-2005. Projet labélisé RITEAU (Ministère de l’Industrie, 677 k€). As5 : Mise au

point d’un procédé biologique de potabilisation des eaux arséniées. Partenaires : IRH

Environnement, BEFS-PEC, LMCP UMR 7590 (G. Morin).

• 2004-2006. Projet ECODYN (AC, FNS, ECCO, 30 k€). « Processus de transfert et

écotoxicité de l’arsenic et des métaux associés dans un hydrosystème en aval d’un drainage

minier. Contrôles physico-chimiques et microbiologiques ». Partenaires : UMR 7590-CNRS-

Universités Paris 6 et 7-IPG (G. Morin), LCABIE, UMR 5034, CNRS Université de Pau (O.

Donard), LEM, Université de Pau (R. Durand), CB UPR 9043, Marseille (V. Bonnefoy),

INERIS (J-M. Porcher), BRGM (M. Motelica), ECOLAG, UMR 5119 CNRS (C. Aliaume)

• 2004-2006. Projet PICS CNRS (21 k€), Université de Huelva, Espagne). « Signature

de l’activité bactérienne dans les précipités riches en fer des drainages miniers acides ».

Partenaires: Departamento de Geologia, Universidad de Huelva, Espagne (JM. Nieto)

• 2006-2007. PAI Protea (Ministères des Affaires Etrangères et de l’Education

Nationale, de l’Enseignement Supérieur et de la Recherche, 10 k€) avec l’Afrique du Sud.

« Metal and metalloid biotransformations in South African acid mine drainage systems”.

Partenaires : Department of Biotechnology (D. Cowan), Department of Chemistry (L. Petrik)

University of Western Cape, Capetown, Afrique du Sud

• 2006-2008. Projet EC2CO-3BIO (INSU, CNRS, 40 k€) « Biologie, biominéraux et

biotransformations dans les eaux acides minières ». Partenaires : IMPMC, UMR CNRS7590,

Paris (G. Morin), IPREM, UMR 5254, CNRS- Université de Pau (R. Duran)

• 2007-2008. Coordinatrice pour HSM du P2R Safe-Water (Afrique du Sud, 15 k€).

« Study of the metal and metalloid biotransformations in South African acid mine drainage”.

Partenaires : Department of Biotechnology (D. Cowan), Department of Chemistry (L. Petrik)

University of Western Cape, Capetown, Afrique du Sud

• 2007-2009. Coordinatrice du projet EC2CO-MicroBien (INSU CNRS, 90 k€)

« Impact des microorganismes sur les transformations des métaux et métalloïdes dans des

drainages miniers riches en sélénium ». Partenaires : IMPMC, UMR CNRS 7590, Paris (G.

Morin), IPREM, UMR 5254, Pau (R. Duran)

• 2007-2010. Projet ANR RARE, programme blanc (Agence Nationale pour la

Recherche, 166 k€) « Reactivity of an arsenic-rich ecosystem: an integrated genomics

approach ». Partenaires : GMGM, UMR 7156 de Strasbourg (P. Bertin) ; IMPMC, UMR

7590, Paris (G. Morin) ; IPREM, UMR 5254, Pau (R. Duran)

• Depuis 2009- aujourd’hui. OSU OREME (INSU, INEE, 12 k€/an). Tâche

d’Observation 1 (TO1, environ 10 k€/ans) « Suivi des processus hydrobiogéochimiques de

transfert des métaux et métalloïdes issus des activités minières sur le site de Carnoulès ».

Labelisée par l’OSU OREME (http://www.oreme.univ-montp2.fr/spip.php?rubrique41).

Système d’Observation Pollution et adaptabilité biologique en aval des anciens sites miniers

• 2009-2011. Bourse de thèse présidente environnée de l’UM2 (Université Montpellier

II, 30 k€, A. Volant). « Etude des processus microbiens et géochimiques de mobilisation et de

piégeage des éléments métalliques issus des activités minières ».

• 2010-2011. Projet FRB MIGR’AMD (Fondation pour la Recherche sur la

Biodiversité, 40 k€). « Microbial biogeography of acid mine drainage: a study of genetic

7

diversity and species diversity from an evolutionary perspective ». Partenaires : IPREM,

UMR 5254, Pau (B. Lauga). Responsable du projet pour HSM

• 2011-2012. Projet Ec2co Microbien. (INSU CNRS, 38 k€) « Rôle des bactéries du

genre Thiomonas dans les transformations de polluants métalliques au sein d’écosystèmes

miniers ». Partenaires : GMGM, UMR 7156, Strasbourg (F. Ploetze) et LSMBO, UMR 7509,

Strasbourg (C. Carapito). Responsable du projet pour HSM

• 2011-2013. Projet MISTRALS (INSU CNRS). Mediterranean integrated studies at

regional and local scales. Aide au montage d’un réseau sur les activités minières dans le

bassin méditerranéen – Interactions contaminants métalliques / écosystèmes - interfaces avec

la santé, l’environnement et la société (Coordinateur : P. Doumas, UMR BPMP Montpellier).

Membre du comité de pilotage

• 2012-2013. Coordinatrice du Projet Ec2co ECODYN/MICROBIEN (INSU CNRS, 60

k€). « Etude des interactions plantes-microorganismes dans un contexte de réhabilitation de

sites miniers au Maroc: mécanismes adaptatifs et effets sur le devenir des polluants

métalliques ». Partenaires : LMBM, Rabat (L. Sbabou, J. Aurag, A. Filali-Maltouf) ; LPBV,

Rabat (A. Smouni, M. Fahr) ; AMPERE, Lyon-LSTM, Montpellier (E. Navarro) ; UMR

BPMP, (P. Doumas) ;

• 2012-2014. Projet de coopération CNRS-CNRST avec le Maroc (CNRS, 4 k€).

« Dynamique des métaux et métalloïdes et processus biogéochimiques mis en jeu dans les

lacs de carrière du district minier de Zeïda (Maroc) ». Partenaire : Laboratoire d'Ingénierie

Géologique de Meknes (A. Dekayir)

• 2014-2017. Projet ANR ECO-TS IngECOST-DMA (ANR, Programme Eco-

technologies & Eco-Services, 850 k€). « Ingénierie écologique appliquée à la gestion intégrée

de stériles et drainages miniers acides riches en arsenic ». Partenaires : BRGM, Orléans (F.

Battaglia-Brunet, C. Joulian) ; IMPMC, UMR 7590, Paris (G. Morin) ; Sol Environnement,

Nanterre (A. Esnault) ; IRH, Gennevilliers (G. Grapin)

8

III ENCADREMENT D’ETUDIANTS ET ENSEIGNEMENT

ENCADREMENT D’ETUDIANTS

Licence et IUT

• Julie Mougeot (2008) 2ème

année DUT Génie Biologique option Analyses Biologiques et

Biochimiques. UM2, Montpellier

• Sandrine Gavalda (2009) 2ème



• Cédric Bocquet (2009) 1ère

année BTS Analyses Biologiques et Biotechnologie.

Castelnaudary

• Blandine Luce (2009-2010, 19 oct au 19 fév). Licence professionnalisante Géosciences,

Traitement et Prévention des Pollutions 5GTPT). UM2, Montpellier

• Aurélia Aidi (2010) 2ème



• Coencadrement L. Rubini (2011) 2ème

année DUT Génie Biologique, option Analyses

Biologiques et Biochimiques. UM2, Montpellier

• Co-encadrement M. Dufour (2012) 2ème



• Co-encadrement L. Causse (2013) 2ème



• Encadrement Keltoum Ouassal (2013) Licence en Sciences de la Vie, module PFE. Faculté

des sciences. Université Mohammed V, Rabat, Maroc

Masters

• Noémie Pascault (2006) Master 2 BGAE (Biologie, Géologie, Agroressources et

Environnement). Spécialité BIMP (Biodiversité et Interactions Microbiennes et Parasitaires),

parcours SM (Systèmes Microbiens). UM2, Montpellier

Publication : Bruneel et al., 2008. Archaeal diversity in a Fe-As rich acid mine drainage at Carnoulès

(France). Extremophiles. 12, 563-571

• Amélie Bardil (2007) Master 1 BGAE TEE, R2E (Terre, Eau et Environnement/Recherches

en Eau). UM2, Montpellier

• Amélie Bardil (2008) Master 2 BGAE, TEE, R2E (Terre, Eau et Environnement/Recherches

en Eau). UM2, Montpellier

Publication : Bruneel et al., 2011. Characterization of the active bacterial community involved in natural

attenuation processes in arsenic-rich creek sediments. Microbial Ecology. 61, 793-810

• Anouk Favri (2008) Master 2 Pro Gestion des Ressources en Eaux. Parcours dans le cadre

de la formation permanente. UM2, Montpellier

• Camila Cordier (2010) Master 2 BGAE. Spécialité BIMP (Biodiversité et Interactions

Microbiennes et Parasitaires), parcours SM (Systèmes Microbiens), UM2, Montpellier

• Encadrement d’une étudiante chilienne, V. Verdejo Parada (2011) Master 1. Mention

BGAE, spécialité BIMP, UM2, Montpellier

• Encadrement Ikram Dahmani (2013) Master 2 BIOGECO (Biodiversité, Gestion et

Conservation). Faculté des sciences, Université Mohammed V, Rabat, Maroc

• Coencadrement Najoua Mghazli. 2015. Master 2 Production Végétale. Faculté des sciences,

Université Mohammed V, Rabat, Maroc

9

Thèses

• Co-encadrement, pour la partie microbiologie, de la thèse de Doctorat d’Ingénieur du CNRS

de Marion Egal (2005-2008). Directrice de thèse : F. Elbaz Poulichet (DR CNRS chimiste à

HSM). Encadrement par C. Casiot. Encadrement personnel effectif : 5%

• Co-encadrement de thèse d’Aurélie Volant (2009-2012) soutenue le 12/12/12. Ecole

Doctorale SIBAGHE. Directeurs thèse : F. Elbaz Poulichet (DR CNRS chimiste, HSM) et P.

Bertin (Professeur au laboratoire GMGM, Strasbourg). Encadrement personnel effectif: 90%

Publications : Bruneel et al. (2011) Characterization of the active bacterial community involved in natural

attenuation processes in arsenic-rich creek sediments. Microbial Ecology. 61, 793-810

Volant et al. (2012) Archaeal diversity: temporal variation in the arsenic-rich creek sediments of Carnoulès

Mine, France. Extremophiles. 16, 645-657

Volant et al. (2014) Diversity and spatiotemporal dynamics of bacterial communities: physicochemical and

others drivers along an acid mine drainage. FEMS Microbiology Ecology. 90, 247-263

• Co-encadrement thèse Ikram Dahmani. Décembre 2013. Faculté des sciences. Université

Mohammed V, Rabat, Maroc. Directeur de thèse : J. Aurag (LMBM, Rabat), co-encadrement

avec L. Sbabou (LMBM, Rabat) et E. Navarro (AMPERE, Lyon - LSTM, Montpellier).

Encadrement personnel effectif : 60%

ACTIVITES D’ENSEIGNEMENT

Université de Pau et des Pays de l’Adour ; Master 2. Module écologie moléculaire

bactérienne. (3 heures/an, de 2004-2006)

Université Montpellier 2 ; DEA SEEC – Etude du rôle des microorganismes dans le

transfert de la pollution minière (2 heures/an, 2005-2008)

Université Montpellier 2 ; M2R Fenec, L3Pro GPTP. Sortie terrain sur l’ancien site minier

de Carnoulès (Gard) (3 à 9 h/an 2006-2011).

Université Paris VI ; DEA puis M2R Sciences de l’Univers, Ecologie, Environnement –

Parcours Hydrologie, Hydrogéologie, Stage terrain et visite de l’ancien site minier de

Carnoulès (4 à 8 heures/an, de 2003-2011)

Université Mohamed V de Rabat, Faculté des Sciences, Masters PV & BioGéCo (Module

de Microbiologie du sol) (4 à 8 heures/an, 2012-2015)

10

IV PUBLICATION ET COMMUNICATION

SYNTHESE DE LA PRODUCTION SCIENTIFIQUE

Bibliométrie ISI (mai 2015)

Results found 31

Sum of the Times Cited without self-citations 690

Average Citations per Item 26

h-index 17

Journal Nb d’articles IF 2013

The ISME Journal 1 9.267

PLoS Genetics 1 8.167

Environmental Microbiology 1 6.24

Environmental Science and Technology 3 5.481

Water Research 1 5.323

Geochimica et Cosmochimica Acta 2 4.250

Applied and Environmental Microbiology 2 3.952

FEMS Microbiol Ecol 2 3.875

Chemosphère 1 3.499

Vaccine 1 3.485

Chemical Geology 2 3.482

Microbial Ecology 1 3.118

Science of the Total Environment 1 3.163

Environmental Chemistry 1 3.035

Journal of Applied Microbiology 1 2.386

Extremophiles 2 2.174

Applied Geochemistry 2 2.021

Aquatic Geochemistry 1 1.809

Geomicrobiology Journal 1 1.804

Environmental Science: Processes and Impacts. 1

11

PUBLICATIONS

Revues à Comité de lecture dans des revues indexées (ISI & Pubmed)

1. Bruneel O, Personné J-C, Casiot C, Leblanc M, Elbaz-Poulichet F, Mahler BJ, Le

Flèche A, Grimont PAD (2003) Mediation of arsenic oxidation by Thiomonas sp. in acid mine

drainage (Carnoulès, France). Journal of Applied Microbiology. 95, 492-499

2. Morin G, Juillot F, Casiot C, Bruneel O, Personné J-C, Elbaz-Poulichet F, Leblanc M,

Ildefonse P, and Calas G (2003) Bacterial formation of tooeleite and mixed arsenic(III) or

arsenic(V)-Iron(III) gels in the Carnoulès acid mine drainage, France. A XANES, XRD, and

SEM study. Environmental Science and Technology. 37, 1705-1712

3. Casiot C, Morin G, Juillot F, Bruneel O, Personné JC, Leblanc M, Duquesne K,

Bonnefoy V, Elbaz-Poulichet F (2003) Bacterial immobilization and oxidation of arsenic in

acid mine drainage (Carnoulès creek, France). Water Research. 37, 2929-2936

4. Duquesne K, Lebrun S, Casiot C, Bruneel O, Personné J-C, Leblanc M, Elbaz-Poulichet

F, Morin G, and Bonnefoy V (2003) Immobilization of arsenite and ferric iron by

Acidithiobacillus ferrooxidans in acid mine drainage. Applied and Environmental

Microbiology. 69, 6165-6173

5. Casiot C, Leblanc M, Bruneel O, Personné J-C, Koffi K, Elbaz-Poulichet F (2003)

Geochemical processes controlling the formation of As-rich waters within a tailings

impoundment. Aquatic Geochemistry. 9, 273-290

6. Casiot C, Bruneel O, Personné J-C, Leblanc M, Elbaz-Poulichet F (2004) Arsenic

oxidation and bioaccumulation by the acidophilic protozoan, Euglena mutabilis, in acid mine

drainage (Carnoulès, France). Science of the Total Environment. 320, 259-267

7. Mévelec MN, Bout D, Desolme B, Marchand H, Magne R, Bruneel O, Buzoni-Gatel D

(2005) Evaluation of protective effect of DNA vaccination with genes encoding antigens

GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital

toxoplasmosis in mice. Vaccine. 23, 4489-4499

8. Casiot C, Lebrun S, Morin G, Bruneel O, Personné JC, Elbaz-Poulichet F (2005)

Sorption and redox processes controlling arsenic fate and transport in a stream impacted by

acid mine drainage. Science of the Total Environment. 347, 122-30

9. Bruneel O, Duran R, Koffi K, Casiot C, Fourçans A, Elbaz-Poulichet F, Personné J-C

(2005) Microbial diversity in a pyrite-rich tailings impoundment (Carnoulès, France).

Geomicrobiology Journal. 22, 249 - 257

10. Bruneel O, Duran R, Casiot C, Elbaz-Poulichet F, Personné JC (2006) Diversity of

microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France. Applied and

Environmental Microbiology. 72, 551-556

11. Casiot C, Pedron V, Bruneel O, Duran R, Personné J-C, Grapin G, Drakides C, Elbaz-

Poulichet F (2006) A new bacterial strain mediating As oxidation in the Fe-rich biofilm

naturally growing in a groundwater Fe treatment pilot units. Chemosphère. 64, 492-496

12. Bruneel O, Pascault N, Egal M, Bancon-Montigny C, Goni M, Elbaz-Poulichet F,

Personné J-C, Duran R (2008) Archaeal diversity in a Fe-As rich acid mine drainage at

Carnoulès (France). Extremophiles. 12, 563-571

13. Egal M, Elbaz-Poulichet F, Casiot C, Motelica-Heino M, Negrel P, Bruneel O, Nieto

JM, Sarmiento AM (2008) Iron isotopes in acid mine waters and iron-rich solids from the

Tinto-Odiel Basin (Iberian Pyrite Belt, Southwest Spain). Chemical Geology. 253, 162–171

12

14. Benzerara K, Morin G, Yoon TH, Miot J, Tyliszczak T, Casiot C, Bruneel O, Farges

F, and Brown Jr GE (2008) Nanoscale study of As transformations by bacteria in an acid mine

drainage system. Geochimica and Cosmochimica Acta. 72, 3949-3963

15. Casiot C, Egal M, Bruneel O, Cordier M-A, Bancon-Montigny C, Gomez E, Aliaume

C, Elbaz-Poulichet F (2009) Hydrological and geochemical controls on metals and arsenic in

a Mediterranean river contaminated by acid mine drainage (the Amous River, France);

preliminary assessment of impacts on fish (Leuciscus cephalus). Applied Geochemistry. 24,

787-799

16. Egal M, Casiot C, Morin G, Parmentier M, Bruneel O, Lebrun S, Elbaz-Poulichet F

(2009) Kinetic control on the formation of tooeleite, schwertmannite and jarosite by

Acidithiobacillus ferrooxidans strains in an As(III)-rich acid mine water. Chemical Geology.

265, 432-441

17. Arsène-Ploetze F, Koechler S, Marchal M, Coppee J-Y, Chandler M, Bonnefoy V,

Barakat M, Barbe V, Battaglia -Brunet F, Brochier-Armanet C, Bruneel O, G. Bryan C,

Cleiss J, Heinrich-Salmeron A, Hommais F, Joulian C, Krin E, Lieutaud A, Lièvremont D,

Michel C, Muller D, Ortet P, Proux C, Siguier P, Roche D, Rouy Z, Salvignol G, Slyemi D,

Talla E, Weiss S, Weissenbach J, Médigue C, Bertin PN (2010) Structure, function and

evolution of the Thiomonas spp. genome inferred from sequencing and comparative analysis.

PLoS Genetics. 6 (2) e1000859

18. Egal M, Casiot C, Morin G, Elbaz-Poulichet F, Cordier MA Bruneel O (2010) An

updated insight into the natural attenuation of As concentrations in Reigous Creek (southern

France). Applied Geochemistry. 25, 1949–1957

19. Bruneel O, Volant A, Gallien S, Chaumande B, Casiot C, Carapito C, Bardil A, Morin

G, Brown Jr GE, Personné JC, Le Paslier D, Schaeffer C, Van Dorsselaer A, Bertin PN,

Elbaz-Poulichet F, Arsène-Ploetze F (2011) Characterization of the active bacterial

community involved in natural attenuation processes in arsenic-rich creek sediments.

Microbial Ecology. 61, 793-810

20. Bertin PN, Heinrich-Salmeron A, Pelletier E, Goulhen- Chollet F, Arsène-Ploetze F,

Gallien S, Lauga B, Casiot C, Calteau A, Vallenet D, Bonnefoy V, Bruneel O, Chane-Woon-

Ming B, Cleiss-Arnold J, Duran R, Elbaz-Poulichet F, Fonknechten N, Giloteaux L, Halter D,

Koechler S, Marchal M, Mornico D, Schaeffer C, Thil Smith AA, Van Dorsselaer A,

Weissenbach J, Médigue C and Le Paslier D (2011) Metabolic diversity between main

microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics. The

ISME Journal. 5, 1735-1747. Cet article a fait l’objet d’une note dans la revue Sciences (2011,

vol 332, p1128)

21. Casiot C, Egal M, Bruneel O, Verma N, Parmentier M, Elbaz-Poulichet F (2011)

Predominance of aqueous Tl(I) species in the river system downstream from the abandoned

Carnoulès mine (Southern France). Environmental Science & Technology. 45, 2056-2084

22. Volant A, Desoeuvre A, Casiot C, Lauga B, Delpoux S, Morin G, Personné JC, Héry

M, F Elbaz-Poulichet, Bertin P and Bruneel O (2012) Archaeal diversity: temporal variation

in the Arsenic-Rich Creek Sediments of Carnoulès Mine, France. Extremophiles. 16, 645-657

23. Giloteaux L, Duran R, Casiot C, Bruneel O, Elbaz-poulichet F and Goñi-urriza M

(2013) A survey of sulfate reducing bacteria in a heavily arsenic contaminated acid mine

drainage (Carnoulès, France). FEMS Microbiol Ecol. 83 724–737

13

24. Maillot F, Morin G, Juillot F, Bruneel O, Casiot C, Ona-Nguema G, Wang Y, Lebrun

S, Aubry E, Vlaic G, Brown GE Jr (2013) Structure and reactivity of As(III)- and As(V)-rich

schwertmannites and amorphous ferric arsenate sulfate from the Carnoulès acid mine

drainage, France: comparison with biotic and abiotic model compounds and implications for

As remediation. Geochimica et Cosmochimica Acta. 104, 310-329

25. Resongles E, Casiot C, Elbaz-Poulichet F, Freydier R, Bruneel O, Piot C, Delpoux S,

Volant A, Desoeuvre A (2013) Fate of Sb(V) and Sb(III) species along a gradient of pH and

oxygen concentration in the Carnoulès mine waters (Southern France)". Environmental

Science: Processes and Impacts. 15, 1536-1544

26. Adra A, Morin G, Ona-Nguema G, Maillot F, Casiot C, Bruneel O, Lebrun S, Juillot

F, Brest J (2013) Arsenic Scavenging by Al-Substituted Ferrihydrites in a Circumneutral pH

River Impacted by the Acid Mine Drainage of Carnoulès, Gard, France. Environmental

Science and Technology. 47, 12784-12792

27 Héry M, Casiot C, Resongles E, Gallice Z, Bruneel O, Desoeuvre O, Delpoux S (2014)

Release of arsenite, arsenate and methyl-arsenic species from streambed sediment impacted

by acid mine drainage : a microcosm study. Environmental Chemistry. 11, 514-524

28 Volant A, Bruneel O, Desoeuvre A, Héry M, Casiot C, Bru N, Delpoux S, Fahy A,

Javerliat F, Bouchez O, Duran R, Bertin PN, Elbaz-Poulichet F and Lauga B (2014) Diversity

and spatiotemporal dynamics of bacterial communities: physicochemical and others drivers

along an acid mine drainage. FEMS Microbiology Ecology. 90, 247-263

Publications soumises

Doumas P, Munoz M, Banni M, Becerra S, Bruneel O, Casiot C, Cleyet-Marel J-C,

Gardon J, Noak Y, Sappin-Didier V. Polymetallic pollution from abandoned mines in

Mediterranean regions: a multidisciplinary approach of environmental risks. Soumis à

Regional Environmental Change

Publications en cours de soummission

Idir Y, Sbabou L, Bruneel O, Filali-Maltouf A and Aurag J. Characterization of root-

nodule bacteria isolated from Hedysarum spinosissimum L, growing in mining sites of

Northeastern region of Morocco. Sera soumis à Environmental Science and Pollution

Research

Publication dans des revues non indexées

Casiot C, Héry M, and Bruneel O (2012) Pollution by mine drainage: towards biological

treatment? In: Water at the Heart of Science. IRD Edition, Marseille

Benyassine EM, Dekayir A, Héry M, Delpoux S, Desoeuvre A, Bruneel O, Benhassou H,

Rouai M, Casiot C (2013) Contrasted arsenic speciation in two alkaline pit lakes from the

abandoned Pb mining area of Zeida (Moulouya, Morocco). International Journal Clean-Soil,

Air, Water. Special Focus Issue on Emerging Pollutants in Euro-Mediterranean and MENA

Countries

14

COMMUNICATIONS, CONFERENCES ET POSTER

Communication orale (5 dernières années)

Bruneel O, Casiot C, Personné J-C, Volant A, Vadapalli VRK, Petrik L, Cowan DA,

Morin G, Duran R, and Elbaz-Poulichet F. Impact des microorganismes sur les

transformations des métaux et métalloïdes dans des drainages miniers d’Afrique du Sud.

Proceeding, réunion de restitution du programme Ec2co. Toulouse, France. 22-26 November

2010

Bertin PN, Heinrich-Salmeron A, Pelletier E, Goulhen- Chollet F, Arsène-Ploetze F,




Weissenbach J, Médigue C and Le Paslier D. Diversity of metabolic interactions inside an

arsenic-rich microbial ecosystem revealed by meta- and proteo-genomics. BAGECO11, 11th

Conference on Bacterial Genetics and Ecology. Corfu, Greece. 29 May - 2 June 2011

Morin G, Ona-Nguema G, Juillot F, Maillot F, Wang Y, Egal M, Bruneel O, Casiot C,

Elbaz-Poulichet F, Calas G, Brown JR. How biogenic nano-iron oxides can control the fate of

pollutants. Goldschmidt. Prague, République Tchèque. 14-19 août 2011

Casiot C, Delpoux S, Desoeuvre A, Volant A, Egal M, Resongles E, Hery M, Freydier R,

Elbaz-Poulichet F, Cadot E, Gardon J, Bruneel O. Spéciation et processus de transfert de

métaux et métalloïdes dans les eaux minières: exemple du site de Carnoulès dans le Gard.

Premières rencontres du Réseau "Environnements Miniers Méditerranéens".Montpellier,

France. 14-16 mai 2012

Bruneel O, Desoeuvre A, Volant A, Héry M, Casiot C, Delpoux S, Freydier R, Elbaz –

Poulichet F. Impact des microorganismes sur le transfert des contaminants métalliques dans

les environnements miniers. Premières rencontres du Réseau "Environnements Miniers

Méditerranéens". Montpellier, France. 14-16 mai 2012

Casiot C, Bruneel O, Hery M, Delpoux S, Desoeuvre A, Volant A, Resongles E, Freydier

R, Elbaz-Poulichet F. Speciation and transfer processes of metals /metalloids in mining water

: exemple of studies at the Carnoulès mining site (Gard). 4th

SPECIATION seminar;

Biological, environmental and nuclear speciation. Montpellier, France. May 29-31, 2012

Bruneel O, Volant A, Dahmani I, Sbabou L, Navarro I, Héry M, Désœuvre A, Casiot C,

Filali-Maltouf A. Study of diversity using next generation sequencing. 4ème Congrès de

l'Association Marocaine de Microbiologie (AMM) et 16ème Congrès de l'Association

Africaine pour la Fixation Biologique de l'azote (AABNF) sur le thème BIOFERSOL,

Biofertilisation des sols et développement durable en Afrique. Maroc, Rabat. 03-07 novembre

2014

Dekayir A, Benyassine M, Casiot C, Hery M, Bruneel O, El Hachimi ML, Rouai M.

Contamination des eaux de lacs de carrières de la mine abandonnée de Zeida (Maroc).

Colloque SICMED. Tunisie, Tunis. 18-20 novembre 2014

Poster (5 dernières années)

Bruneel O, Casiot C, Personné J-C, and Elbaz-Poulichet F. The Carnoulès mine (Gard,

France). Generation of as-rich acid mine drainage and natural attenuation processes. 5ème

15

Colloque International « Contamination Métallique : Impact sur l’Environnement, la Santé et

la Société ». Oruro, Bolivie. 13-15th October 2010

Elbaz-Poulichet F, Casiot C, Bruneel O, Egal M, Morin G, Miot J, Benzerara K, Duran R,

Goni-Urriza, M.; Giloteaux, L. Biologie, biominéraux et biotransformations dans les eaux

acides minières – 3BIO. Colloque de restitution EC2CO. Toulouse, France. 23-25 novembre

2010

Bertin P.N., Heinrich-Salmeron A., Pelletier E., Goulhen- Chollet F., Arsène-Ploetze F.,




Weissenbach J, Médigue C and Le Paslier D. Diversity of metabolic interactions inside an

arsenic-rich microbial ecosystem revealed by meta- and proteo-genomics. Colloque

Restitution ANR “Des molécules aux écosystèmes”. Montpellier France, 13-14th

September

2011

Lauga B, Volant A, Bruneel O, Fahy A, Laoudi S, Casiot C, Nieto JM and Duran R.

MIGRAMD : Microbial biogeography of Acid Mine Drainage: a study of genetic diversity

and species diversity from an evolutionary perspective. Colloque FRB "Les Ressources

Génétiques face aux nouveaux enjeux environnementaux, économiques et sociétaux".

Montpellier, France. 21-22 September 2011

Lauga B, Volant A, Bruneel O, Fahy A, Laoudi S, Casiot C, Nieto M and Duran R.

Microbial biogeography of Acid Mine Drainage: a study of specific diversity and molecular

diversity, in Colloque Jacques Monod "Génomique écologique intégrative". Roscoff, France.

15-19 octobre 2011

Javerliat F, Volant A, Laoudi S, Bruneel O, Fahy A, Casiot C, Iniguez V, Nieto JM, Duran

R and Lauga B. Microbial biogeography of Acid Mine Drainage: a study of genetic diversity

and species diversity from an evolutionary perspective, Colloque Génomique

Environnementale. Lyon, France. 28-30 November 2011

Volant A, Bruneel O, Desoeuvre A, Casiot C, Bru N, Delpoux S, Héry M, Javerliat F,

Fahy A, Elbaz-Poulichet F, Duran R, Bertin P and Lauga B. Spatio-temporal dynamics of

bacterial community in the very As-rich creek waters of Carnoulès mine, France. In Ecole

Thématique Expert Génomique Environnementale. Aussois, France. 23-27 Avril 2012

Volant A, Bruneel O, Desoeuvre A, Casiot C, Bru N, Delpoux S, Héry M, Javerliat F,

Fahy A, Elbaz-Poulichet F, Duran R, Bertin P and Lauga B. Spatiotemporal dynamics of

bacterial community in the very As-rich creek waters of Carnoulès mine, France. ISME.

Copenhague, Danemark, 19-24 August 2012

Benyassine EM, Dekayir A, Héry M, Delpoux S, Desoeuvre A, Bruneel O, Benhassou H,

Rouai M, Casiot C.Contrasted arsenic speciation in two alkaline pit lakes from the abandoned

Pb mining area of Zeida (Moulouya, Morocco). International Symposium On Emerging

Pollutants in Irrigation Waters: Origins, Fate, Risks, and Mitigation. Tunisia : Hammamet.25-

28 November 2013

16

V ACTIVITE DE RECHERCHE

PREAMBULE

Lors de mon stage de DEA, effectué en 1997 au sein du CJF INSERM 93-09,

Immunologie des Maladies Infectieuses (Tours), j’ai travaillé sur la vaccination génique

contre la toxoplasmose en utilisant le gène SAG1. Suite à ce stage et désirant depuis

longtemps faire de la coopération mais n’ayant pas trouvé d’opportunités en biologie, je suis

partie 2 ans en tant que professeur des écoles en CE2, à Djibouti, dans le cadre d’une

coopération civile d’aide au développement. Cette expatriation ayant été extrêmement

enrichissante et afin de pouvoir trouver du travail plus facilement dans la coopération, j’ai

entrepris, à mon retour en France en 2000, une formation plus appliquée, un DESS «

Diagnostics, Prévention et Traitements en Environnement ». Déjà très intéressée par l’Institut

de Recherche pour le Développement, j’ai effectué mon stage de fin d’étude de 6 mois en

avril 2002 au laboratoire HydroSciences Montpellier, UMR 5569 (IRD, CNRS, Université de

Montpellier 1 et 2). Ce stage a porté sur l’identification des microorganismes présents dans les

eaux de drainage de la mine de Carnoulès (Gard) et sur l’isolement d’espèces actives sur

l’arsenic et le fer. La finalité de ces travaux était, à terme, d’aider au développement de

procédés passifs de bio-réhabilitation des effluents miniers et industriels en utilisant ces

microorganismes. Très intéressée par ce sujet et par les potentialités d’application de ce

travail, j’ai finalement poursuivi cette étude par une thèse au sein de cette même UMR. Après

ma thèse, soutenue en avril 2004, j’ai été recrutée à l’IRD en octobre de la même année pour

travailler sur l’implication des microorganismes dans les environnements miniers. Mon

programme de recherche s’intitule : «Etude des processus microbiens et géochimiques de

transfert des métaux et métalloïdes issus des activités minières».

L'écologie microbienne suscite un engouement très important car les microorganismes,

bien qu’invisibles à l’œil nu, sont essentiels à la vie sur terre. Ces microorganismes catalysent

en effet les transformations uniques et indispensables aux cycles biogéochimiques de la

biosphère de part leurs activités métaboliques. Ils produisent les composants essentiels de la

planète et représentent le plus grand réservoir de nutriments terrestre, comme le nitrogène et

le phosphore et séquestrent également environ 50% du carbone total des organismes vivants

(Whitman et al., 1998). Ils sont également les principaux recycleurs de matières en

décomposition, rendant disponible différents types de composés sous forme organique,

permettant ainsi la survie et le fonctionnement des écosystèmes (Whitman et al., 1998 ;

Falkowski et al., 2008). Parce que les microorganismes sont présents dans les 3 domaines du

vivant (Archaea, Bacteria et Eukarya) et qu’ils représentent les groupes les plus diversifiés

d’organismes sur Terre, une connaissance de leur diversité est primordiale pour la

compréhension du fonctionnement des écosystèmes et des processus planétaires (Pace, 1997 ;

Behnke et al., 2011).

17

L'exploitation minière est vitale pour l'économie mondiale, mais l'extraction des composés

métalliques génère de grandes quantités de déchets. Actuellement, le volume est estimé à

plusieurs milliers de millions de tonnes par an, mais est en augmentation exponentielle en

raison de la demande qui ne cesse de croître et de l'exploitation de gisements de faibles

teneurs (Hudson-Edwards and Dold, 2015). En l’absence d’une gestion extrêmement

rigoureuse des sites miniers, ces derniers sont une source de nuisance importante, en raison de

la présence de composés très toxiques comme le plomb, l’arsenic ou le mercure Leur

accumulation tout au long de la chaîne alimentaire génère des problèmes importants pour la

végétation, la santé animale et humaine.

Lorsque des minéraux sulfurés sont présents dans ces déchets, ils peuvent former, en

présence d’eau et d’oxygène, des effluents acides, riches en métaux et métalloïdes, appelés

Drainages Miniers Acides (DMA) (Langmuir, 1997). Ces drainages de mine sont considérés

comme l’une des plus importantes et pernicieuses forme de pollution des eaux provenant de

l’activité minière à travers le monde et représentent d’importants impacts environnementaux

et sociaux économique (Hallberg, 2010) avec des coûts de traitements estimés à plusieurs

milliards de dollars. Même s’il est très difficile d’estimer l’impact des DMA à travers le

monde, il a été suggéré que plus de 12 000 km de cours d’eau étaient touchés par les DMA

rien qu’au Royaume Uni (Hallberg, 2010). Le problème de ces DMA est leur potentiel de

menace à long terme, avec une production généralement étalée sur des dizaines, voire des

centaines d’années après la fermeture des mines (Younger, 1997 ; Hallberg, 2010). Bien que

ces milieux soient très hostiles en raison des conditions extrêmes de vie en termes de pH et de

concentration en métaux et métalloïdes toxiques, de nombreux microorganismes (Bactéries,

Archaea et Eucaryotes), naturellement présents, sont capables de s’y développer (Baker and

Bandfiel, 2003 ; Jonhson and Hallberg, 2003). Ces microorganismes adaptés jouent un rôle

essentiel car ils sont impliqués dans les mécanismes biogéochimiques contrôlant le

comportement des métaux et métalloïdes, qui sont présents dans l’environnement sous

différentes formes chimiques qui n’ont ni la même toxicité, ni la même mobilité. Les

réactions d’oxydoréduction ou de méthylation sont généralement très lentes et nécessitent la

plupart du temps une catalyse qui est bien souvent assurée par les microorganismes. Par

exemple, le rôle clé de l’activité des microorganismes (et notamment ceux qui oxydent le fer)

est connu depuis longtemps dans les réactions d’oxydation de la pyrite à l’origine de

l’apparition des DMA (Edwards et al., 2000a ; Sand et al., 2001 ; Vera et al., 2013). Selon

certains auteurs, l’activité microbienne serait à l’origine d’environ 75% de la production des

DMA (Edwards et al., 2000b ; Baker and Banfield, 2003). Ces mêmes organismes qui

oxydent le fer sont également susceptibles de promouvoir, dans l’eau, la formation d’oxydes

de fer qui favorisent l’immobilisation des métaux en les coprécipitant ou en les adsorbant

(Johnson and Hallberg, 2003, 2005 ; Johnson, 2014). Les bactéries sulfato-réductrices sont

aussi capables d’immobiliser des métaux en favorisant la précipitation directe de sulfures

métalliques généralement insolubles (Johnson and Hallberg, 2005). De plus, certains

processus métaboliques vont également modifier la mobilité de l’élément toxique et/ou sa

toxicité. Par exemple, la forme oxydée As(V) produite par Thiomonas sp. est considérée

comme 60 fois moins toxique pour les organismes supérieurs que la forme réduite As(III) à

pH acide. Ces quelques exemples illustrent le rôle des microorganismes dans les processus de

18

précipitation, complexation, adsorption, remise en solution et la distribution des différentes

formes chimiques en solution.

De plus, les stériles miniers sont généralement constitués de particules très fines facilement

transportées par la pluie et le vent, polluants les terres agricoles, les cours d’eau ou les puits

environnants et générant un problème de santé publique majeur pour les populations alentours

(Mendez and Maier, 2008). Depuis une quinzaine d’années, des travaux se sont intéressés à

l’utilisation de plantes pour limiter l’impact de cette pollution ; les déchets pouvant être

immobilisés par la mise en place d’un couvert végétal (phytostabilisation) ou être accumulés

dans les tissus végétaux (phytoextraction, Ma et al., 2011). Alors que l'établissement d'un

couvert végétal sur ces déchets minier reste un défi, les microorganismes peuvent fortement

accélérer le processus de phytostabilisation en influençant la croissance des plantes grâce à

différents mécanismes (fixation d’azote, solubilisation du phosphate, production de

phytohormones, etc., Rajkumar et al., 2012). Ils peuvent aussi intervenir directement sur la

mobilisation/immobilisation des métaux et métalloïdes dans le sol (production de

sidérophores, d’enzymes, etc. ou transformation rédox de ces éléments (Ma et al., 2011 ;

Rajkumar et al., 2012).

Les microorganismes jouent ainsi un rôle primordial dans ces environnements. Leur

connaissance présente donc un intérêt fondamental majeur pour la gestion et la remédiation

des sites contaminés.

Mon programme de recherche s’inscrit dans le cadre de l’axe 1 (Biogéochimie,

Contaminant, Santé) de l’UMR HydroSciences Montpellier qui aborde les questions de

pollution et de toxicité pour les écosystèmes aquatiques. Cet axe s’intéresse également aux

aspects de bioréhabilitation et de recyclage des eaux. L’étude des pollutions d’origine minière

a commencé il y a maintenant une 20aine

d’années au laboratoire. D’abord principalement

centrée sur les aspects purement géochimiques puis microbiologiques, cette équipe s’intéresse

maintenant également à l’impact de ces polluants métalliques sur la santé grâce au

recrutement d’un médecin et d’une géographe épidémiologiste. Au sein de cette équipe, je

m’intéresse à la partie microbiologie et principalement au rôle des microorganismes dans le

transfert des polluants inorganiques. Ce travail inclut à la fois de la microbiologie classique

par isolement mais aussi de la biologie moléculaire et maintenant de la génomique. L’équipe

de microbiologie comprend actuellement une Assistante Ingénieure depuis 2010 ainsi qu’une

maître de conférences recrutée en janvier 2011.

19

TRAVAUX ANTERIEURS

J’ai commencé ma première activité de recherche en 1997 au sein du CJF INSERM 93-09,

Immunologie des Maladies Infectieuses (Tours), à l’occasion de mon stage de DEA sur la

vaccination génique contre la toxoplasmose en utilisant le gène SAG1. La toxoplasmose est

une maladie infectieuse très répandue qui touche les animaux à sang chaud dont l’Homme en

raison de la présence d’un parasite protozoaire, Toxoplasma gondii. Généralement bénigne

chez l’homme et asymptomatique dans 90% des cas, ce parasite peut menacer la vie lors

d’une immunodépression ou peut avoir de graves conséquences pour le fœtus lors de la

contamination d’une femme pendant la grossesse (primo-infection) en raison de la

transmission transplacentaire du parasite. Durant mon stage, des essais vaccinaux, utilisant de

l’ADN codant une des protéines de Toxoplasma gondii, le gène SAG1 (vaccination à ADN),

ont été réalisés chez la souris qui présente des formes de toxoplasmose très proches de la

toxoplasmose humaine. Cette étude a montré une bonne réponse du système immunitaire de la

souris avec la production d’anticorps mais un taux de survie très faible lors de l’immunisation

intramusculaire1.

Depuis mon stage de DESS en avril 2001, je m’intéresse à l’implication des

microorganismes dans les biotransformations et processus de transfert des métaux et

métalloïdes dans les drainages miniers acides. Ce programme de recherche a pour objectif de

mieux comprendre les processus biogéochimiques qui contrôlent les transferts de métaux et

métalloïdes, en particulier l’arsenic, et d’étudier de manière pluridisciplinaire et intégrée le

fonctionnement de ces environnements extrêmes. Il se situe en effet à l’interface de la

microbiologie, de la géochimie, mais également de l’hydrogéologie et de la minéralogie. En

microbiologie, pour l’étude du chantier de Carnoulès, ce programme de recherche fédère

différentes compétences apportées par plusieurs équipes d’autres laboratoires comme la

métagénomique ou la métaprotéomique (collaboration étroite avec l’EEM de Pau (R. Duran,

B. Lauga) et le laboratoire GMGM de Strasbourg (P. Bertin, F. Ploetze) ou la minéralogie

(partenariat avec G. Morin, IMPMC, IPGP de Paris). Ces recherches incluent également la

caractérisation physicochimique approfondie de ces environnements extrêmes par les

chimistes du laboratoire (C. Casiot, F. Elbaz-Poulichet, MA. Cordier puis S. Delpoux). Ces

approches combinées permettent d’obtenir une vision globale et intégrée des processus

complexes qui conditionnent les interactions entre les microorganismes et leur

environnement.

1 Mévelec MN, Bout D, Desolme B, Marchand H, Magne R, Bruneel O, Buzoni-Gatel D (2005) Evaluation of

protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF

plasmid, against acute, chronical and congenital toxoplasmosis in mice. Vaccine. 23, 4489-4499

20

Introduction à l’étude des drainages miniers acides

L’intérêt pour l’étude de ces écosystèmes extrêmes est multiple. Comme nous l’avons vu,

ces microorganismes présentent tout d’abord un grand intérêt pour la gestion des déchets

miniers et leur connaissance est primordiale pour mieux gérer leurs impacts sur

l’environnement et est également critique pour pouvoir continuer l’exploitation des ressources

minérales, dont la demande ne cesse de croitre à travers le monde (Hallberg, 2010). Les

microorganismes qui peuplent ces écosystèmes extrêmes sont généralement constitués de

communautés simplifiées par rapport aux environnements plus hospitaliers (Tyson et al.,

2004 ; Denef et al., 2010). Ceci est dû notamment aux pressions de sélection qu’impose

l’adaptation des microorganismes à ces environnements, ainsi que par le nombre limité de

sources d’énergie disponible dans le milieu (Baker and Banfield, 2003). Ces environnements

sont donc colonisés par des espèces dites spécialistes, généralement peu abondantes, ce qui en

font d’excellents modèles pour étudier la dynamique des microorganismes dans le temps et/ou

l’espace, d’identifier les paramètres qui les gouvernent, d’étudier leurs capacités d’adaptation,

de mieux comprendre leurs interactions et d’explorer les fonctions qu’elles exercent (Denef et

al., 2010). Les DMA représentent également des habitats fragmentés, qui possèdent chacun

des conditions physicochimique (T°, pH, concentration en oxygènes) et/ou des concentrations

en métaux et métalloïdes différents, permettant ainsi d’aborder des questions particulières de

biogéographie (Hallberg, 2010 ; Kuang et al., 2012). L’étude de la diversité de ces

environnements extrêmes suscite également un intérêt important du fait que ces écosystèmes

peuvent représenter un réservoir de nouvelles biomolécules ayant un intérêt biotechnologique.

Enfin, les similarités qui existent entre la minéralogie de ces environnements, comme celui du

Rio Tinto en Espagne, et de la planète Mars (vaste dépôts de sulfates et d’oxydes de fer) ont

conduit à l’idée que les propriétés de ces acidophiles pourraient être similaires à ceux

susceptibles d’être retrouvés sur Mars (Amils et al., 2007).

Parmi les éléments toxiques des DMA, l’arsenic pose un problème particulier parce qu’il

est fortement assimilable par les organismes vivants du fait que ses propriétés chimiques sont

très voisines de celles du phosphore et du soufre, qui sont des éléments essentiels à la vie

(Yammura and Amachi, 2014). Chez l’être humain, il est toxique et induit de nombreuses

pathologies dont des cancers et ne devrait pas dépasser 10 µg.l-1

selon l’OMS (Yamanaka et

Okada, 1994 ; McClintock et al., 2012 ; Jiang et al., 2013). L’arsenic présent dans l’eau de

boisson représente un problème mondial majeur qui touche plusieurs millions de personnes,

en particulier au Bangladesh où 35-77 millions de personnes sont concernés (Nordstrom,

2000; Argos et al., 2010 ; Yunus et al., 2011) mais cela touche également de nombreux pays

comme les Etats-Unis, la Chine, le Mexique, l’Espagne ou le Canada, etc. (Jiang et al., 2013).

Malgré sa faible abondance dans la croûte terrestre (0.0001%), il est largement distribué dans

l’environnement où il est souvent associé avec les minerais métalliques sulfurés comme le

cuivre, le plomb ou l’or, etc. (Oremland and Stolz, 2003). Dans les sols, il est généralement

retrouvé à des concentrations inférieures à 15 mg.kg-1

(Yammura and Amachi, 2014).

Bien que l’arsenic existe sous 4 états d’oxydation différents (V, III, 0, -III) avec une

multitude de formes organiques et inorganiques, l’arséniate (As(V)) et l’arsénite (As(III) sont

les formes inorganiques prédominantes dans l’environnement (Ormeland and Stolz, 2005),

avec l’As(III) considéré comme plus toxique que l’As(V) (Lièvremont et al., 2009 ; Yammura

21

and Amachi, 2014). L’As(V) est généralement présent sous forme d’oxyanions chargés

négativement (H2AsO4-/ HAsO4

2-) à pH modéré et a ainsi tendance à être fortement adsorbé

sur la surface de nombreux minéraux chargés positivement, comme les oxydes et hydroxydes

de fer et d’aluminium. L’As(III) est quand à lui généralement présent sous une forme non

chargée (H3AsO30) dans l’environnement et est donc habituellement moins adsorbé et donc

plus mobile que l’As(V) (Yammura and Amachi, 2014). Dans les environnements aérobies,

l’As(V) est souvent la forme prédominante alors qu’en conditions anoxiques, c’est la forme

As(III) qui prédomine.

Certains microorganismes ne sont pas seulement résistant à l’As mais le métabolisent

activement via des réactions de méthylation, déméthylation, oxydation ou réduction,

modifiant ainsi les formes redox de l’As et utilisant certaines de ces étapes pour générer de

l’énergie (Oremland and Stolz, 2005 ; Stolz et al., 2010). A ce jour, de nombreux

microorganismes, principalement des bactéries capables d’oxyder ou de réduire l’As, ont été

isolés d’environnements contaminés par l’arsenic (Oremland and Stolz, 2003 ; Lièvremont et

al., 2009). La réduction de l’arséniate comprend une voie de détoxification (gène arsC) ainsi

que la respiration (gènes arrA/B). L’organisation de l’opéron ars varie fortement entre les

taxons et les gènes de base inclus arsR, arsB et arsC tandis que arsD et arsA peuvent

également parfois être trouvés (Oremland and Stolz, 2003). Les gènes arrA/B codent une

enzyme réductase active durant la respiration anaérobie, utilisant l’As(V) comme accepteur

final d’électron (Costa et al., 2014). L’oxydation microbienne de l’As(III), décrite pour la

première fois en 1918, peut être médiée par 2 enzymes distinctes, Aio (comprenant gènes aox,

aso et aro) très étudié et Arx récemment décrite par Zargar et al. (2012). L’oxydation aérobie

de l’As(III) est catalysée par une arsénite oxidase qui utilise l’O2 comme accepteur terminal

d’électrons et qui est codée par les gènes aioB/A (Lett et al., 2012 ; Costa et al., 2014). ArxAB

est détectée chez des bactéries oxydant As(III) en conditions anoxiques, où la réduction du

nitrate ou du chlorate est couplée à l’oxydation de l’As(III) (Oremland et al., 2009 ; Sun et al.,

2010 ; Costa et al., 2014). Certains membres du genre Ectothiorhodospira sont également

capables d’utiliser l’As(III) comme donneur d’électrons pour la croissance phototrophe

anoxygénique (Kulp et al., 2008). Parce que ces processus de réduction d’As(V) ou

d’oxydation d’As(III) affectent directement la spéciation et la mobilité de l’As, l’activité

microbienne joue un rôle clé dans les cycles biogéochimiques de ce métalloïde et peuvent être

utilisés pour dépolluer les sols et les eaux pollués par l’arsenic (Yammura and Amachi, 2014 ;

Costa et al., 2014 ; Sarkar et al., 2014).

L’ancien site minier de Carnoulès a constitué pour le laboratoire HydroSciences un cadre

privilégié pour l’étude des interactions entre les microorganismes et les polluants métalliques

et notamment l’arsenic (Leblanc et al., 1996). L’intérêt dans ce site réside également dans le

fait qu’un système de remédiation naturel est présent où, près de 99% de l’arsenic va

précipiter et être piégé dans des minéraux de fer le long des 1,5 km du Reigous, petit ruisseau

alimenté par les drainages miniers acides du stérile de Carnoulès (Leblanc et al., 1996). Enfin,

la proximité géographique de ce site avec le laboratoire HydroSciences est un facteur non

négligeable étant donné les nombreux allers-retours nécessaires pour étudier cet

environnement sur le long terme. Ce site atelier est, depuis 2009, un site d’observation de

22

l’Observatoire des Sciences de l’Univers OREME (tâche d’observation intitulé « Suivi des

processus hydrobiogéochimiques de transfert des métaux et métalloïdes issus des activités

minières sur le site de Carnoulès »). Les connaissances acquises sur ce site avec mes

collègues du laboratoire HSM, M. Leblanc, (géologue), J.-C. Personné puis A. Desoeuvre (AI

depuis 2010) et M. Héry en 2011 (microbiologistes) ; F. Elbaz-Poulichet et C. Casiot

(géochimistes) en association avec G. Morin (minéralogiste à l’IMPMC, Paris) et en

collaboration avec 2 laboratoires de microbiologie, l’EEM de Pau (R. Duran, B. Lauga) puis

le laboratoire GMGM de Strasbourg (P. Bertin, F. Ploetze) ont permis de mieux comprendre

cet écosystème et ont ainsi contribué à fédérer différents groupes de recherches sur ce site.

Description du site minier de Carnoulès

La mine de Carnoulès est située dans les Cévennes dans le Sud de la France et a été

définitivement fermée en 1962.

Figure 1. Localisation et carte du site minier de Carnoulès. D’après Bruneel et al., 2005

23

Au Sud-Est du Massif Central, le long des Cévennes, un horizon conglomératique de 3 à 5

m d’épaisseur contenant de la marcasite (et/ou de la pyrite), de la galène, de la barytine et

accessoirement de la sphalérite, des sulfo-arséniures (proustite, arsénopyrite) et des sulfure

d’antimoine (freibergite) est présent au niveau de la mine de Carnoulès (Leblanc et al., 1996).

Le gisement de 2.5 Mt contenait 3.5% Pb et 0.8% Zn et a été principalement exploité à ciel

ouvert puis définitivement abandonné en 1962. Le stérile actuel, d’environ 1.2 Mt qui est

confiné derrière une digue, comporte les déchets d’après traitement qui contiennent encore

environ 0.7% de Pb et 10% de sulfure de Fe, (Leblanc et al., 1996).

Figure 2. Schéma simplifié du dépôt de stériles miniers de Carnoulès avec en (a) la localisation du

forage instrumenté, des carottages réalisés sur le site (T1, T4) et du système de drainage et en (b) une

coupe dans le dépôt montrant les différents horizons, la couverture d’argile en surface, les sables gris

fins et riches en pyrite, les sables grossiers et le socle composé de quartzites du Trias (b). D’après

Casiot et al., 2003a

Ce stérile a une superficie de 5500 m2 et une épaisseur de 10 à 24 m. Il est recouvert d’une

couche d’argile de 0.3 m d’épaisseur. En dessous, il est constitué majoritairement de sables à

pyrite contenant 75% de quartz et entre 5 et 15 % de pyrite qui contient de 1 à 4% d’As. Les

minéraux secondaires incluent le K-feldspath, la biotite, la barytine et la galène (Alkaaby et

al., 1985). Ces matériaux sont généralement très fins (taille moyenne des grains de 30 µm) et

peu perméables, excepté près du fond où une couche de 2 à 3 mètres d’épaisseur contient du

matériel ferrugineux relativement grossier (200 µm) (Koffi et al., 2003). L’oxydation des

sulfures est limitée dans la partie supérieure du dépôt, contrairement à ce qui est généralement

constaté dans d’autres stériles miniers et est probablement dû à la présence d’une couverture

argileuse peu perméable et à la faible conductivité hydraulique du matériau qui limite

l’infiltration des eaux de pluie (Koffi et al., 2003). Dans la partie inférieure du dépôt au

contraire, les sulfures sont partiellement oxydés en liaison avec la présence d’un drain et la

circulation d’eaux à la base du stock, dans une zone à matériaux plus grossiers très

probablement en raison de la présence de sources enterrées présentes sous le stérile (Koffi et

24

al., 2003). Le niveau de l’eau se situe entre 1 et 10 m sous la surface en fonction de la

localisation dans le stérile et de la saison.

L’eau qui circule dans le stock de déchet donne naissance au ruisseau du Reigous dont la

source apparaît à la base de la digue qui retient les déchets. La masse d’arsenic contenu dans

ce stock de stériles est estimée à 3000 t. Compte tenu de la masse annuelle d’As rejetée par la

source acide (6 t), la durée de vie du système est estimée à au moins 500 ans (Leblanc et al.,

2002). Les études physicochimiques ont montré que le débit à la source est relativement faible

(0.2 à 1 l.s-1

) mais ces eaux coulent toute l’année. Elles sont pratiquement anoxiques à la

source mais en quelques dizaines de mètres, on observe une augmentation de la concentration

en oxygène. Les flux annuels d’arsenic, calculés au cours de 2 années hydrologiques aux

caractéristiques différentes, varient de 2 à 6 t. Les concentrations en As diminuent rapidement

en aval, juste avant le confluent avec l’Amous, elles sont en moyenne de 6 mg.l-1

avec de très

fortes variations saisonnières (Leblanc et al., 1996). Ces diminutions sont à attribuer en partie

à des dilutions avec de petits rus latéraux mais surtout à la précipitation de l’arsenic et à la

formation de sédiments riches en fer et en arsenic. Les variations saisonnières du système du

Reigous sont fortement marquées : en période d’étiage, les sédiments arséniés s’accumulent

mais, en période de fortes pluies (printemps, automne), les sédiments sont érodés et

transportés, entraînant une forte augmentation du flux d’arsenic avec un transport

essentiellement sous forme particulaire (Leblanc et al., 2002).

A mon arrivée au laboratoire HydroSciences dans le cadre de mon stage de DESS en 2002,

les travaux de Leblanc et al. (1996) avaient permis de mettre en évidence à Carnoulès, dans le

ruisseau du Reigous qui draine le site, la formation de précipités contenant près de 20%

d’arsenic autour de structures bactériennes, mais les processus géochimiques et

microbiologiques à l’origine de la formation de ces solides n’étaient pas connus. Durant ce

stage, sous l’encadrement de Jean Christian Personné, j’ai isolé une vingtaine de colonies

bactériennes dans les eaux du stock de déchets miniers ainsi que dans les eaux le long du

Reigous et j’ai commencé leurs études en laboratoire et en particulier, leurs activités sur

l’oxydation du fer et de l’arsenic. La grande majorité de ces souches ont été identifiées

comme étant des bactéries des genres Thiomonas et Acidithiobacillus ferrooxidans. Ce travail

a permis de décrire plusieurs souches de Thiomonas et de montrer pour la première fois que

des souches pures de Thiomonas étaient capables d’oxyder l’arsenic2.

Suite à ce travail, j’ai débuté en décembre 2002 une thèse intitulée « Contribution à l'étude

des mécanismes couplés géochimiques et bactériologiques de transfert de la pollution minière

sur le site de Carnoulès (Gard) » sous l’encadrement de Jean Christian Personné et de

François Elbaz Poulichet, ma directrice de thèse. Bien qu’apportant des informations très

2 Bruneel O, Personné J-C, Casiot C, Leblanc M, Elbaz-Poulichet F, Mahler BJ, Le Flèche A, Grimont PAD. (2003)

Mediation of arsenic oxidation by Thiomonas sp. in acid mine drainage (Carnoulès, France). Journal of Applied

Microbiology. 95, 492-499

25

intéressantes sur le métabolisme des souches isolées, les techniques classiques d’isolement de

souches pures et la caractérisation de leurs activités en laboratoire ne permettent pas de

comprendre un écosystème étant donné que près de 99% des organismes ne peuvent être pour

l’instant isolés par des approches culturales (Rappé and Giovannoni, 2003). J’ai donc

rapidement été amenée à travailler avec le Laboratoire d’Ecologie Moléculaire (R. Duran, EA

3525, Ecologie Moléculaire Microbiologie de l’Université de Pau) pour mettre en œuvre une

approche moléculaire qui n’était pas disponible, à l’époque, au Laboratoire HydroSciences.

Processus de génération du drainage minier acide riche en As de Carnoulès

Les eaux de drainage de mines sont générées par l’exposition des minerais sulfurés, telles

que la pyrite (FeS2) à l’oxygène et à l’eau (Johnson and Hallberg, 2003 ; Vera et al., 2013).

De nombreux métaux sont présents sous forme de minerais sulfurés, comme la galène (PbS)

ou la sphalérite et sont également souvent associés à la pyrite qui est le minerai sulfuré le plus

commun. Le fer ferrique (Fe(III)) est le principal oxydant des minerais sulfurés (Baker and

Banfield, 2003) :

FeS2 + 14 Fe3+

+ 8 H2O → 15 Fe2+

+ 2 SO42−

+ 16 H+

La régénération du Fe(III), selon l’équation ci-dessous, est l’étape limitante de l’oxydation

des minerais et nécessite de l’oxygène (Singer and Stumm, 1970) :

14Fe2+

+ 3.5 O2 + 14H+ → 14Fe

3+ + 7H2O

A pH supérieur à 4, l’oxydation du fer ferreux se produit chimiquement en présence

d’oxygène ou biologiquement mais à des pH inférieures à 4, le taux d’oxydation chimique est

très lent, voir négligeable et c’est l’activité des microorganismes oxydant le fer qui va avoir

un rôle pivot dans la génération des DMA (Baker and Bandfield, 2003 ; Vera et al., 2013). De

plus, en raison des faibles pH rencontrés dans ces environnements (jusque -3 comme dans la

mine de Richmond aux Etats Unis (Californie, Nordstrom et al 2000), la solubilité des métaux

est plus importante et les DMA contiennent donc généralement de très fortes concentrations

en métaux et métalloïdes qui vont varier en fonction de la minéralogie de la roche d’origine

(Hallberg, 2010).

Des études réalisées au sein du piézomètre S5, situé approximativement au centre du stérile

minier en 2001 et 2002, ont montré de très fortes variations de la chimie sur une année qui

semblaient être liées au niveau de la nappe et aux concentrations en oxygène dissous3. En

période de remontée de la nappe, le niveau d’oxygène est très élevés (7-9 mg.l-1

), le pH est

acide (1.8), et de très fortes concentrations de fer (proche 20000 mg.l-1

) et d’As (jusque 12000

mg.l-1

, concentrations parmi les plus importantes au monde) ont été relevées avec les espèces

oxydantes qui dominent (As(V) et Fe(III)). Ces teneurs très élevées ont été attribuées à la

dissolution de phases secondaires, en particulier des hydroxysulfates de fer contenant jusque

10% d’As, présents dans le stock de déchets. A l’inverse, lorsque le niveau de la nappe

diminue et que le milieu devient pratiquement anoxique (DO = 0.5 mg.l-1

), le pH remonte

autour de 4 et les concentrations en As et en Fe diminuent fortement et se stabilisent (autour

3 Casiot C, Leblanc M, Bruneel O, Personné J-C, Koffi K, Elbaz-Poulichet F (2003) Geochemical processes

controlling the formation of As-rich waters within a tailings impoundment. Aquatic Geochemistry. 9, 273-290

26

de 3000 mg.l-1

pour Fe et 750 mg.l-1

pour As) avec As et Fe principalement sous forme

réduite As(III), Fe(II).

Dans le cadre de ma thèse et pour tenter de mieux comprendre la génération de ces eaux

acides et riches en métaux ainsi que les variations associées, j’ai initié l’étude des bactéries

présentes par des approches moléculaires ciblant l’ARNr 16S par les techniques de clonage-

séquençage. Ces analyses nous ont permis de mettre en évidence que la diversité était faible

comparée à des eaux non polluées avec un total de 5 taxons identifiés ici4.

Table 1. Inventaire des fragments d’ADNr 16S des clones présents en octobre 2001 (S5Oct) et janvier

2002 (S5Jan) dans les eaux du stock de déchets miniers, groupés selon l’analyse RFLP et l’analyse

phylogénétique. D’après Bruneel et al., 2005

Ce travail a également montré que ce sont curieusement des groupes proches des bactéries

sulfato-réductrices (BSR, Desulfosarcina variabilis) qui dominent et ceci principalement

lorsque les concentrations en oxygène sont élevées et le pH très bas alors que ces bactéries

sont pourtant connues pour préférer les conditions anoxiques. En octobre, quand le taux

d’oxygène est faible, on trouve des organismes dont les séquences sont affiliées à

Desulfosarcina variabilis (représentant environ 27% du nombre total de clones) associés à des

séquences apparentées à Acidithiobacillus ferrooxidans, Thiobacillus et Acidimicrobium alors

qu’en janvier, lorsque les conditions sont très oxygénées et le pH très acide, la communauté

bactérienne est composée essentiellement de Desulfosarcina variabilis (représentant environ

95%) associée à Acidithiobacillus ferrooxdians et Thiobacillus spp.

4 Bruneel O, Duran R, Koffi K, Casiot C, Fourçans A, Elbaz-Poulichet F, Personné J-C (2005) Microbial diversity in

a pyrite-rich tailings impoundment (Carnoulès, France). Geomicrobiology Journal. 22, 249 - 257

27

Etude du système de remédiation présent sur le site de Carnoulès

Contrairement aux composés organiques qui peuvent être dégradés en composés simples et

sans risque pour la santé comme le CO2 ou l’H2O du fait de leur minéralisation, la

remédiation des métaux et métalloïdes implique seulement leur retrait de la solution dans le

milieu aquatique (Bahar et al 2013). Cette remédiation de l’eau est due à des réactions

biotiques et abiotiques qui font que ces composés toxiques deviennent insolubles et

précipitent, s’accumulant dans des sédiments composés généralement d’une variété

d’(oxyhydr)oxydes et d’hydroxysulfates de fer tel que la jarosite, la schwertmannite ou la

ferrihydrite (Johnson and Hallberg, 2005; Johnson, 2014). Ces processus de précipitation

résultent en grande partie de l’oxydation et de la précipitation du fer, qui est souvent le

principal métal soluble présent dans le DMA, et de l’adsorption d’autres métaux et

métalloïdes comme le Pb, l’U ou As sur les minéraux sulfurés formés (Hallberg, 2010).

Comme l’oxydation abiotique du Fe(II) est un processus très lent dans les eaux acides, les

microorganismes oxydants le fer qui catalysent ces réactions jouent un rôle pivot dans les

processus de remédiation (Rowe and Johnson, 2008; Hallberg, 2010; Johnson, 2014).

A Carnoulès, les études de Leblanc et al. (1996, 2002) avaient révélé la présence d’un

système de remédiation naturel efficace dans les eaux du Reigous qui permettait de limiter les

concentration de l’As en aval du système. Pour identifier les processus chimiques et

microbiologiques qui influencent le transfert de l’As dans le Reigous, des échantillons d’eau

ont été prélevés lors de 8 campagnes de prélèvements en 2001. Les stations de prélèvement

étaient situées dans les 30 premiers mètres du ruisseau où aucun apport latéral d’eau n’avait

été observé.

Figure 3. Coupe montrant la localisation des stations de prélèvements dans les 30 premiers mètres du

ruisseau du Reigous (1, A, C, E, F, 2). Le temps d’écoulement des eaux entre les stations 1 et 2 est

d’environ 1 heure. D’après Casiot et al., 2003b

Les analyses physicochimiques réalisées dans le ruisseau du Reigous qui draine le site ont

montré que l’arsenic en solution est essentiellement sous forme réduite (As(III)) à la source

du Reigous5. Sur les 30 premiers mètres, 20 à 60% de l’arsenic coprécipite en liaison avec

5 Casiot C, Morin G, Juillot F, Bruneel O, Personné JC, Leblanc M et al. (2003b) Bacterial immobilization and

oxidation of arsenic in acid mine drainage (Carnoulès creek, France). Water Res. 37, 2929-2936

28

l’oxydation du Fe(II) en Fe(III). Les formes méthylées sont absentes. Le taux de précipitation

est variable selon les saisons. Il semble plus important pendant la saison humide lorsque la

teneur en oxygène dans les eaux à la source est plus élevée.

Pour tenter de mieux comprendre ce système de remédiation, des études moléculaires

combinées (comprenant des analyses de clonage et séquençage par la méthode de Sanger ainsi

que des analyses t-RFLP) ont été réalisées afin d’identifier les communautés bactériennes

présentes6.

Table 2. Inventaire des fragments d’ADNr 16S des clones présents dans les eaux du ruisseau du

Reigous groupés selon l’analyse RFLP et l’analyse phylogénétique. D’après Bruneel et al, 2006

a abondance relative des clones dans chaque librairies

Ces analyses ont montré, comme au sein du stérile, une diversité faible avec

l’identification de 2 à 4 taxons par échantillons. De plus, comme attendu en raison de la

chimie de l’eau, les résultats ont mis en évidence que les eaux du ruisseau acide étaient

largement dominées par des bactéries impliquées dans le cycle du fer et du soufre. Les

séquences affiliées à la bactérie neutrophile qui oxyde le Fe, Gallionella ferruginea, sont

largement dominante. Cette bactérie pourrait jouer un rôle important dans la remédiation

naturelle observée dans le ruisseau acide en immobilisant l’As par coprécipitation avec le

Fe(III).

La structure et la spéciation de l’As dans les sédiments du Reigous ont été également

caractérisées par des analyses spectroscopiques et minéralogiques (XRD, XANES et SEM)7.

Cette étude a mis en évidence des variations spatiales et temporelles des précipités formés

dans le ruisseau. Pendant la saison humide, les précipités présents dans les 10 premiers mètres

du ruisseau consistent essentiellement en tooéléite (un minéral rare de Fe6(AsO3)4-

(SO4)(OH)4•4H2O) associée à des précipités amorphes d’As(III)-Fe(III). Pendant la saison

sèche, la formation d’un oxyhydroxyde de Fe(III)-As(V) amorphe prédomine.

6 Bruneel O, Duran R, Casiot C, Elbaz-Poulichet F, Personné JC (2006) Diversity of microorganisms in Fe-As-rich

acid mine drainage waters of Carnoulès, France. Applied and Environmental Microbiology. 72, 551-556 7 Morin G, Juillot F, Casiot C, Personné JC, Elbaz-Poulichet F, Leblanc M, Ildefonse P, Calas G (2003) Bacterial

formation of tooeleite and mixed arsenic(III) or arsenic(V)-iron(III) gels in the Carnoulès Acid Mine Drainage,

France. A XANES, XRD, and SEM study. Environmental Science and Technology. 37, 1705-1712

29

Dans la poursuite des études réalisées dans le cadre du DESS, le rôle des bactéries isolées

(Thiomonas et Acidithiobacillus ferrooxidans) dans les réactions de précipitation de l’As et du

Fe a été testé grâces à des études en laboratoire. Six souches (B1 à B6) isolées à partir de

l’eau du ruisseau ont été inoculées individuellement dans l’eau de la source. B1, B2, B3 et B6

sont des souches du genre Thiomonas et B4 et B5 sont des Acidithiobacillus ferrooxidans. En

parallèle, les précipités formés par les souches B1 à B6 ont été analysés.

Figure 3. Essais en laboratoire présentant le pourcentage d’As total (AsT), de Fe(II) et d’As(III)

éliminés après une semaine d’incubation de différentes souches de microorganismes (B1 à B6) isolées

à partir de l’eau du Reigous avec les concentration en As(V) en solution en fin d’expérience. S1: eau

de la source avant incubation et SA: témoin stérile. D’après Casiot et al., 2003b

Ces études ont montré que 3 souches de Thiomonas ont la capacité d’oxyder l’As(III) dans

l’eau du Reigous: B2, B3 et B6. C’est la souche B6, qui du fait de l’oxydation simultanée de

l’arsenic et du fer, entraîne le plus grand battement d’As (87%)5.

Les précipités du témoin abiotique, tout comme dans ceux des bactéries B1, B2, B3, B4 et

B6, sont constitués essentiellement d’hydroxydes sulfates ferriques d’As(V)7. Une souche

d’Acidithiobacillus ferrooxidans (bactérie B5) permet la formation de tooéléite nanocristalline

associée à un mélange de composés d’oxyhydroxydes d’As(III)/As(V)-Fe(III) amorphes.

Des études avec d’autres souches d’Acidithiobacillus ferrooxidans ont également montrées

que ce genre était capable de faire précipiter rapidement l’arsenic avec le Fe(III) en milieux

synthétiques sous forme de schwertmannite8.

La bioremédiation de l’arsenic peut s’appuyer sur l’activité des microorganismes qui ont la

capacité de détoxifier, mobiliser ou immobiliser l’As à travers différents processus comme

l’oxydation, la réduction, la biométhylation, la sorption ou la complexation (Oremland et al.,

2005 ; Bahar et al., 2013). Etant donné que l’As(III) est fortement toxique et mobile dans

l’environnement, une stratégie de remédiation intéressante consiste généralement à le

convertir en As(V), forme moins toxique et mobile qui a tendance à se fixer sur différents

types de matrices (Bahar et al., 2013).

8 Duquesne K, Lebrun S, Casiot C, Bruneel O, Personné J-C, Leblanc M, Elbaz-Poulichet F, Morin G, and

Bonnefoy V (2003) Immobilization of arsenite and ferric iron by Acidithiobacillus ferrooxidans in acid mine

drainage. Applied and Environmental Microbiology. 69, 6165-6173

30

Aux cotés des bactéries, les eucaryotes, présents dans ces drainages miniers acides, ont

également développé des stratégies de résistance vis-à-vis de cet élément toxique dans

l’environnement. Euglena mutabilis est un protozoaire photosynthétique, communément

rencontré dans les eaux minières acides qui semble bien adapté aux conditions extrêmes qui y

règnent (Brake et al., 2001). Cet organisme peut jouer un rôle important dans les DMA en

contribuant à l’apport d’oxygène par leur activité photosynthétique, en séquestrant le fer et

probablement d’autres métaux par précipitation intracellulaire et en apportant de la matière

organique (Brake et al., 2001). A Carnoulès, les euglènes sont présentes en grand nombre

dans le Reigous et sont visibles grâce à la présence de tapis verts caractéristiques, pouvant

atteindre une épaisseur d’environ 1 cm. L’étude de la dynamique saisonnière de ces biofilms a

montré qu’elle n’est pas liée aux variations des concentrations en polluants métalliques mais à

l’effet de l’érosion mécanique du sédiment du fait des fortes précipitations9. Cultivés en

milieu synthétique en présence de 0.2 à 300 mg/l d’As(III), les euglènes issues du site de

Carnoulès accumulent l’As à l’intérieur de leurs cellules sous forme d’arsénite et d’arséniate

dont les concentrations varient en fonction des concentrations en As(III) du milieu de culture.

L’arsenic est également adsorbé à la surface de la cellule sous forme d’As(V).

9 Casiot C, Bruneel O, Personné J-C, Leblanc M, Elbaz-Poulichet F (2004) Arsenic oxidation and bioaccumulation

by the acidophilic protozoan, Euglena mutabilis, in acid mine drainage (Carnoulès, France). Science of the Total

Environment. 320, 259-267

31

L’étude de cet écosystème s’est poursuivie après ma thèse dans le cadre de plusieurs

projets de recherche. Une étude sur les Archaea a été réalisée suite aux résultats de l’étude

moléculaire réalisée sur les bactéries présentes dans les eaux souterraines au sein du stock de

déchets miniers. Historiquement, on pensait en effet que les bactéries représentaient les

principaux microorganismes impliqués dans les processus de lixiviation à l’origine de la

formation des DMA mais il a été montré depuis plusieurs années maintenant que les Archaea

sont elles aussi susceptibles de jouer un rôle majeur, de part de leurs capacités à oxyder le fer

dans les processus de génération et/ou de remédiation des DMA (Edwards et al., 2000c ;

Baker and Banfield, 2003 ; Bini, 2010).

Table 3. Inventaire des fragments d’ADNr 16S des clones présents dans les eaux au sein du stock de

déchets miniers et dans le DMA du Reigous. D’après Bruneel et al., 2008

a abondance relative des clones dans chaque librairie

Cette étude a révélé que l’ensemble des séquences retrouvées était affilié au phylum des

Euryarchaeota, tandis que les Crenarchaeota n’étaient pas du tout présentes10

. Ce travail a

10

Bruneel O, Pascault N, Egal M, Bancon-Montigny C, Goni M, Elbaz-Poulichet F, Personné J-C, Duran R (2008)

Archaeal diversity in a Fe-As rich acid mine drainage at Carnoulès (France). Extremophiles. 12, 563-571

32

également montré que la structure des communautés d’Archaea dans l’aquifère au sein du

stock de déchet minier était très différente de celle présente dans les eaux du ruisseau du

Reigous drainant le site. Au sein de la nappe qui draine le stock de déchets, les séquences

majoritaires sont proches de Ferroplasma acidiphilum, un microorganisme acidophile, sans

paroi cellulaire, oxydant le fer et connu pour son rôle majeur dans le lessivage (Golyshina and

Timmis, 2005). Dans les eaux du Reigous, par contre, ce sont des séquences affiliées à un

groupe de Thermoplasmatales non cultivé, le clone YAC1, qui est largement dominant.

Une étude en collaboration avec le laboratoire EEM de Pau s’est également intéressée aux

bactéries sulfato-réductrice présentent sur ce site pour essayer de déterminer l’influence des

paramètres environnementaux sur la structure de ces communautés par analyse t-RFLP et

étude des gènes dsrAB11

. Ce travail réalisé sur une période de 3 ans a permis de mettre en

évidence la présence prédominante de la famille Desulfobulbaceae dans le système et a

montré que la dynamique des bactéries sulfato-réductrices semble être liée aux fluctuations

spatio-temporelles du pH, du fer et des formes spécifiques de l’arsenic.

Pour tenter de mieux comprendre le fonctionnement du système de remédiation présent

dans le drainage minier acide de Carnoulès, les concentrations en arsenic et métaux ont été

suivis sur une période de plus de 4 ans sur l’ensemble du ruisseau. Cette étude a mis en

évidence que les variations saisonnières semblaient être liées aux précipitations avec une

augmentation des concentrations durant les mois secs12

. Environ 30% de l’As initialement

présent en solution se trouve sous forme d’As(III) qui coprécipite avec le fer dans les 40

premiers mètres du ruisseau. La minéralogie de ces précipités varie spatialement et

saisonnièrement. Dans les 40 premiers mètres, on trouve des composés amorphes d’As(V)-

As(III)-Fe(III) associés à de la tooéléite alors que plus en aval, ces phases d’oxyde de Fe sont

remplacées par de la schwertmannite et de la ferrihydrite12

.

Des travaux en collaboration avec l’IMPMC de Paris, ont permis de montrer que ces

minéraux d’arsenic se forment généralement en étroite association avec des cellules

bactériennes dans le milieu extracellulaire ou dans le périplasme des cellules ainsi qu’autour

d’abondantes vésicules organiques d’origine inconnue13

.

Les conditions de formation de ces minéraux de fer très riches en As(III) et As(V) et

l’implication des bactéries dans ces processus ont été par ailleurs étudiées en laboratoire. Des

bactéries du genre Acidithiobacillus ferrooxidans inoculées dans l’eau du Reigous conduisent

à la formation d’un assemblage de minéraux (schwertmannite, tooéléite) qui est différent de

celui obtenu en conditions abiotiques où l’on trouve généralement de la jarosite. De plus, la

proportion des minéraux formés semble différer selon la souche d’Acidithiobacillus

11

Giloteaux L, Duran R, Casiot C, Bruneel O, Elbaz-poulichet F and Goñi-urriza M (2013) A survey of sulfate

reducing bacteria in a heavily arsenic contaminated acid mine drainage (Carnoulès, France). FEMS Microbiol

Ecol. 83 724–737 12

Egal M, Casiot C, Morin G, Elbaz-Poulichet F, Cordier MA Bruneel O (2010) An updated insight into the natural

attenuation of As concentrations in Reigous Creek (southern France). Applied Geochemistry. 25, 1949–1957 13

Benzerara K, Morin G, Yoon TH, Miot J, Tyliszczak T, Casiot C, Bruneel O, Farges F, and Brown Jr GE (2008)

Nanoscale study of As transformations by bacteria in an acid mine drainage system. Geochimica et

Cosmochimica Acta. 72, 3949-3963

33

ferrooxidans et la taille de l’inoculum de départ14

. Ces résultats semblent montrer que les

bactéries peuvent influencer la composition minéralogique du précipité formé en intervenant

sur la cinétique d’oxydation du fer, principalement durant les premiers stades d’incubation. La

tooéléite, par exemple, ne semble se former que lorsque la cinétique d’oxydation du fer est

lente et que le rapport As(III)/Fe(III) est élevé (≥ 0.8) avec des concentrations en Fe(III) du

même ordre de grandeur que celles d’As(III).

Pour mieux comprendre l’implication des microorganismes du genre Thiomonas, une

collaboration avec le laboratoire GMGM de Strasbourg a permis de séquencer le génome de

l’une de ces souches, Thiomonas sp. 3As, ce qui a permis de révéler les adaptations

spécifiques de cet organisme lui permettant de survivre et de résister aux concentrations

élevées de métaux et métalloïdes dans ces environnements extrêmes15

. De plus, 8 souches

différentes incluant 5 souches de la même espèce, ont également été comparées par

hybridation génomique comparative. Le génome du genre Thiomonas semble avoir évolué à

travers le gain ou la perte d’ilots génomiques, comme ceux conférant la résistance à l’As

(opéron ars) par exemple. Cette capacité a permis à cette espèce de s’adapter à son

environnement et suggère aussi que l’environnement influence l’évolution génomique de ces

bactéries. Ces résultats soulignent de plus la variabilité très importante qui peut exister à

l’intérieur d’un même groupe taxonomique, élargissant le concept d’espèces.

Etude des sédiments présents dans le DMA du Reigous

En raison de la précipitation des éléments toxiques en solution, présent en grande quantité

dans ces DMA, les sédiments de ces cours d’eau agissent comme des puits et accumulent de

fortes quantités de composés métalliques toxiques. Cependant, ces éléments peuvent

également être relargués dans l’eau en fonction de changements dans la chimie des sédiments,

de l’évolution du régime hydrologique ou de l’activité microbienne et peuvent ainsi

représenter une source potentielle de métaux et de métalloïdes toxiques (Park et al., 2006;

Butler, 2011; Héry et al., 2014). Etudier les microorganismes présents et leurs fonctions dans

de tels écosystèmes est donc également très important pour comprendre le devenir des

polluants.

Toujours en collaboration avec le GMGM de Strasbourg (F. Ploetze), une étude s’est

intéressée aux populations actives de ces écosystèmes présentes à la fois dans l’eau et les

sédiments du ruisseau du Reigous. L’utilisation d’une méthode de clonage-séquençage nous a

permis d’identifier les différentes populations présentes et une étude de métaprotéomique

14

Egal M, Casiot C, Morin G, Parmentier M, Bruneel O, Lebrun S, Elbaz-Poulichet F( 2009). Kinetic control on the

formation of tooeleite, schwertmannite and jarosite by Acidithiobacillus ferrooxidans strains in an As(III)-rich

acid mine water. Chemical Geology. 265, 432-441 15

Arsène-Ploetze F, Koechler S, Marchal M, Coppee J-Y, Chandler M, Bonnefoy V, Barakat M, Barbe V, Battaglia

-Brunet F, Brochier-Armanet C, Bruneel O, G. Bryan C, Cleiss J, Heinrich-Salmeron A, Hommais F, Joulian C, Krin

E, Lieutaud A, Lièvremont D, Michel C, Muller D, Ortet P, Proux C, Siguier P, Roche D, Rouy Z, Salvignol G, Slyemi

D, Talla E, Weiss S, Weissenbach J, Médigue C, Bertin PN (2010) Structure, function and evolution of the

Thiomonas spp. genome inferred from sequencing and comparative analysis. PLoS Genetics. 6 (2) e1000859

34

nous a permis d’identifier les membres actifs de ces environnements16

. Ce travail a été réalisé

en partie dans le cadre de la thèse d’Aurélie Volant (2009-2012, bourse environnée) que j’ai

principalement encadré, intitulée « Etude des communautés microbiennes (Bactéries, Archaea

et Eucaryotes) et de leurs variations spatiotemporelles dans la mine de Carnoulès fortement

contaminée en arsenic ».

Table 4. Inventaire des fragments d’ADNr 16S des clones présents dans les eaux de drainage et les

sédiments au point COWG, 30 mètres en aval de la source dans le ruisseau du Reigous. D’après

Bruneel et al, 2011

Les analyses taxonomiques des banques de gènes codant pour l’ARNr 16S ont permis de

montrer que la diversité bactérienne est plus faible dans l’eau que dans les sédiments avec 11

souches identifiées dans l’eau contre 13 souches dans les sédiments. Un total de 17 groupes

taxonomiques différents ont été identifiés avec seulement 7 genres présents à la fois dans

l’eau et les sédiments. La plupart des ces bactéries étaient affiliées à des β-protéobactéries

telles que Gallionella ou Thiomonas mais également à des γ- protéobactéries (tel que

Acidithiobacillus ferrooxidans), des α-protéobactéries (Acidiphilium), des δ-protéobactéries

(Desulfomonile limimaris), des Nitrospira (Leptospirillum ferrooxidans), des Actinobacteria

et des Firmicutes. Il s’agit majoritairement d’espèces trouvées communément dans les DMA

avec une majorité impliquée dans les cycles du fer, de l’arsenic et du soufre. Les bactéries

impliquées dans l’oxydation de l’As(III) sont affiliées à Thiomonas, celles impliquées dans

l’oxydation de Fe(II) sont affiliées à Gallionella, At ferrooxidans, Ferrimicrobium,

Leptospirillum, Sideroxydans lithotrophicus, et Ferrovum myxofaciens alors que la réduction

16

Bruneel O, Volant A, Gallien S, Chaumande B, Casiot C, Carapito C, Bardil A, Morin G, Brown Jr GE, Personné

JC, Le Paslier D, Schaeffer C, Van Dorsselaer A, Bertin PN, Elbaz-Poulichet F, Arsène-Ploetze F (2011)

Characterization of the active bacterial community involved in natural attenuation processes in arsenic-rich

creek sediments. Microbial Ecology. 61, 793-810

35

du fer a été mise en évidence pour les bactéries des genres Acidiphilium, Desulfuromonas

svalbardensis, Acidocella, Rhodoferax ferrireducens, At ferrooxidans et Ferrimicrobium

acidiphilum. Concernant le cycle du soufre, des populations capables d’oxyder des composés

soufrés inorganiques ont été mis en évidence telles que Thiobacillus, Thiomonas ou At

ferrooxidans. Des bactéries sulfato-réductrices comme Desulfomonile limimaris ou

Desulfuromonas svalbardensi pourraient être impliquées dans la consommation du sulfate.

L'oxydation de l'arsenic associée à l'oxydation du fer et l'oxydation du soufre pourrait

contribuer à la co-précipitation de ces éléments et expliquerait l'atténuation de la

contamination arséniée constatée dans le Reigous. L’étude par métaprotéomique faite au

niveau des sédiments a révélé que les genres oxydants le fer comme Gallionella et

Acidithiobacillus et oxydants l’As comme Thiomonas comptent parmi les membres

métaboliquement actifs de la communauté procaryote du Reigous.

Nous avons également caractérisé la communauté d’Archaea présente dans ces sédiments

et avons étudié sa dynamique temporelle en utilisant la technique de clonage-séquençage du

gène codant pour l’ARNr16S. Les Archaea restent pour l’instant assez mal connues et peu

étudiées en raison notamment de difficultés d’isolement qui font que les connaissances sur

leurs métabolismes ne concernent pour l’instant qu’un nombre restreint de souches. Ainsi, la

diversité de ces organismes et leurs rôles physiologique au sein des DMA sont assez obscur et

les études moléculaires restent indispensables.

36

Figure 4. Arbre phylogénétique basé sur le gène codant pour l'ARNr 16S représentant l'affiliation

taxonomique de la communauté des Archaea présente dans les sédiments du ruisseau du Reigous au

point COWG. Le nombre entre parenthèses indique le nombre de séquences de clones pour la période

d'échantillonnage représenté par un symbole ( Avril 2006, Octobre 2008, Janvier 2009 et

Novembre 2009 ; Volant et al., 2012)

L'affiliation taxonomique des Archaea a montré un faible degré de diversité avec

uniquement 2 phylums détectés: les Thaumarchaeota (contenant la grande majorité des

séquences) et les Euryarchaeota17

. Contrairement aux Archaea retrouvées dans les eaux du

17

Volant A, Desoeuvre A, Casiot C, Lauga B, Delpoux S, Morin G, Personné JC, Héry M, F Elbaz-Poulichet, Bertin

P and Bruneel O (2012) Archaeal diversity: temporal variation in the Arsenic-Rich Creek Sediments of Carnoulès

Mine, France. Extremophiles. 16, 645-657

37

stock de déchet minier et dans les eaux du DMA, nous n’avons pas retrouvé ici de

microorganismes directement impliqués dans le cycle du fer ou du soufre. Un grand nombre

de séquences sont affiliées à Thermogymnomonas acidicola, une Archaea hétérotrophe

retrouvée dans d’autres DMA et qui pourrait jouer un rôle important dans l’écosystème en

utilisant les composés organiques qui peuvent être toxiques pour certains autotrophes

(Hallberg, 2010 ; Yang et al., 2014). Des microorganismes affiliés à des Archaea

méthanogènes, impliquées dans le cycle du carbone, telle que Methanomassiliicoccus

luminyensis, ont également été identifiées. Enfin, des séquences apparentées à Candidatus

Nitrososphaera viennensis et Candidatus nitrosopumilus sp., des Archaea impliquées dans

l’oxydation de l’ammonium, une étape clé du cycle de l’azote, ont été décris. L’ensemble de

ces microorganismes pourrait donc contribuer conjointement avec les bactéries au processus

de remédiation observé in situ. Cette étude a également permis de mettre en évidence des

modifications importantes de la structure et de la composition de la communauté d’Archaea

au cours du temps qui sont probablement liées à des modifications de l’environnement.

Une étude, en collaboration avec l’IMPMC de Paris (G. Morin), s’est également intéressée

aux conditions de formation des minéraux de fer riches en arsénite As(III) et arséniate As(V)

identifiés sur le site, tel que la schwertmannite qui joue un rôle très important dans la rétention

de l’As et la remédiation de ce métalloïde. Ce travail a montré que l’oxydation bactérienne de

l’arsenic, en favorisant la formation d’As(V)-schwertmannite ou d’arséniate ferrique,

améliore grandement l’immobilisation de l’As dans la phase solide18

.

Une autre étude avec l’IMPMC de Paris s’est également intéressée à la structure de la

ferrihydrite, un oxyhydroxyde de fer qui est également impliqué dans la rétention de l’As.

C’est la phase minérale prédominante présente dans les sédiments de la rivière Amous (pH

6−7) qui se forme, après la confluence, après neutralisation avec les DMA du Reigous. Ces

travaux montrent que cet oxyhydroxyde de fer pourrait également jouer un rôle important

dans la séquestration de l’As dans les environnements miniers19

.

Des études géochimiques ont également porté sur le thallium et ont montré que la forme

réduite du thallium Tl(I) est largement prédominante dans le DMA de Carnoulès et est peu

adsorbé sur les particules de ferrihydrite, qui se forment dans la rivière Amous en aval du

Reigous, impliquant une forte mobilité du thallium dans l’hydrosystème aval20

.

18

Maillot F, Morin G, Juillot F, Bruneel O, Casiot C, Ona-Nguema G, Wang Y, Lebrun S, Aubry E, Vlaic G, Brown

GE Jr (2013) Structure and reactivity of As(III)- and As(V)-rich schwertmannites and amorphous ferric arsenate

sulfate from the Carnoulès acid mine drainage, France: comparison with biotic and abiotic model compounds

and implications for As remediation. Geochimica et Cosmochimica Acta. 104, 310-329 19

Adra A, Morin G, Ona-Nguema G, Maillot F, Casiot C, Bruneel O, Lebrun S, Juillot F, Brest J (2013) Arsenic

Scavenging by Al-Substituted Ferrihydrites in a Circumneutral pH River Impacted by the Acid Mine Drainage of

Carnoulès, Gard, France. Environmental Science and Technology. 47, 12784-12792 20

Casiot C, Egal M, Bruneel O, Verma N, Parmentier M, Elbaz-Poulichet F (2011) Predominance of aqueous Tl(I)

species in the river system downstream from the abandoned Carnoulès mine (Southern France). Environmental

Science & Technology. 45, 2056-2084

38

Etude de la diversité fonctionnelle des communautés du Reigous par métagénomique

La diversité fonctionnelle des microorganismes présents au sein du drainage minier acide

de Carnoulès a été étudiée dans le cadre du projet ANR RARE initié par P. Bertin (GMGM de

Strasbourg). Au départ prévu sur l’eau, le manque de matériel biologique a conduit la

réalisation de cette étude dans les sédiments de surface au point COWG. Le séquençage

massif et le réassemblage de l’ADN, réalisé par le Génoscope d’Evry, ont conduit à la

reconstruction de 7 pseudo-génomes microbiens (CARN1 à CARN7) présents dans cet

environnement21

.

Tableau 5. Analyse phylogénétique des pseudogénomes présents dans le sédiment de la mine de

Carnoulès réalisée à l’aide de 27 marqueurs universels ou du gène de l’ARNr 16S avec RDP. D’après

Bertin et al., 2011

(1) Pour le 16S, l’organisme le plus proche a été obtenu par recherche à l’aide du logiciel BLAST sur la base de

donnée NCBI nr. Seuls les microorganismes ayant un pourcentage de similarité >90 sont indiqués. La recherche

des 27 marqueurs universels a été réalisé selon Ciccarelli et al., (2006).

(2) Absence du gène de l’ARNr 16S

Cette analyse a confirmé la présence de souches identifiées par les études antérieures

réalisées par PCR/Clonage/séquençage comme Thiomonas, Acidithiobacillus ferrooxidans,

Thiobacillus sp. ou Gallionella. L’utilisation conjointe de la métagénomique et de la

métaprotéomique a également permis de mettre en évidence les relations entre les

microorganismes ainsi que les fonctions importantes dans cet environnement.

21

Bertin PN, Heinrich-Salmeron A, Pelletier E, Goulhen- Chollet F, Arsène-Ploetze F, Gallien S, Lauga B, Casiot C,

Calteau A, Vallenet D, Bonnefoy V, Bruneel O, Chane-Woon-Ming B, Cleiss-Arnold J, Duran R, Elbaz-Poulichet F,

Fonknechten N, Giloteaux L, Halter D, Koechler S, Marchal M, Mornico D, Schaeffer C, Thil Smith AA, Van

Dorsselaer A, Weissenbach J, Médigue C and Le Paslier D (2011) Metabolic diversity between main

microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics. The ISME Journal. 5,

1735-1747. Cet article a fait l’objet d’une note dans la revue Sciences (2011, vol 332, p1128)

39

Figure 5. Modèle de la communauté bactérienne de Carnoulès mettant en évidence les fonctions

majeures identifiées par le séquençage du métagénome et la métaprotéomique présentes au sein du

sédiment de COWG. Les interactions entre les organismes et les composés biologiques ou chimiques

sont indiquées par des flèches. Les microorganismes CARN sont numérotés de 1 à 7.

Ce travail a permis de mettre en évidence différentes activités et interactions au sein de

cette communauté comme la capacité de certains microorganismes à fixer l’azote

(particulièrement peu abondant dans ce type d’environnement) et le carbone inorganique par

les microorganismes autotrophes permettant le développement des microorganismes

hétérotrophes. La capacité de formation de biofilms, connue pour apporter une meilleure

résistance face aux différents stress environnementaux (Harrison et al., 2007, Marchal et al.,

2012), a été révélée ainsi que la présence de flagelles et de capsules. Les mécanismes

énergétiques impliquants l’arsenic, le fer et le soufre ont été identifiés ainsi que le recyclage et

le transport de la matière organiques : acides aminés, vitamines et nucléosides. En particulier,

ces études ont permis l’identification d'un nouveau phylum, ‘Candidatus Fodinabacter

comunificans’ qui pourrait exercer à Carnoulès un rôle indirect mais important en participant

au recyclage de la matière organique comme les acides aminés ou les nucléosides provenant

notamment de microorganismes eucaryotes retrouvés sur le site (Halter et al., 2012a). Ces

approches génomiques ont ainsi permis de mieux comprendre le rôle des microorganismes

dans l’atténuation naturelle de l’arsenic sur ce site et d’attribuer à des organismes spécifiques,

dont des organismes non encore cultivés, des fonctions importantes.

40

Utilisation des nouvelles technologies de séquençage pour l’étude des DMA

Depuis quelques années, les avancées dans le domaine des techniques de séquençage haut

débit, encore appelé séquençage massif ou de nouvelle génération, ont révolutionné la

biologie moléculaire et ont ouvert une nouvelle aire dans les recherches concernant les études

sur la biodiversité (Sogin et al., 2006; Behnke et al., 2011). En raison de la rapidité

d’obtention et du coût relativement faible (et qui ne cesse de diminuer) pour produire des

millions de séquences, il est maintenant possible d’explorer en profondeur la diversité et la

complexité des communautés microbiennes. Dans les études de diversité, ces techniques

apparaissent comme essentielles car elles permettent d’avoir une profondeur de séquençage et

une vue quasi exhaustive des microorganismes présents. Ces techniques permettent donc de

s’intéresser également aux populations minoritaires qui peuvent malgré tout jouer un rôle

crucial dans les processus biogéochimiques alors que l’on ne pouvait que très difficilement les

étudier avant selon les méthodes traditionnelles de biologie moléculaire comme le clonage-

séquençage (Behnke et al., 2011).

C’est ce type d’analyses qui a été utilisé dans le cadre de 2 études qui se sont intéressées

aux microorganismes (bactéries et eucaryotes) présents dans les sédiments miniers de la mine

de Carnoulès.

Comme nous l’avons vu précédemment, des études antérieures principalement basées sur

des souches isolées, ont suggéré un rôle déterminant de l’activité bactérienne dans la co-

précipitation de l’As avec le Fe(III) et le sulfate et la formation de phases amorphes

d'oxyhydroxydes associées à des minéraux comme la tooéléite, la schwertmannite ou la

ferrihydrite. Cette étude a permis l’analyse de la diversité bactérienne présente dans différents

types de sédiments le long des 1500 m du Reigous, en utilisant, pour la première fois une

approche de pyroséquençage 454 ciblant le gène codant pour l’ARNr 16S. Le but était

d’identifier les communautés bactériennes présentes et d’étudier leurs dynamiques spatiales

en fonction de la structure minéralogique des sédiments (comprenant notamment des sites très

riches en tooéléite et en schwertmannite) permettant de comprendre si la dynamique de ces

communautés est liée aux changements dans les sédiments.

Cette approche a permis de générer un total de 53075 séquences de bonne qualité après

normalisation, conduisant à l'identification de 966 OTU, mettant en évidence une diversité

beaucoup plus importante que précédemment observé. Il est également à noter qu’une grande

majorité de cette diversité est du à la présence d’OTUs rares (371, encore appelés singletons)

et observés une seule fois pour l’ensemble des séquences. Ceci suggère qu’une part

importante de la diversité observée se réfère à des taxons présents à une très faible abondance

donnant naissance au concept de biosphère rare (Pedrós-Alió, 2007). En dépit de l’importance

de cette biosphère rare dans de nombreuses études, son rôle écologique et fonctionnel reste

mal compris actuellement (Galand et al., 2009). Pour certains auteurs, ces organismes

pourraient devenir dominants et actifs suite à des modifications des conditions

environnementales et pourraient permettre aux processus biogéochimiques d’être maintenus

limitant ainsi les effets des modifications de l’environnement (Sogin et al, 2006 ; Behnke et

al., 2011 ; Bachy and Worden, 2014). D'autres études s’interrogent également sur l'exactitude

des estimations de la richesse en OTUs générée par le séquençage à haut débit, qui pourrait

correspondre à des erreurs de séquençage (Huse et al., 2010).

41

Figure 6. Courbes de raréfaction des séquences bactériennes des gènes codants pour l'ARNr 16S

présents dans les sédiments de la mine de Carnoulès et basé sur le nombre d’OTU calculés à 97%

d'identité. Le nombre total de séquences analysées est tracé en fonction du nombre d’OTU observé.

Les courbes de raréfaction ont tendance à atteindre une asymptote pour la plupart des

échantillons, ce qui suggère que la majorité des phylotypes bactériens présents ont été

identifiés, ce qui est confirmé par la couverture très élevée (de 98 à 100%) pour tous les

échantillons.

Table 6. Estimation de la richesse en OTU, des indices de diversité et de la couverture estimée pour

les 5 échantillons de sédiments. Les résultats sont présentés pour les données normalisées,

rééchantillonnées au hasard pour avoir une taille d’échantillon égale entre les sites.

a Les OTUs ont été définis à 97% d’identités

b Somme des probabilités des classes observées calculées comme suit (1 - (n / N)), où n est le nombre de

séquences uniques (singletons) et N est le nombre total de séquences c Prend en compte le nombre et la régularité des espèces

Les valeurs entre parenthèses sont des intervalles de confiance à 95%

Au total, 15 phylums ont pu être identifiés ici pour l’ensemble des échantillons, ce qui est

bien plus important que ceux retrouvés dans les analyses antérieures obtenues par clonage-

séquençage qui n’excédaient généralement pas 5 phylums. L'analyse phylogénétique a révélé

que la grande majorité des séquences (65%) appartenaient au phylum des Proteobacteria avec

0

50

100

150

200

250

300

350

400

450

0 2000 4000 6000 8000 10000 12000

Nu

mb

er

of

OT

Us

Number of sequences

S1

COWG

GALm

GAL

CONF

42

une prédominance des bactéries oxydant de fer, représentées principalement par des

séquences proches de Gallionella ou Acidithiobacillus ferrooxidans. Cette analyse quasi

exhaustive des taxons présents a également révélé la présence de genres abondants encore

jamais détectés auparavant par les analyses de clonage/séquençage, comme les membres des

Comamonas, Stenotrophomonas ou Pseudoxanthomonas avec certains d'entre eux impliqués

dans l'oxydation de l’As, un métabolisme importante impliqué dans la précipitation de As.

Cependant, aucun paramètre évident ne semble lier les communautés à la structure des

sédiments. Ce travail devrait être prochainement soumis dans le journal FEMS Microbiology

Ecology.

Une seconde étude s’est intéressée aux communautés eucaryotes. Les communautés de

bactéries des DMA ont été extensivement étudiées depuis plusieurs 10aines

d’années avec les

premières études de diversité qui remontent au milieu des années 1990 (Goebel et

Stackebrandt, 1994 ; Kuang et al., 2012). Paradoxalement, les communautés eucaryotes de ces

environnements ont été très peu étudiées bien qu’un intérêt croissant leur soit porté de part

leur rôle écologique potentiellement important dans ces écosystèmes. Certains sont par

exemple susceptibles de modifier l’abondance, la composition et l’activité des communautés

microbiennes procaryotes par de nombreux mécanismes comme la prédation (Baker et al.,

2004, 2009; Gadanho et al., 2006). D’autres sont connus pour jouer un rôle important dans le

cycle du carbone et le recyclage des nutriments ce qui est primordiale dans ces

environnements oligotrophes (Baker et al., 2004). Certains peuvent également apporter de

l’oxygène au milieu par leurs activités photosynthétiques ou encore séquestrer des polluants

métalliques dans les matrices extracellulaires ou à l’intérieur de la cellule (Brake et al., 2001).

Concernant la mine de Carnoulès, aucune analyse de diversité n’avait encore été réalisée

sur la communauté eucaryotes au niveau moléculaire bien que des études précédentes se

soient intéressées au protozoaire photosynthétique Euglena mutabilis9 (Halter et al., 2012a,

2012b). L’objectif de ce travail était d’identifier les communautés eucaryotes présentes dans

les sédiments du Reigous et d’étudier leur distribution spatiale le long du ruisseau par

pyroséquençage 454 des gènes codant pour l’ARNr 18S.

43

Table 7. Répartition taxonomiques des séquences eucaryotes présentes dans les sédiments de la mine

de Carnoulès. Le chiffre entre parenthèses représente l'abondance relative (%) des taxons.

*Calculé par rapport au nombre total de séquences présentes dans cette étude.

Autres champignons corresponds aux Blastocladiomycota et à des champignons non classés

Les analyses phylogénétiques ont révélé la présence de 14 taxons essentiellement dominés

par 6 groupes (représentant 91 % des séquences totales) affiliés aux phyla des Ascomycètes,

Basidiomycètes, Alveolates, Stramenopiles, Streptophytes et Chlorophytes. Parmi ces groupes,

les champignons constituaient à eux seuls près de 60 % des séquences et sont apparus être le

groupe majoritaire sur l’ensemble des sédiments prélevés (ce qui est en accord avec les

résultats obtenus par Baker et al. (2009)), suivis dans une moindre mesure par les Alveolates

et les Stramenopiles. La majorité des séquences obtenues dans cette étude se sont révélées être

apparentées à des taxons trouvés précédemment dans d’autres DMA tels que les

Chlorophytes, les Streptophytes ou les Champignons, etc. (Amaral-Zettler et al., 2002, 2011).

Le pyroséquençage a également permis de mettre en évidence de nouveaux taxons non

détectés auparavant dans ce type de milieu tels que les Apusozoaires, les Centroheliozoaires

et les Jakobides. Ces travaux ont également permis de mettre en évidence une structuration

spatiale des communautés eucaryotes qui semble être liée en partie à la physicochimie de

l’eau (arsénite, fer et potentiel redox). Ce travail devrait être prochainement soumis à la revue

Environmental Microbiology.

Projet MIGRAMD et analyse de biogéographie (France, Bolivie et Espagne)

Le projet FRB, MIGRAMD, intitulé : « Microbial biogeography of Acid Mine Drainage: a

study of genetic diversity and species diversity from an evolutionary perspective », porté par

l’EEM de Pau (B. Lauga) nous a permis d’aborder une nouvelle notion, celle de

biogéographie. L’objectif de ce projet était d’évaluer la diversité spécifique des

microorganismes présents dans les DMA de 4 pays (Espagne, Portugal, France et Bolivie)

plus ou moins riches en arsenic et séparés géographiquement pour mieux comprendre leurs

organisations spatiales et leurs répartitions afin d’appréhender les processus qui les mettent en

44

place. Parallèlement à cette approche spatiale, la dynamique spatiotemporelle des

communautés bactériennes a été étudiée dans les eaux le long du continuum du DMA de

Carnoulès afin d’identifier les paramètres physicochimiques structurant l’assemblage des

bactéries. C’est dans le cadre de cette dernière partie que s’est focalisé le travail de thèse

d’Aurélie Volant. La configuration du DMA de Carnoulès est en effet telle que la

contamination du Reigous (qui prend sa source au sein du stock de déchet minier) s'atténue le

long du continuum, mettant en évidence un important gradient spatial des conditions physico-

chimiques. De plus, les fortes contraintes physico-chimiques qui s'exercent sur ces

écosystèmes donnent l'opportunité d'étudier l'effet des pressions de sélection sur la

biodiversité. Les méthodes de séquençages haut-débit, de part la profondeur de séquençage

qu’elles permettent semblaient ici un outil de choix pour aborder ces problématiques.

Concernant l’approche spatiale, douze DMA (eau et sédiments) ont ainsi été échantillonnés

dans 3 régions du monde, l’Amérique du Sud (Bolivie), la péninsule Ibérique (Espagne et

Portugal) et la France (DMA du Reigous à Carnoulès). Leurs principaux paramètres physico-

chimiques (pH, température, conductivité, concentrations en éléments métalliques et sulfates,

etc) ont été caractérisés et la spéciation a été réalisée pour As et Fe). Les gènes codant pour

l'ARNr 16S des bactéries et des Archaea ont été amplifiés par PCR puis séquencés sur

pyroséquençage Roche 454 à la plateforme de génomique de Toulouse. Ce travail, s’est fait

également en association avec l’équipe « Instituto de Biologıa Molecular y Biotecnologıa,

Universidad Mayor de San Andres » de La Paz en Bolivie. Cette partie de l’étude est en cours

d’analyse par l’EEM de Pau.

Concernant l’étude spatio-temporelle de Carnoulès, 6 campagnes de prélèvement ont été

réalisées de novembre 2007 à janvier 2010 au niveau de 5 points de prélèvements, soit 30

échantillons au total22

. Les paramètres physico-chimiques ont été caractérisés et l’étude à été

réalisé en combinant une technique à empreinte moléculaire, la T-RFLP et le pyroséquençage

454 à la plateforme de génomique de Toulouse. Les analyses physico-chimiques ont montré

qu’en moyenne 60% des concentrations en sulfate, 96% de celles en fer et 99% de celles en

arsenic étaient précipitées le long des 1500 mètres du ruisseau du Reigous. Le

pyroséquençage a permis de générer un total de 66016 séquences qui ont permis

l’identification de 6801 OTUs incluant 4629 singletons représentant 68% des séquences.

Vingt trois phylums bactériens ont été identifiés sur l’ensemble des échantillons analysés et

le phylum largement majoritaire (68%) est celui des Protéobactéries. Les 3 OTUs

majoritairement présents sont apparentés aux espèces trouvées précédemment comme

Gallionella ferruginea, Acidithiobacillus ferrooxidans et Thiobacillus sp. et confortent ainsi

les études antérieures. Cette étude a également permis de mettre en évidence des genres

jamais identifiés jusqu’à présent à Carnoulès comme Ignavibacterium, Ralstonia ou

Paludibacter, etc.

22

Volant A, Bruneel O, Desoeuvre A, Héry M, Casiot C, Bru N, Delpoux S, Fahy A, Javerliat F, Bouchez O, Duran

R, Bertin PN, Elbaz-Poulichet F and Lauga B (2014) Diversity and spatiotemporal dynamics of bacterial

communities: physicochemical and others drivers along an acid mine drainage. FEMS Microbiology Ecology. 90,

247-263

45

Figure 7. Analyse Canonique par Correspondance (ACC) corrélant la structure des communautés

bactériennes avec les paramètres physicochimiques présents dans les différents sites incluant l’arsenic

(As), le fer (Fe), la conductivité (Cond), la température (T), l’oxygène dissous (DO), le potentiel redox

(Eh), le pH et le sulfate. La structure des communautés correspond à l’abondance des OTUs obtenus à

partir des données de T-RFLP (a) ou du pyroséquençage (b). Les principaux clusters ont été entourés.

D’après Volant et al., 2014

Une séparation spatiale a également pu être clairement mise en évidence par les données de

pyroséquençage et une analyse canonique des correspondances a identifié 3 paramètres

physicochimiques (la concentration en arsenic, la température et le potentiel redox) comme

des facteurs potentiellement responsables de cette structuration.

Conclusion sur l’étude des drainages miniers acides

En conclusion sur cette partie, de nombreuses questions de recherche ont été abordées et

les résultats ont permis une amélioration notable des connaissances acquises sur ce site,

comme le souligne la vingtaine de publications issues des travaux de notre équipe et de ses

partenaires citées dans les paragraphes précédents. Cela démontre également l’intérêt de ces

travaux pluridisciplinaires qui ont permis d’obtenir une vision globale et intégrée des

processus complexes qui conditionnent les interactions entre les microorganismes et leur

environnement.

La combinaison d’études de terrain et d’expériences en laboratoire a permis des avancées

importantes dans la compréhension des processus d’oxydation et de précipitation de Fe et As

et le rôle des microorganismes dans ces processus est désormais mieux compris. L’aptitude de

souches, isolées du site, comme Acidithiobacillus ferrooxidans et Thiomonas à oxyder le fer

ou l’arsenic a été montré en laboratoire, ainsi que leurs rôles dans la formation de minéraux

particuliers. Ces résultats démontrent également qu’en dépit des conditions extrêmes qui

règnent à Carnoulès, des communautés microbiennes complexes (Bactéries, Archaea et

Eucaryotes) coexistent, interagissent et influencent directement ou indirectement le cycle de

46

certains métaux et métalloïdes et en particulier l’arsenic. Les résultats obtenus soulignent

également l’intérêt de l’utilisation des nouvelles techniques de séquençage permettant de

mettre en évidence une diversité majoritairement sous estimée par les techniques classiques

de clonage-séquençage mais ouvre aussi la voie à de nombreuses interrogations concernant

par exemple le rôle écologique des nombreux taxons rares mis en évidence.

Un Projet ANR ECO-TS IngECOST-DMA, « Ingénierie écologique appliquée à la gestion

intégrée de stériles et DMA riches en arsenic » a été accepté sur Carnoulès en 2014 pour une

durée de 4 ans. Il a pour but la mise en place et l’étude d’un système de bioremédiation

(intégrant un système utilisant la capacité des bactéries qui oxydent le fer à précipiter les

polluants associé à un système utilisant la capacité des bactéries sulfato-réductrices à former

des sulfures de métaux insolubles) qui pourra s’appuyer sur les connaissances déjà acquises

sur ce site et qui permet d’aborder ici des études de remédiation appliquées.

47

TRAVAUX ACTUELS

Depuis mon expatriation en février 2012 au sein du Laboratoire de Microbiologie et de

Biologie Moléculaire de l’Université Mohammed V de Rabat au Maroc dirigé par le

professeur Karim Filali-Maltouf, je m’intéresse à une thématique un peu différente qui est

l’étude des interactions plantes-microorganismes dans la mise en place d’un couvert végétal.

Ce travail a été initié à l’origine dans le cadre du Laboratoire Mixte International «

Biotechnologie Végétale et Microbienne (Directeurs K. Filali-Maltouf et G. Béna) » dans

l’axe thématique «Identification de plantes et microorganismes tolérants aux polluants

métalliques dans les sites miniers marocains». Ce travail a été réalisé en grande partie dans le

cadre d’un projet Ec2co (2012-2013) sur l’«Etude des interactions plantes-microorganismes

dans un contexte de réhabilitation de sites minier: mécanismes adaptatifs et effets sur le

devenir des polluants métalliques» dont je suis porteur.

Comme nous l’avons vu, les activités minières sont très polluantes et ont un impact

important sur l'environnement et la santé que ce soit lors de l’extraction du minerai, de sa

transformation ou du fait de la production de milliers de tonnes de déchets. Ces déchets sont

généralement composés de particules très fines et souvent riches en divers composés toxiques.

L'activité minière a été l'un des piliers de l'économie marocaine et a entrainé l’accumulation

de milliers de tonnes de résidus pour la plupart abandonnés à l'air libre. Les conditions

climatiques du bassin méditerranéen (vents violents et périodes de pluies intenses qui

succèdent à des périodes très sèches) favorisent le lessivage et la dissémination des polluants

et rendent difficile l’installation d’un couvert végétale. Plusieurs technologies ont été

développées pour dépolluer les sols contaminés par les polluants métalliques comme leur

extraction par des moyens chimiques ou physiques ou encore l’élimination physique du sol

qui est confiné dans des sites d'enfouissement. Mais ces techniques sont souvent très

coûteuses à la fois d’un point de vue économique et environnemental et peuvent fortement

altérer les qualités physiques, chimiques et biologiques des sols (Glick, 2010). Depuis une

quinzaine d’années, des travaux se sont intéressés à l’utilisation de plantes pour dépolluer ces

sols. Les polluants peuvent en effet être soit stabilisés dans le sol pour les rendre moins

bioassimilables (phytostabilisation), soit être accumulés dans les tissus végétaux

(phytoextraction) ou encore être transformés en formes volatiles (phytovolatilisation) (Khan,

2005 ; Kavamura and Esposito, 2010). Le principal obstacle à ces techniques est dû au fait

que la plupart des résidus miniers sont de mauvais substrats pour la croissance des plantes en

raison à la fois de la présence de métaux toxiques en concentrations élevées, de la présence

parfois de pH acides, de la salinité souvent élevée, du manque de matière organique et de

nutriments essentiels, de la mauvaise structure du sol et d’une mauvaise rétention de l’eau

(Kid et al., 2009 ; de-Bashan et al., 2010). Ces résidus restent généralement dépourvus de

couverture végétale pendant des décennies ou plus (Mendez and Maier, 2008 ; de-Bashan et

al., 2010). Pour remédier ces environnements extrêmes et en raison des concentrations élevées

en polluants métalliques, c’est la phytostabilisation qui est généralement préférée, c'est-à-dire

la création d’une couverture végétale qui va limiter l’érosion éolienne et hydrique en

stabilisant et précipitant les éléments métalliques au niveau des racines tout en limitant leur

accumulation dans les feuilles (de-Bashan et al., 2010, Bolan et al., 2014).

Alors que l'établissement d'un couvert végétal sur des sols contaminés par des polluants

chimiques reste un défi, les microorganismes peuvent fortement accélérer le processus de

48

phytostabilisation (de-Bashan et al., 2010, Ma et al., 2011). La croissance des plantes est en

effet fortement influencée par les microorganismes qui peuvent intervenir à plusieurs niveaux

(fixation d’azote, solubilisation du phosphate, production de phytohormones ou

d’antibiotiques, etc.). Certains microorganismes ont de plus la capacité à agir sur la

mobilisation/immobilisation des métaux et métalloïdes dans le sol par la production de

sidérophores, d’enzymes, ou d’acides organiques, etc. composés qui peuvent modifier ces

éléments par acidification, chélation, précipitation, oxydoréduction ou méthylation, etc.

(Rajkumar et al., 2012). De nombreux microorganismes sont en effet connus pour favoriser la

croissance des plantes (effet PGPB, Plant Growth Promoting Bacteria, de-Bashan et al., 2010,

Das et al., 2014). Plusieurs études ont ainsi montré que des rhizobactéries appartenant aux

genres Achromobacter, Arthrobacter, Azotobacter, Bacillus, Pseudomonas, ou Serratia

favorisaient la croissance des plantes dans des environnements contaminés par des métaux et

métalloïdes (Ma et al., 2011). Parmi ces microorganismes, on peut distinguer les

microorganismes telluriques, présents dans le sol; les rhizobactéries localisées à proximité

immédiate des racines et les bactéries endophytes qui colonisent les tissus internes des plantes

sans causer d’infections.

Contrairement à la pollution par les mines dans les régions tempérées, il n’existe que très

peu d'études sur l'impact environnemental des activités minières dans les régions arides et

semi-arides (González et al., 2011). Les sites que nous étudions dans le cadre de ce projet se

situent dans le district minier de la ville d’Oujda, au Nord-Est du Maroc près de la frontière

algérienne.

Figure 8. Situation géographique des régions étudiées et localisation des stations de prélèvements.

D’après Smouni et al., 2010

Les sites d’études comprennent les digues de lavage des mines abandonnées de Pb et Zn de

Touissit et Boubker ainsi que les scories de l’ancienne fonderie de Oued El Heimer. Ces

déchets qui représentent plusieurs millions de tonnes constituent des digues de très grande

superficie.

49

Figure 9. Description des sites de Oued El Heimer, Touissit et Boubker. A) Scories

plombifères déposées sur de grandes surfaces aux abords de la fonderie. B) Digue de sable

aux abords du village de Touissit. C) Digue de sable à proximité d’un champ de blé dans la

région de Boubker. D’après Smouni et al., 2010

Malgré un climat austère et une forte teneur en polluants, une flore tolérante parvient à s’y

développer. Ces plantes, ainsi que les microorganismes associés sont à priori adaptés aux

conditions édapho-climatiques de ces régions et présentent donc une ressource pour le

développement de stratégies de réhabilitation, notamment par la phytostabilisation et la mise

en place d’un couvert végétal qui limiterait l’érosion éolienne et hydrique.

L’intérêt pour cette région est multiple. D’une part, le périmètre étudié est fortement

impacté par une pollution polymétallique aussi bien au niveau des terres agricoles que des

cours d’eau et des puits (Smouni et al., 2010). L’index de pollution des échantillons prélevés

dans ces environnements est généralement très élevé du fait de la présence simultanée de

plusieurs polluants (As, Cd, Cu, Ni, Pb et Zn) avec de très importantes teneurs en Pb, Zn et As

(respectivement jusque 7 g/kg, 2 g/kg et 187 mg/kg) (Smouni et al., 2010). Enfin, la présence

de sites divers et originaux (stériles couverts et digues nues, revégétalisés ou non, de façon

naturelle ou par action humaine) permet de comparer leurs impacts sur l’association plantes-

A

B

C

50

microorganismes qui les a colonisés sur une échelle de temps variée. Cette situation constitue

ainsi une source d’une incroyable diversité floristique et microbienne.

L’objectif de ce projet de recherche consiste à étudier à la fois les plantes et les

microorganismes associés et doit permettre : (i) d’étudier les mécanismes de résistance et

d’accumulation notamment pour le Pb et le Zn de 2 plantes endémiques (une plante

hyperaccumulatrice de plomb, Hirschfeldia incana et une légumineuse Hedysarum

spinosissimum) (ii) d’identifier les communautés microbiennes capables de se développer sur

ces différents environnements et de mieux comprendre leurs mécanismes de résistance et

d’adaptation et enfin (iii) d’isoler de nouveaux microorganismes à la fois rhizosphériques et

symbiotiques (présents dans les nodules de H spinosissimum) et de mieux appréhender leurs

mécanismes d’action sur la croissance des plantes et la mobilisation/immobilisation des

métaux et métalloïdes. Cette étude combinée des plantes et des microorganismes devrait

permettre à terme de proposer une collection de plantes et de microorganismes résistants à ces

polluants et susceptibles d’être des outils efficaces pour établir un programme de

phytoremédiation. Elle pourrait ainsi avoir un impact sociétal important en accélérant

significativement les processus de réhabilitation de ces zones contaminées.

C’est un travail pluridisciplinaire qui associe à la fois des végétalistes, des microbiologistes

et une géochimiste (P. Moulin, Ingénieur IRD, US IMAGO). Il a été réalisé en collaborations

avec des laboratoires marocains : le Laboratoire de Microbiologie et Biologie Moléculaire

(LMBM, L. Sbabou et J. Aurag) dans lequel s’effectue actuellement mon expatriation ainsi

que le Laboratoire de Physiologie et Biotechnologie Végétale (LPBV, A. Smouni, M. Fahr)

de l’Université de Rabat. Ce projet comprend également l’implication de partenaires français:

le Laboratoire des Symbioses Tropicales et Méditerranéennes (AMPERE-LSTM, E. Navarro)

ainsi que le laboratoire de Biochimie et Physiologie Moléculaire des Plantes de Montpellier

(BPMP, P. Doumas, F. Auguy). Ce travail s’inscrit également dans le cadre du réseau

SICMED «Environnements Miniers Méditerranéens » coordonné par P. Doumas (BPMP,

Montpellier).

Dans le cadre de ce projet de recherche, mon travail a essentiellement porté sur l’étude de

la diversité des microorganismes et sur leur rôle dans le transfert des métaux et métalloïdes

dans ces environnements extrêmes. Ce travail est réalisé dans le cadre de la thèse de I.

Dahmani qui a débutée en décembre 2013 et que je coordonne avec 2 encadrants marocains, J.

Aurag, L. Sbabou ainsi que E. Navarro. Ces travaux comprennent l’utilisation de nouvelles

techniques de séquençage associant études taxonomiques et études fonctionnelles. L’analyse

des données de pyroséquençage réalisée sur 24 sols miniers en triplicat (associant sols nus et

sols rhizosphériques) est en cours par bioinformatique à l’aide du logiciel Mothur

(http://www.mothur.org/wiki). Cette technique de pyroséquençage haut débit, permet

d'appréhender de manière la plus exhaustive possible la diversité microbienne globale et nous

permet également de pouvoir accéder à la biosphère “rare” qui semble jouer un rôle important

dans l’adaptation à ces environnements pollués.

51

L’étude fonctionnelle par métagénomique (travail en collaboration avec E. Navarro,

AMPERE-LSTM) nous permettra d’étudier la résistance des microorganismes et les

mécanismes importants impliqués dans de tels écosystèmes, comme la fixation du CO2

atmosphérique ou le métabolisme de l’azote, etc. La comparaison des métagénomes issus de

divers environnements permettra une analyse de type fonctionnel (présence/absence/diversité

de gènes impliqués dans les fonctions du sol). Les échantillons de l’analyse métagénomique

ont été choisis parmi les 24 échantillons utilisés pour le pyroséquençage et comprennent

l’étude des sols qui font l’objet d’isolements pour l’étude des bactéries PGPB (travail effectué

par des membres de l’équipe LMBM) afin de pouvoir comparer l’activité des bactéries isolées

à celles de l’analyse fonctionnelle.

L’analyse des données du pyroséquençage a commencé a donné ses premiers résultats. Un

des soucis rencontré lors de cette étude a été la génération d’un fichier conséquent (près de 5

Go pour le fichier ssh) qui n’a pas permis de réaliser l’ensemble des opérations sur ordinateur

et il a fallu l’utilisation d’un serveur à distance au niveau du laboratoire de Montpellier pour

finaliser les analyses.

Les séquences brutes générées par la technique 454 GS-FLX Titanium auprès de la

plateforme de séquence MR DNA (Molecular Research LP, Texas, EU, http://mrdnalab.com)

ont été analysées en utilisant la version 1.33.2 du logiciel mothur, (http://www.mothur.org)

(Schloss et al., 2009). Ces séquences ont été traitées par la commande "shhh.flows" en

utilisant l'algorithme PyroNoise (Quince et al. 2009, 2011). Le prétraitement des séquences

non alignées a inclus la suppression des codes barres, des deux amorces, de toutes les

séquences ambigües (contenant au moins un nucléotide «N», ainsi que toutes celles qui

contenaient plus de 8 homopolymères). Les séquences identiques (100%) ont ensuite été

regroupées pour accélérer le traitement des données et les séquences représentatives ont été

alignées sur la base de données de référence SILVA (bactéries et archées) en utilisant

l'algorithme de Needleman-Wunsch (Needleman & Wunsch, 1970). Les séquences mal

alignées ont ensuite été éliminées. Une autre étape de criblage (pré-cluster) a été appliquée

pour réduire les erreurs dues au pyroséquençage, par regroupement des séquences qui ne

présentent qu’une base de différence sur 100 pb par rapport à une séquence de référence

présente en plus grand nombre dans le groupe (Huse et al., 2010). Les séquences chimériques

ont été détectées et supprimées en utilisant le programme Uchime Chimera (Edgar et al.,

2011) et les séquences d’Archaea ou les organites des organismes eucaryotes comme les

chloroplastes ont également été retirés de l'ensemble de données.

Ces analyses sont en cours et je ne m’étendrai donc pas trop dessus. Les premiers résultats

montrent que l’étude des 72 échantillons de sols (24 sites en triplicat) a permis d’obtenir un

total de 1545801 séquences brutes ayant une longueur moyenne d’environ 400 pb. Après le

nettoyage et l’ensemble des traitements, 743501 séquences de bonne qualité (d’environ 172

pb) ont été récupérées. Après normalisation, 101 016 séquences correspondant à 6966 OTUS

dont 3640 OTUs rares (représentant 52% des séquences) ont pu être identifiés.

52

Table 8. Estimation de la richesse en OTUs, des indices de diversité et de la couverture estimée pour

les 5 échantillons de sédiments. Les résultats sont présentés pour les données normalisées,

rééchantillonnées au hasard pour avoir une taille d’échantillon égale entre les sites.

Sampling stations N° reads

after quality

filtering

N° of

normalized

reads

N° of

OTUa

Singletons Good's

coverageb

Chao1 Richness Shannon

diversityc

MINE DE BOUBKER

OMF12BoGrRh1a 16966 1403 428 62 82% 934 (785; 1146) 5.14 (5.06; 5.23)

OMF12ToGrRh1b 15417 1403 397 40 85% 715 (617; 857) 5.06 (4.98; 5.15)

OMF12ToGrRh1c 16215 1403 433 62 82% 855 (732; 1028) 5.18 (5.09; 5.26)

OMF12BoGrRh3a 16543 1403 396 43 84% 769 (655; 933) 4.92 (4.83; 5.02)

OMF12BoGrRh3b 9265 1403 362 33 87% 617 (535; 737) 4.86 (4.77; 4.95)

OMF12BoGrRh3c 6182 1403 368 25 87% 623 (540; 745) 4.99 (4.90; 5.07)

OMF12BoGrNu4a 5796 1403 132 13 98% 178 (151; 240) 4.23 (4.17; 4.28)

OMF12BoGrNu4b 10148 1403 147 18 97% 198 (169; 266) 4.36 (4.31; 4.42)

OMF12BoGrNu4c 5781 1403 123 13 98% 170 (141; 248) 4.27 (4.22; 4.32)

OMF12BoGHRh5a 15933 1403 363 43 85% 663 (569;800) 4.77 (4.68; 4.86)

OMF12BoGHRh5b 17654 1403 390 50 84% 761 (648;923) 4.92 (4.84; 5.01)

OMF12BoGHRh5c 6858 1403 362 31 86% 624 (541; 746) 4.83 (4.74; 4.91)

OMF12BoGHNu6a 1479 1403 10 1 100% 12 (10; 25) 1.39 (1.36; 1.43)

OMF12BoGHNu6b 2548 1403 27 2 100% 33 (28;66) 2.79 (2.75; 2.83)

OMF12BoGHNu6c 1403 1403 26 10 99% 92 (48; 223) 2.20 (2.15; 2.24)

OMF12BoHeRh13a 12336 1403 291 27 88% 697 (550; 927) 4.56 (4.48; 4.64)

OMF12BoHeRh13b 9130 1403 280 32 89% 514 (431; 642) 4.42 (4.33; 4.51)

OMF12BoHeRh13c 16533 1403 273 26 89% 526 (434; 669) 4.40 (4.31; 4.49)

OMF12BoHeRh15a 16622 1403 336 45 86% 760 (618;973) 4.70 (4.61; 4.78

OMF12BoHeRh15b 4431 1403 326 36 88% 555 (477;673) 4.75 (4.66; 4.83)

OMF12BoHeRh15c 8442 1403 322 32 88% 534 (463; 642) 4.66 (4.57; 4.74)

MINE DE TOUISSIT

OMF12ToGrRh17a 13495 1403 426 39 82% 862 (734; 1043) 5.04 (4.95; 5.13)

OMF12ToGrRh17b 9853 1403 443 48 82% 901 (768; 1088) 5.10 (5.01; 5.19)

OMF12ToGrRh17c 5823 1403 407 35 84% 760 (653; 913) 4.96 (4.87; 5.06)

OMF12ToGrNu18a 14943 1403 213 39 92% 396 (323; 517) 3.93 (3.84; 4.01)

OMF12ToGrNu18b 15623 1403 233 42 91% 441 (361; 573) 4.01 (3.92; 4.10)

OMF12ToGrNu18c 5824 1403 211 28 93% 322 (277; 399) 4.00 (3.92; 4.09)

OMF12ToGrRh19a 11327 1403 444 50 82% 815 (708; 965) 5.12 (5.03; 5.21)

OMF12ToGrRh19b 10389 1403 408 36 85% 728 (629; 870) 5.12 (5.03; 5.21)

OMF12ToGrRh19c 9623 1403 429 46 84% 740 (647; 873) 5.20 (5.12; 5.29)

OMF12ToGrNu20a 11811 1403 307 45 88% 641 (526; 818) 4.48 (4.39; 4.57)

OMF12ToGrNu20b 11922 1403 310 43 88% 656 (536; 839) 4.48 (4.39; 4.57)

OMF12ToGrNu20c 8779 1403 309 51 88% 595 (497; 746) 4.48 (4.38; 4.57)

OMF12ToHeRh21a 13553 1403 465 75 77% 1312 (1071; 1650) 4.92 (4.82; 5.02)

OMF12ToHeRh21b 11746 1403 449 80 80% 1019 (857; 1246) 4.92 (4.82; 5.02)

OMF12ToHeRh21c 11950 1403 444 73 81% 864 (744; 1032) 4.99 (4.90; 5.09)

OMF12ToHeNu22a 5971 1403 449 49 81% 863 (745; 1028) 5.20 (5.12; 5.29)

OMF12ToHeNu22b 15394 1403 440 54 81% 1026 (853; 1271) 5.11 (5.02; 5.19)

OMF12ToHeNu22c 12936 1403 443 56 81% 954 (807; 1162) 5.11 (5.02; 5.20)

OMF12ToHeRh23a 4035 1403 532 58 78% 1067 (923; 1264) 5.62 (5.55; 5.69)

OMF12ToHeRh23b 9998 1403 552 70 78% 967 (858; 1115) 5.72 (5.65; 5.79)

OMF12ToHeRh23c 7476 1403 552 61 78% 975 (864; 1126) 5.69 (5.62; 5.76)

OMF12ToGrRh25a 1775 1403 385 28 86% 681 (585; 821) 4.98 (4.89; 5.07)

OMF12ToGrRh25b 3785 1403 375 28 87% 576 (512; 669) 4.87 (4.78; 4.96)

53

OMF12ToGrRh25c 3707 1403 414 44 84% 773 (665; 929) 5.09 (5.01; 5.18)

OMF12ToGrNu26a 11614 1403 448 53 82% 931 (787; 1136) 5.28 (5.20; 5.36)

OMF12ToGrNu26b 10035 1403 434 47 83% 750 (658; 881) 5.18 (5.10; 5.27)

OMF12ToGrNu26c 8394 1403 430 50 83% 924 (774; 1140) 5.14 (5.06; 5.23)

OMF12ToGrRh27a 13447 1403 445 74 82% 792 (693; 929) 5.08 (4.99; 5.17)

OMF12ToGrRh27b 6646 1403 415 51 84% 697 (613; 816) 5.02 (4.93; 5.11)

OMF12ToGrRh27c 19157 1403 458 62 81% 905 (779; 1081) 5.13 (5.04; 5.22)

SITE DE OUED EL HEIMER

OMF12OhGrRh29a 18557 1403 243 26 91% 457 (374; 594) 4.40 (4.33; 4.48)

OMF12OhGrRh29b 6486 1403 270 25 89% 523 (431; 666) 4.49 (4.41; 5.57)

OMF12OhGrRh29c 14358 1403 292 27 88% 576 (477; 728) 4.56 (4.48; 4.64)

OMF12OhGrNu30a 6869 1403 106 4 96% 212 (157; 327) 2.43 (2.33; 2.53)

OMF12OhGrNu30b 7341 1403 174 27 93% 368 (285; 513) 2.86 (2.75; 2.98)

OMF12OhGrNu30c 5523. 1403 112 13 96% 245 (175; 392) 2.25 (2.13; 2.36)

OMF12OhGrRh31a 7651 1403 541 105 74% 1350 (1136; 1641) 5.31 (5.22; 5.41)

OMF12OhGrRh31b 12625 1403 565 119 73% 1370 (1164; 1646) 5.42 (5.33; 5.51)

OMF12OhGrRh31c 9757 1403 556 108 74% 1345 (1141; 1619) 5.37 (5.27; 5.46)

OMF12OhHeRh33a 14507 1403 586 110 73% 1377 (1177; 1644) 5.68 (5.60; 5.76)

OMF12OhHeRh33b 19202 1403 589 113 72% 1363 (1169; 1621) 5.66 (5.58; 5.74)

OMF12OhHeRh33c 19009 1403 572 107 74% 1371 (1164; 1651) 5.64 (5.56; 5.72)

OMF12OhHeNu34a 8970 1403 475 67 80% 1007 (856; 1217) 5.38 (5.30; 5.45)

OMF12OhHeNu34b 9217 1403 485 86 79% 1003 (860; 1201) 5.37 (5.29; 5.45)

OMF12OhHeNu34c 16679 1403 510 91 78% 1053 (905; 1255) 5.49 (5.41; 5.57)

OMF12OhHeRh35a 5205 1403 492 68 80% 869 (766; 1009) 5.44 (5.36; 5.51)

OMF12OhHeRh35b 16874 1403 536 100 75% 1238 (1056; 1485) 5.46 (5.38; 5.54)

OMF12OhHeRh35c 16636 1403 527 126 75% 1260 (1065; 1525) 5.49 (5.41; 5.57)

OMF12OhHeNu36a 1904 1403 404 57 86% 637 (565; 742) 5.34 (5.27; 5.41)

OMF12OhHeNu36b 4089 1403 464 63 82% 838 (732; 986) 5.45 (5.38; 5.52)

OMF12OhHeNu36c 5329 1403 425 39 85% 661 (590; 762) 5.35 (5.28; 5.42 a OTUs définis avec un seuil de 97% de similarités entre les séquences

b Somme des probabilités des classes observées calculées selon (1 - (n/N)), où n représente le nombre de

singleton et N est le nombre total de séquences c Prend en compte le nombre et la régularité des espèces

Les valeurs entre parenthèses représentent les intervalles de confiance à 95%

L’indice de diversité de Shannon varie fortement entre les échantillons et est compris entre

1.39 et 5.72 et le taux de couverture est compris entre 72 et 100%.

Figure 10. Composition des différents phylums basés

codants pour les ARNr16S des bactéries

est basée sur la classification RDP

Soixante dix-sept pourcent des

phylums ont pu être identifiés. Les

plus importants représentant respectivement

suivi par les Bactéroidetes (7%), les

groupe TM7 (environ 2%). Le

plus abondants sont Sphingomonas

Pseudonocardia. Les genres

stériles miniers (Schippers et al.,

souvent retrouvé dans la phyllosphere

protection de plantes comme

al., 2011). Plusieurs études ont également montrées l’effet

sur la croissance des plantes dans des environnements contaminés par des métaux et

métalloïdes (Ma et al., 2011).

Un important travail d'analyses statistiques et bioinformatiq

afin de décrire la diversité spécifique de chaque site, de comparer les assemblages microbiens

entre les sites, de révéler les relations inter

environnementaux déterminant la compositio

des différents phylums basés sur la classification des séquences des gènes

des bactéries présents dans les 3 sites étudiés. L'affiliation des

est basée sur la classification RDP.

sept pourcent des séquences ont pu être classées au niveau du

ont pu être identifiés. Les Actinobacteria et les Protéobactéria sont les phylums les

plus importants représentant respectivement environ 35 et 25% de l’ensemble des séquences

(7%), les Acidobactéria (5%), les Gemmatimonadetes

groupe TM7 (environ 2%). Le reste des phylums représente moins de 1%.

Sphingomonas, Arthrobacter, Rubrobacter,

Sphingomonas et Arthrobacter ont déjà été détecté

stériles miniers (Schippers et al., 2010, Chen et al., 2013). Le genre

phyllosphere de différentes plantes et pourrait être impliqué dans la

comme Arabidopsis thaliana contre certains pathogènes (

Plusieurs études ont également montrées l’effet bénéfique du genre


Un important travail d'analyses statistiques et bioinformatiques est maintenant nécessaire

décrire la diversité spécifique de chaque site, de comparer les assemblages microbiens

, de révéler les relations inter-spécifiques et d’identifier les facteurs

environnementaux déterminant la composition de la communauté.

54

sur la classification des séquences des gènes

L'affiliation des séquences

au niveau du phylum et 17

sont les phylums les

l’ensemble des séquences

Gemmatimonadetes (3%) et le

moins de 1%. Les 5 genres les

, Nocardioides et

déjà été détectés dans des

Le genre Sphingomonas est

de différentes plantes et pourrait être impliqué dans la

contre certains pathogènes (Innerebner et

bénéfique du genre Arthrobacter


ues est maintenant nécessaire

décrire la diversité spécifique de chaque site, de comparer les assemblages microbiens

spécifiques et d’identifier les facteurs

55

VI. PROJET DE RECHERCHE

Ces thématiques sur lesquelles j'ai acquis aujourd'hui certaines connaissances vont définir

le cadre de mes recherches pour les années à venir.

Je souhaite développer 3 grands volets dans le cadre de l’étude sur les environnements

miniers:

- Caractériser la diversité taxonomique et fonctionnelle en utilisant les nouvelles

technologies de séquençages associées à des méthodes tels que la métagénomique ou la

métatranscriptomique afin d’explorer la structure et la dynamique des communautés

microbiennes (Bactéries, Archaea et Eucaryotes) et obtenir ainsi une vue d'ensemble de leurs

potentiels métaboliques dans le but de mieux comprendre le fonctionnement de ces

écosystèmes particuliers,

- Continuer l’étude des interactions plantes-microorganismes initiée lors de mon

expatriation qui permettent d’aider à la mise en place de solutions pratiques dans le cas de la

mise en place d’un couvert végétal,

- Poursuivre l’isolement de microorganismes afin de pouvoir les étudier en laboratoire ce

qui permet d’avoir une meilleure connaissance de leurs réelles capacités métaboliques.

Caractérisation de la diversité taxonomique et fonctionnelle

Ces dernières années, les nouvelles technologies de séquençage ont fortement accélérées

les recherches en biologie et microbiologie et ont permis la production de très grands volumes

de séquences en diminuant fortement les prix, comparées aux méthodes de séquençage

traditionnelles (Knief et al., 2014). Ces développements récents permettent maintenant de

répondre à des questions qui n’étaient pas concevables il y a seulement quelques années en

raison essentiellement des limitations techniques et financières comme par exemple : qu’elles

sont les communautés microbiennes présentes, que font elles, comment arrivent t’elles à se

développer dans ces environnements, comment évoluent t’elles en fonction des perturbations

et des changements de leurs environnements, comment interagissent elles entre elles et avec

leur environnement, comment peuvent elles affecter le développement des plantes (Knief et

al., 2014)? Le recours aux disciplines « méta-omiques », permet en effet d’avoir une meilleure

compréhension de l'écologie microbienne. La métagénomique est par exemple devenue une

des disciplines scientifiques les plus actives. Cette approche permet désormais l’analyse de

communautés microbiennes qui semblaient largement hors de portée il y a encore quelques

années comme les organismes non cultivés et permet d'obtenir une vue d'ensemble du

potentiel métabolique des communautés présentes. Des études comme la

métatranscriptomique basées sur l'expression des gènes sont également très intéressantes pour

apporter de nouvelles connaissances sur la dynamique fonctionnelle des communautés

microbiennes, mieux identifier les facteurs environnements qui régulent leurs activités et

mettre en exergue les fonctions phares essentielles pour le fonctionnement de la communauté

(Gifford et al., 2011 ; Carvalhais et al., 2012).

Cependant, il faut malgré tout bien garder à l’esprit que, comme la plupart des techniques

de biologie, ces technologies ne sont pas non plus exempts de biais. Concernant les études de

diversité par les techniques de séquençage à haut débit (pyroséquençage ou technologie

56

Illumina, etc.), il est à noter par exemple la faible taille des amplicons générés pour l’instant

ainsi que le taux important d’erreurs de séquençage, que l’utilisation de nouveaux algorithmes

tente de corriger (Huse et al., 2010 ; Knief, 2014). Il reste également les biais inhérents à la

biologie moléculaires comme ceux au niveau de l’extraction de l’ADN ou ceux générés lors

de l’étape de PCR. L’utilisation des séquenceurs de 3ème

générations permettront peut être de

limiter ce dernier biais dans les prochaines années. Concernant la métagénomique par

exemple, même un séquençage « en profondeur » d’un environnement ne permet d’accéder

qu’à une petite fraction de la variabilité génétique réellement présente en identifiant

principalement les membres les plus abondants (Gilbert and Dupont, 2011).

Je souhaite, dans la poursuite de mes travaux, continuer à m’investir dans l’étude des

drainages miniers acides. Comme déjà présenté précédemment, les conditions de vie extrêmes

de ces environnements (pH acides, concentrations élevés en métaux et métalloïdes toxiques

qui diffèrent d’un site à un autre) et les communautés simplifiées qui les caractérisent

permettent d’aborder un certain nombre de questions fondamentales et contribuent à mieux

comprendre la structure des communautés microbiennes et leurs profils de diversité. Mais

c’est surtout l’aspect appliqué qui m’intéresse en raison de l’implication de ces organismes

dans les processus de génération et/ou de remédiation de ces DMA qui peuvent avoir des

applications concrètes dans de nombreux pays du Sud, en accord avec les objectifs de l’IRD.

Une meilleure caractérisation de la diversité génétique mais aussi fonctionnelle de ces

communautés microbiennes ainsi que l’étude de leurs interactions entre elles et avec leur

environnement est en effet une étape essentielle à la compréhension du fonctionnement de ces

écosystèmes pour pouvoir développer à terme des stratégies pour remédier à ces pollutions.

Pour ce faire, il est également indispensable de prendre en compte l’ensemble de la

communauté microbienne comprenant les organismes procaryotes et eucaryotes car, jusqu’à

présent, la majorité des études réalisées sur ces écosystèmes se sont focalisées sur les

bactéries.

L’ancien site minier de Carnoulès, par ses caractéristiques comme les concentrations

exceptionnelles en As ainsi que par la présence d’un gradient spatial de pollution résultant de

processus naturels de remédiation demeurera un site d’étude important pour mes travaux,

notamment dans le cadre de projets comme l’ANR ECO-TS IngECOST-DMA ou au travers

de l’observatoire OSU OREME.

Les projets de recherche que je souhaite développer au cours des prochaines années

concerneront surtout les pays du Sud avec par exemple le Maroc, en continuation avec les

travaux lancés depuis 3 ans maintenant dans le cadre de mon expatriation et pays avec lequel

je souhaite poursuivre les collaborations dans le futur. L’étude des DMA va se faire par

exemple dans le cadre d’un projet débuté récemment concernant les drainages de mine non

pérennes présents dans un nouveau chantier, la mine de Kettara au Maroc qui se fait en

association avec l’équipe de recherche E2G (R. Hakkou) de la Faculté des Sciences et

Techniques de Guéliz à Marrakech.

La mine de Kettara, située à environ 30 km au nord-est de Marrakech a été exploitée pour

sa pyrrhotite de 1964 à 1981 et a produit 5.2 Mt de pyrrhotite concentré contenant une

57

moyenne de 29% en poids de sulfures ce qui a généré d’importants stériles miniers répartis,

sans aucune protection, sur de très grande surfaces autour de la mine (Lghoul et al., 2014).

C’est l’une des mines qui pose le plus de problèmes autour de Marrakech en raison

notamment de la présence de ces DMA qui se forment à chaque pluie importante et qui

expose directement la population du village minier de Kettara avoisinant, qui comprends

environ 2000 habitants. Le climat est semi aride avec une moyenne de 250 mm de pluies

annuelles qui surviennent généralement sur de courtes périodes et avec une forte intensité.

Figure 10. Présentation de la mine de Kettara: (a) localisation, (b) effluent de DMA, and (c) minéraux

secondaires, (d) vue panoramique. D’après Lghoul et al. 2014

Cette mine a été extensivement étudiée comme en atteste les nombreuses publications

depuis quelques années (Hakkou et al., 2008a, 2008b, 2009 ; Lghoul et al., 2014 et références

citées). Ces travaux ont été réalisés en grande partie dans le cadre d’une chaire de recherche

IDRC maroco-canadienne, entre l’équipe de recherche E2G (R. Hakkou) de la Faculté des

Sciences et Techniques de Marrakech et l’Institut de recherche sur les mines et

l’environnement (UQAT) situé à Québec au Canada.

Cette étude microbiologique, se fait en partenariat avec l’équipe de recherche E2G à

Marrakech dans le cadre du Master 2 de N. Mghazli qui vient de débuter (co-encadrement

avec L. Sbabou du LMBM de Rabat) et qui a pour but d’identifier les communautés

procaryotes (Bactéries et Archaea) présentes dans ces déchets miniers pour tenter d’identifier

les communautés responsables de la génération de ces drainages miniers acides. Les

prélèvements ont été réalisés sur 9 points répartis sur l’ensemble du site et une analyse par

séquençage Illumina est en cours.

58

Un autre aspect de la mobilisation des métaux et métalloïdes concerne le cas particulier de

l’As qui fait actuellement peser une lourde menace sur la santé de nombreuses personnes à

travers le monde et ce, principalement dans les pays du Sud, où plusieurs millions de

personnes consomment des eaux de boisson contaminées par ce toxique (Nordstrom, 2000 ;

Jiang et al., 2013). Comme nous l’avons vu, les microorganismes sont fortement impliqués

dans les processus de transfert de ce polluant dans l’environnement. Des travaux sur l’étude

des transferts de l’As (d’origine minière ou géogénique, présent naturellement dans la roche)

vers le milieu aquatique et les impacts sanitaires associés sont traités dans le cadre du LMI

Picass-Eau (« Prédire l’Impact du Climat et des usAges sur les reSSources en Eau en Afrique

SUbsaharienne"). Ce projet prévoit notamment d’aborder la question de la mobilisation de

l’arsenic vers la ressource en eau sur le bassin du Nakambé au Burkina Faso. Il s’agit de

déterminer les facteurs qui influencent la variabilité spatiale de l’arsenic (facteurs

géologiques, hydrogéologiques, physico-chimiques et microbiologiques) dans les aquifères de

la région de Ouahigouya dans le Nord du Burkina Faso et d’étudier l’implication des

microorganismes dans ces systèmes et l’impact sur la santé des populations exposés.

Ces travaux de recherche seront développés dans le cadre de l’ANR BALWASA

(Basement aquifers for a local water service in Africa), si elle est acceptée. Cet ANR a été

déposé cette année par P. Genthon, un hydrogéologue de HydroSciences. Ces travaux se

feront plus spécifiquement dans le cadre du Work Package n° 3 intitulé « Arsenic

contamination and health near Ouahigouya » en collaboration notamment avec F. Lalanne de

l’Institut International d’Ingénierie de l’Eau et de l’Environnement et de P. Genthon.

Etude des interactions plantes–microorganismes dans un contexte de phytoremediation

et de réhabilitation des environnements miniers au Maroc

Le travail initié dans le cadre du projet Ec2co va se poursuivre dans les prochains mois

avec un volet important concernant l’analyse métagénomique des populations bactériennes

présentes dans ces environnements et se fera dans le cadre de la thèse de Ikram Dahmani en

collaboration avec Isabelle Navarro (AMPERE, Lyon-LSTM, Montpellier). Cette nouvelle

approche permettra de mieux comprendre comment les communautés bactériennes s’adaptent

à leur environnement et interagissent avec lui (identification des gènes de résistance ou

d’oxydation à l’arsenic, etc.) et comment elles peuvent affecter le développement des plantes

(fixation d’azote par exemple, etc.).

D’autres études sur la revégétalisation pourront également se mettre en place, par exemple

sur la mine de Kettara, dans le cadre d’un projet de recouvrement de ces déchets utilisant des

déchets de mines de phosphates, basiques, qui est à l’étude actuellement (Lghoul et al. 2014)

afin de limiter les infiltrations d’eau et la formation de DMA. Si ce revêtement est mis en

place sur l’ensemble du site, ce qui est prévu dans le cadre de la chaire, une couverture

végétale devra être apportée sur le long terme. Des études de diversité et de métagénomiques

permettraient d’identifier les communautés de microorganismes présentes et de mieux

comprendre leurs interactions avec les plantes. Ce travail est à combiner également avec

l’isolement de souches bactériennes pour étudier leurs activités bénéfiques sur les plantes

naturellement présentes (solubilisation du phosphate, production de sidérophores ou d’auxine,

59

fixation d’N, etc.) afin de pouvoir proposer à terme une collection de plantes et de

microorganismes résistants à ces polluants et susceptibles d’être des outils efficaces pour

établir un programme de phytoremédiation.

Isolement de microorganismes et étude en laboratoire de leurs capacités métaboliques

Bien que les méthodes moléculaires apportent des informations indispensables du fait

qu’un faible pourcentage de microorganismes de l’environnement peuvent actuellement être

cultivés en laboratoire, les méthodes culturales restent indispensables pour une meilleure

connaissance des organismes et de leurs interactions réelles avec l’environnement en utilisant

des études physiologiques.

Des études d’isolement réalisées à Carnoulès sur les sédiments du Reigous (Delavat et al.,

2012), ont par exemple souligné l’importance du maintient des méthodes culturales pour

l’identification précise et la compréhension du rôle fonctionnel des microorganismes dans

leurs environnements. Les études réalisées sur les souches de Thiomonas et d’Aciditiobacillus

ferrooxidans ont également bien montré l’intérêt de ces travaux pour la compréhension de

leur rôle réel dans l’environnement et le système de remédiation présent à Carnoulès. De plus,

les méthodes culturales permettent de contourner certains biais inhérents aux approches

moléculaires comme la résistance de certaines bactéries à la lyse cellulaire ou bien la

difficulté à détecter les microorganismes appartenant à la biosphère rare.

Dans le cadre d’une collaboration avec le laboratoire GMGM de Starsboug (P. Bertin), un

projet Ec2co devrait être soumis cette année qui va spécifiquement s’intéresser, entre autre, à

l’étude des microorganismes difficiles à cultiver. Ces travaux vont se focaliser plus

spécifiquement sur des organismes comme Gallionella ferruginea que nous n’avons pas pu

isoler pour l’instant malgré de nombreux essais ou encore pour tenter de cultiver le

pseudogénome CARN1 qui semble avoir un rôle important au sein de l’écosystème et qui est

retrouvé en assez grand nombre dans l’eau et les sédiments et détecté depuis l’utilisation des

méthodes de séquençage à haut débit. Ce projet à pour but de trier par cytométrie les

microorganismes selon des critères taxonomiques et/ou fonctionnels avant d’en séquencer le

génome afin d’étudier le métabolisme de ces microorganismes ; d’isoler par des approches de

culture in situ des populations non cultivées et enfin de déterminer la dynamique et l'activité

des populations microbiennes en fonction des variations contrôlées des paramètres physico-

chimiques.

60

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Volant A, Bruneel O, Desoeuvre A, Héry M, Casiot C, Bru N, Delpoux S, Fahy A,




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67

VIII ANNEXES : SELECTION DE 5 PUBLICATIONS

Bruneel O, Duran R, Casiot C, Elbaz-Poulichet F, Personné JC (200) Diversity of



Bruneel O, Pascault N, Egal M, Bancon-Montigny C, Goni M, Elbaz-Poulichet F,

Personné J-C, Duran R (2008) Archaeal diversity in a Fe-As rich acid mine drainage at


Bruneel O, Volant A, Gallien S, Chaumande B, Casiot C, Carapito C, Bardil A, Morin

G, Brown Jr GE, Personné JC, Le Paslier D, Schaeffer C, Van Dorsselaer A, Bertin PN,

Elbaz-Poulichet F, Arsène-Ploetze F (2011) Characterization of the active bacterial

community involved in natural attenuation processes in arsenic-rich creek sediments.

Microbial Ecology. 61, 793-810

Volant A, Desoeuvre A, Casiot C, Lauga B, Delpoux S, Morin G, Personné JC, Héry M,

Elbaz-Poulichet F, Bertin P and Bruneel O (2012) Archaeal diversity: temporal variation in

the Arsenic-Rich Creek Sediments of Carnoulès Mine, France. Extremophiles. 16, 645-657

Volant A, Bruneel O, Desoeuvre A, Héry M, Casiot C, Bru N, Delpoux S, Fahy A,




APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2006, p. 551–556 Vol. 72, No. 10099-2240/06/$08.00�0 doi:10.1128/AEM.72.1.551–556.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Diversity of Microorganisms in Fe-As-Rich Acid Mine DrainageWaters of Carnoules, France

O. Bruneel,1* R. Duran,2 C. Casiot,1 F. Elbaz-Poulichet,1and J.-C. Personne1

Laboratoire Hydrosciences Montpellier, UMR5569, Universite Montpellier 2, Place E. Bataillon, Case MSE,34095 Montpellier cedex 05,1 and Laboratoire d’Ecologie Moleculaire-Microbiologie, EA 3525,

Universite de Pau et des Pays de l’Adour, Avenue de l’Universite, IBEAS, BP 1155,F-64013 Pau cedex,2 France

Received 14 March 2005/Accepted 28 September 2005

The acid waters (pH 2.7 to 3.4) originating from the Carnoules mine tailings contain high concentrations ofdissolved arsenic (80 to 350 mg · liter�1), iron (750 to 2,700 mg · liter�1), and sulfate (2,000 to 7,500 mg ·liter�1). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of thedissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek havebeen characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA genelibrary analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eightypercent of the sequences obtained are related to sequences from uncultured, newly described organisms orrecently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recoveredare mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related touncultured TrefC4 affiliated with Gallionella ferruginea, a neutrophilic Fe-oxidizing bacterium, are dominant.The description of the bacterial community structure and its dynamics lead to a better understanding of thenatural remediation processes occurring at this site.

The processing of sulfide-rich ores in the recovery of basemetals, such as copper, lead, zinc, and gold, has produced largequantities of pyrite wastes (20). When exposed to rain, thismaterial generates acid mine drainage (AMD) which containslarge amounts of sulfate, iron, arsenic, and heavy metals. De-spite their toxicity, such waters host organisms, both pro-karyotes and eukaryotes, which are able to cope with the pol-lution (2, 33). Some of them have the capacity to modify thephysicochemical conditions of the water either by detoxifica-tion or by metabolic exploitation. For example, efficient oxida-tion of As by bacteria has been reported in AMD or in chem-ically somewhat similar waters like those from hot springs (3, 7,21, 25, 30). Because of their elevated Fe concentration, thedevelopment of iron-oxidizing bacteria is favored in AMD (16)where Acidithiobacillus ferrooxidans and Leptospirillum fer-rooxidans are often observed (2).

Owing to its ability to oxidize Fe, the bacterial consortium inAMD plays a major role in the immobilization of the elementsthat exhibit a strong affinity for solid Fe oxide phases such asSr, Cs, Pb, U (14), and As (8, 24). In addition, the ability ofseveral bacterial strains in AMD to oxidize As further contrib-utes to reduction of its toxicity in water, because As(III) isconsidered to be more toxic than As(V) (28) and becausearsenate adsorbs more strongly than arsenite to Fe(III) oxidesand hydroxides at acidic pH (5, 26).

Owing to their tolerance of heavy metals and the ability ofsome to promote transformations that make some metals less

toxic, bacteria in acid mine waters may be useful in AMDbioremediation or that of some other industrial effluents. Inorder to develop remediation processes or optimize them, fur-ther knowledge of the bacteria living in the extreme environ-ment of AMD is required.

This study aims to investigate the microbial community of asmall creek (the Reigous, present at Carnoules, France). TheCarnoules mine (Fig. 1) has been inactive since 1962. Its ex-ploitation has left about 1.5 megatons of tailings containing0.7% Pb, 10% FeS2, and 0.2% As. The tailings are containedbehind a dam. Water percolating through the tailings emergesat the base of the dam, forming the head of the Reigous creek.The head waters of the creek are characterized by low pH (2.7to 3.4) and high concentrations of As (100 to 350 mg · liter�1),Fe (750 to 2,700 mg · liter�1), and SO4

2� (2,000 to 7,500 mg ·liter�1).

The As and Fe behavior in creek water has been intensivelystudied (8, 22, 23, 24). As(III) is the dominant As type, whereasFe occurs as Fe(II). Along the first 30 m of the creek (about 1 hof residence time), the bacterially mediated oxidation of Fe(II)leads to the coprecipitation of 20 to 60% of the dissolved As.The precipitate which contains up to 22% of As is mainlycomposed of As(III)-Fe(III) oxy-hydroxide in the wet seasonwhile As(V)-Fe(III) oxy-hydroxide compounds predominate inthe dry season. Several phenotypes of Acidithiobacillus ferrooxi-dans have been isolated, and their role in the oxidation ofFe(II) and the coprecipitation of As has been demonstrated inlaboratory experiments (8, 11). Additionally, Bruneel et al. (7)isolated at this site three different strains of Thiomonas spp.closely related to Thiomonas sp. strain Ynys1 able to promoteAs oxidation in laboratory conditions.

The present study combines terminal-restriction fragment

* Corresponding author. Mailing address: Laboratoire Hydro-sciences Montpellier, UMR5569, Universite Montpellier 2, Place E.Bataillon, Case MSE, 34095 Montpellier cedex 05, France. Phone:33-4-67-14-36-59. Fax: 33-4-67-14-47-74. E-mail: [email protected].

551

length polymorphism (T-RFLP) analysis in order to investigatethe dynamics of the bacterial communities and 16S rRNA genelibrary analysis to identify the dominant bacterial group.

MATERIALS AND METHODS

Sampling procedure and physicochemical determinations in situ. Water sam-ples for molecular analysis of microbial populations were collected in October2002 and January 2003 in the spring and at two other locations in the creek overa distance of 30 m (Fig. 1). A volume of 200 ml of water was filtered through asterile 0.22-�m nucleopore filter. These filters were then transferred to a tube,frozen in liquid nitrogen, and stored at �20°C until further analysis.

DNA isolation. Genomic DNA was extracted from filtered water using theUltraClean Soil DNA Isolation kit according to the recommendation of themanufacturer (MoBio Laboratories, Inc.). All extracted genomic DNA sampleswere stored at �20°C until further processing.

T-RFLP analysis. Primers 8F (5�-AGAGTTTGATCCTGGCTCAG-3�) and1489R (5�-TACCTTGTTACGACTTCA-3�) (19, 31) were used for T-RFLPanalysis to assess the bacterial community structures. Forward (8F) and reverse(1489R) primers were fluorescently labeled with tetrachlorofluorescein phos-phoramidite and hexachlorofluorescein phosphoramidite (E.S.G.S. CybergeneGroup), respectively. The PCR amplification mixture contained 12.5 �l Hot StartTaq polymerase master mix (QIAGEN), 0.5 �l of each primer (20 �M), and 10ng of DNA template. A final volume of 50 �l was adjusted with distilled water.16S rRNA gene amplification reactions were cycled in a PTC200 thermocycler(MJ Research) with a hot start step at 94°C for 15 min followed by 35 cycles of94°C for 1 min, 52°C for 1.5 min, and 72°C for 1 min, with a final extension stepat 72°C for 10 min. The amount of PCR product was determined by comparisonto known concentrations by the “dots method” (Smartlader; Eurogentec) aftermigration on agarose gel. PCR products were purified with the GFX PCR DNApurification kit (Amersham-Pharmacia).

Purified PCR products (600 to 700 ng) were digested with 12 U of enzymeHaeIII or HinfI (New England Biolabs). The lengths of terminal-restrictionfragments (T-RFs) from the digested PCR products were determined by capil-lary electrophoresis on an ABI prism 310 (Applied Biosystems). About 50 ng ofthe digested DNA from each sample was mixed with 10 �l of deionized form-amide and 0.25 �l of 6-carboxytetramethylrhodamine size standard, denatured at94°C for 2 min, and immediately chilled on ice prior to electrophoresis. After aninjection step of 10 s, electrophoresis was carried out for up to 30 min, applyinga voltage of 15 kV. T-RFLP profiles were performed using GeneScan software(ABI).

Dominant operational taxonomic units represent T-RFs whose fluorescencewas higher than 100 fluorescence units for at least one sample. Predictive diges-tions were made on the RDP web site (http://rdp.cme.msu.edu/html/index.html)using the T-RFLP Analysis Program.

Cloning and restriction analysis. To further characterize the bacterial popu-lations inhabiting the creek in each sampling period and sampling point, thebacterial diversity was analyzed by cloning PCR amplified 16S rRNA genes. ForS1 and COWG, libraries were constructed for each sampling period. For COWA,a library was constructed only for October, since the comparison between Oc-tober and January T-RFLP profiles showed mainly a disappearance of T-RFs inJanuary. Bacterial 16S rRNA genes were amplified with unlabeled 8F and 1489Rprimers. These PCR products were cloned in Escherichia coli TOP10 using thepCR2.1 Topo TA cloning kit (Invitrogen, Inc.). Cloned 16S rRNA gene frag-ments were amplified using the primers TOP1 (5�-GTGTGCTGGAATTCGCCCTT-3�) and TOP2 (5�-TATCTGCAGAATTCGCCCTT-3�), located on the vec-tor and surrounding the inserted PCR fragment, and then were digested with theenzyme HaeIII or HinfI. Restriction profiles were analyzed using 2.5% agarosegel electrophoresis (small-fragment resolution agarose; QA agarose; QBiogene,Inc.). Sixty clones from each library were analyzed and grouped according totheir RFLP patterns (HaeIII and HinfI digestion). Only sequences from domi-nant groups were determined

16S rRNA gene sequencing. Partial sequences of the 16S rRNA gene (from 8to 336 according to E. coli numbering) were determined by the dideoxy nucle-otide chain termination method using a BigDye cycle sequencing kit (AppliedBiosystems) on an ABI PRISM 310 Genetic analyzer (Applied Biosystems).DNA sequence analyses were performed via the infobiogen server (http://www.infobiogen.fr) by using the FASTA, BLAST, ALIGNN, and CLUSTALW pro-grams (1, 13, 29). Phylogenetic trees were constructed by using the PHYLIPcomputer package (13). The confidence level of the phylogenetic tree topologywas evaluated by performing 100 bootstrap replications with the SEQBOOKprogram.

RESULTS

Bacterial community structures. The results of the T-RFLPanalysis of bacterial community structure are presented in Fig.2. The average T-RF number was relatively small (about 10)both in October and January, reflecting low bacterial diversity.The bacterial population characterized by a 216-bp (� 2 bp)T-RF was generally the most abundant, except at station S1 inOctober. Abundance of this 216-bp T-RF generally increasedbetween October and January, except at station COWG, wherethe variations were minor.

Composition of bacterial communities. The most represen-tative sequences of the dominant clones are summarized inTable 1, and the phylogenetic analysis of all the obtainedsequences are presented in Fig. 3 to 5.

The most abundant sequence types are positioned within thebeta subdivision of the Proteobacteria (Table 1). They wererecovered at all stations during both sampling periods, ac-counting for 5 to 28% of the clones in October and more than65% in January. Numerous clone sequences of this group dis-played around 95% homology with a sequence isolated froman acid- and iron-rich stream in the United Kingdom (Gen-Bank accession no. AY766002) (unpublished data). The phy-logenetic analyses (Fig. 3) did not allow affiliation of the clonesequences with any representative of the subdivision. The clos-

FIG. 1. Map of the Carnoules mining site and location of samplingstations. Sampling stations were Reigous spring (S1), 3 m downstreamof the spring (COWA), and 30 m downstream of the spring (COWG).

552 BRUNEEL ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 2. Seasonal comparison of bacterial community T-RFLP fingerprints from the AMD of Carnoules, France, in October and January samples.

FIG. 3. Phylogenetic analysis of 16S rRNA gene sequences affiliated with the Gallionella division from the AMD of Carnoules, France. Clonenames in boldface correspond to sequences found in October (Oct) and January (Jan) within the three stations along the Reigous Creek, S1,COWA, and COWG.

553

est relative (91%) is Gallionella ferruginea, a neutrophilic iron-oxidizing bacterium.

The sequences representing the second-most abundant typeare positioned within the delta subdivision of the Proteobacte-ria (Table 1, Fig. 4). These sequences were more abundant inOctober, representing 10, 8, and 5% of the clones at S1,COWA, and COWG, respectively, than in January, with 5 and1% at S1 and COWG. In October, all the clones were similar(more than 90% similarity) to clones found in an AMD at IronMountain (4). In contrast, the clones of January were similar(94% similarity) to those found in a forested wetland impactedby sulfate-rich waters from coal piles (6). As for the mainsequence, the phylogenetic analyses did not allow the affilia-

FIG. 4. Phylogenetic analysis of 16S rRNA gene sequences affili-ated with the Desulfobacterium division from the AMD of Carnoules,France. Clone names in boldface correspond to sequences found inOctober (Oct) and January (Jan) within the three stations along theReigous Creek, S1, COWA, and COWG.

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tion of the clone sequences with any representative of thesubdivision. The closest relative was Desulfobacterium indoli-cum, a sulfate-reducing bacterium (18). For the clone fromCOWG in January that was phylogenetically distant from theothers (Fig. 4), the closest relative is Desulfomonile tiedjei, asulfate-reducing bacterium (12).

The next most abundant sequence types, representing 8% ofthe clones at S1 in October and 4% at COWG in January(Table 1), were firmly positioned in the Acidithiobacillus fer-rooxidans group (Fig. 5). Three sequences are related (99%similarity) to uncultured A. ferrooxidans KF/GS-JG36-22 (27)isolated in waste piles of a uranium mine, and one sequencewas related to A. ferrooxidans DSM 2392 (Fig. 5).

The next group, representing 8% of the clones at S1 inOctober, was associated with the Actinobacteria group, with94% similarity with sequences recovered in forested wetlandexposed to coal effluent (6). The phylogenetic analysis couldnot affiliate the sequence with any isolated bacterium (data notshown).

The last sequence found, detected only in January atCOWG, representing 5% of the clones, was firmly positionedin the Thiobacillus group with 91% similarity with T. plum-bophilus DSM 6690. These strains were isolated from a ura-nium mine, and they grew by oxidation of H2S, galena (PbS),and H2 (10).

Finally, sequences closely related to 16S rRNA genes from achloroplast of Euglena spp. were also detected (data notshown). This was not surprising, since the 16S rRNA gene ofchloroplasts is closely related to the bacterial 16S rRNA geneand therefore can be amplified by primers 8F and 1489R.Moreover, this is consistent with previous work indicating thatthese organisms are able to accumulate and oxidize As in thecell (9).

DISCUSSION

In the Reigous creek, the low bacterial diversity as revealedby molecular-based methods is consistent with the results ofBaker and Banfield (2) in a similar environment. This mayreflect the limited number of different electron donors andacceptors available in AMD and the toxicity of heavy metalsand low pH.

Numerous sequences in the libraries are related to se-quences previously found in AMD, indicating that the clonelibraries were not contaminated. Nevertheless, 80% of thesequences could not be closely related to cultured organisms,suggesting that they may constitute new taxa. As long as thebacterial strains were not isolated, their physiological role inthe creek ecology will remain uncertain.

Both molecular methods revealed that the dominant popu-lation (216-bp [�2 bp] T-RF) can be related to Gallionellaferruginea sequences, as indicated by predictive digestion (217bp) and 16S rRNA gene library analyses. Gallionella ferrugineais a neutrophilic bacterium that oxidizes Fe. It has been shownto efficiently remove Fe, As(III), and As(V) in water (17). It ispossible that an acid-tolerant relative of this bacterium has theability to oxidize iron under acid pH conditions. In the creek,the abundance of this population was much more significant inJanuary (more than 65%) than in October (less than 30%).Such variations are consistent with the occurrence of higher Feand As precipitation rates in the rainy seasons than in otherseasons, as reported by Casiot et al. (8). In addition to theGallionella ferruginea sequences, the library analyses show thepresence of other uncultured bacterial groups related to the Fecycle, such as the Actinobacteria group. Members of this group,previously reported in AMD, are iron-oxidizing, heterotrophic,

FIG. 5. Phylogenetic analysis of 16S rRNA gene sequences affili-ated with the Acidithiobacillus division from the AMD of Carnoules,France. Clone names in boldface correspond to sequences found inOctober (Oct) and January (Jan) within the three stations along theReigous Creek, S1, COWA, and COWG. Strains in boldface (CC1, B5,B4, and B9) represent the bacteria isolated in the Carnoules Creek.

VOL. 72, 2006 DIVERSITY OF MICROORGANISMS IN MINE DRAINAGE 555

acidophilic bacteria capable of autotrophic growth. Some ofthem may play a synergistic role, removing organic carbon (4).Finally, A. ferrooxidans constituted a minor group in the Fe-oxidizing bacterial population contrary to expectations fromprevious findings based on isolation and culturing techniques(8).

With respect to bacteria involved in S cycling, the sequencesrecovered, in addition to A. ferrooxidans, are related to mem-bers of the Desulfobacterium genera, which contains sulfate-reducing bacteria (18). As the water of the Carnoules creek isfully oxygenated, the presence of bacteria from this group,which is characterized by anaerobic respiration, may be sur-prising. Nevertheless, this is in agreement with several studiesthat have recently reported sulfate- and iron-reducing bacteriaunder acidic conditions (15, 32).

Considering the small population, sequence analyses indi-cate the presence of bacteria from the � subdivision of theProteobacteria affiliated with the Thiobacillus group. A memberof this group was recently described as a galena and hydrogenoxidizer (11). A Thiomonas sp., which has been isolated andshown to be very active in the oxidation of As (7), was notdetected by molecular techniques, probably reflecting its lowabundance.

Analyses of the most abundant clones strongly indicate thatfurther efforts have to be exerted to fully understand AMDsystems. They include isolation and identification of the organ-isms represented by these clones in order to define their eco-physiological roles.

ACKNOWLEDGMENTS

This study was financed by the project GEOMEX from the CNRS,the project Environnement Vie et Societes (CNRS-INSU), the ACI-Ecologie Quantitative, and the ACI-ECCODYN (French Ministry ofResearch).

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ORIGINAL PAPER

Archaeal diversity in a Fe–As rich acid mine drainageat Carnoules (France)

O. Bruneel Æ N. Pascault Æ M. Egal Æ C. Bancon-Montigny ÆM. S. Goni-Urriza Æ F. Elbaz-Poulichet Æ J.-C. Personne ÆR. Duran

Received: 26 November 2007 / Accepted: 9 March 2008

� Springer 2008

Abstract The acid waters (pH = 2.73–3.4) that originate

from the Carnoules mine tailings (France) are known for

their very high concentrations of As (up to 10,000 mg l-1)

and Fe (up to 20,000 mg l-1). To analyze the composition

of the archaeal community, (their temporal variation inside

the tailing and spatial variations all along the Reigous

Creek, which drains the site), seven 16S rRNA gene

libraries were constructed. Clone analysis revealed that all

the sequences were affiliated to the phylum Euryarchaeota,

while Crenarchaeota were not represented. The study

showed that the structure of the archaeal community of the

aquifer of the tailing stock is different to that of the Rei-

gous Creek. Irrespective of the time of sampling, the most

abundant sequences found inside the tailing stock were

related to Ferroplasma acidiphilum, an acidophilic and

ferrous-iron oxidizing Archaea well known for its role in

bioleaching. Inversely, in Reigous Creek, a sequence

affiliated to the uncultured Thermoplasmatales archaeon,

clone YAC1, was largely dominant. This study provides a

better understanding of the microbial community associ-

ated with an acid mine drainage rich in arsenic.

Keywords Microbial diversity � Arsenic �Acid mine drainage � Mine tailings

Introduction

The processing of sulfide-rich ores in the recovery of base

metals such as copper, lead, zinc, and gold, has produced

large quantities of pyrite wastes (Langmuir 1997). When

exposed to rain, this material generates acid mine drainage

(AMD) which contains large quantities of sulfate, iron,

arsenic and heavy metals. Despite their toxicity, these

waters are colonized by iron- and sulfur-oxidizing pro-

karyotes and form stable microbial communities with

obligate acidophilic eukaryotes (fungi, yeasts, algae and

protozoa; Johnson 1998; Zettler et al. 2002). The metabolic

activities of such communities lead to solubilization

(leaching) of the heavy metals from the sulfidic ores and

pollution of surface and subsurface waters fed by the run-

off.

For several decades, bacteria-like Acidithiobacillus or

Leptospirillum have been considered to be the principal

acidophilic sulfur- and iron-oxidizing microorganisms in

AMD. They were believed to be responsible for pyrite

oxidation and for the release of associated metals. How-

ever, during the last 10 years, several studies have

evidenced the presence of archaeal communities in acidic

waters (Edwards et al. 2000; Dopson et al. 2004). Previ-

ously, Archaea were renowned for their ability to inhabit

extreme environments and specialized niches but their

widespread presence in non-extreme environments, such as

marine and terrestrial soils, was also recently revealed

(Chaban et al. 2006).

Archaeal communities are often better adapted to low

pH, high concentrations of total and ferrous iron and other

Communicated by J.N. Reeve.

O. Bruneel (&) � N. Pascault � M. Egal � C. Bancon-Montigny �F. Elbaz-Poulichet � J.-C.Personne

Laboratoire Hydrosciences Montpellier, UMR 5569,

IRD, CNRS, Universites Montpellier 1 et 2,

Universite Montpellier 2, Place E. Bataillon,

Case MSE, 34095 Montpellier Cedex 05, France

e-mail: [email protected]

M. S. Goni-Urriza � R. Duran

Equipe Environnement et Microbiologie

UMR CNRS 5254, IPREM, EEM, Universite de Pau

et des Pays de l’Adour, Avenue de l’Universite, IBEAS,

BP 1155, 64013 Pau Cedex, France

123

Extremophiles

DOI 10.1007/s00792-008-0160-z

metals, and moderately elevated temperatures than classi-

cal bioleaching mesophilic bacteria (Acidithiobacillus spp.

and Leptospirillum spp.). Archaea were seen as numeri-

cally significant members in these environments (Bond

et al. 2000; Edwards et al. 2000; Johnson and Hallberg

2003). Furthermore, it has been suggested that Archaea

could play a major role in the generation of AMD (Baker

and Banfield 2003) with oxidation of iron. Some members

of the Archaea that respire As(V) like Pyrobaculum

aerophilum and Pyrobaculum arsenaticum have been dis-

covered (Huber et al. 2000; Oremland and Stolz 2003).

Furthermore, Pyrobaculum arsenaticum, forms realgar

(As2S2) as a precipitate under organotrophic conditions in

the presence of thiosulfate and arsenate. These findings

suggest that Archaea may play a significant role in the

biogeochemical cycling of arsenic (Huber et al. 2000;

Chaban et al. 2006).

Highly acidic environments are relatively scarce world-

wide and are generally associated with mining activities.

The oxidation by meteoric water of the pyrite-rich wastes

from the abandoned Pb–Zn Carnoules mine generates low

pH (2.7–3.4) water containing high concentrations of As

and Fe, up to 10,000 and up to 20,000 mg l-1, respectively

(Casiot et al. 2003a). We previously characterized the

bacterial communities and showed that populations related

to sulfate-reducing bacteria and Gallionella ferruginea

seem to play a key role in AMD functioning (Bruneel et al.

2005, 2006). To know how a system is structured and how it

functions, we first have to address the diversity of the whole

community. We used a molecular phylogenetic approach to

characterize the microbial structure and infer a corre-

sponding ecosystem function where appropriate. The aim of

the present study was to investigate the archaeal community

in water samples from an AMD very rich in As, to improve

our understanding of the implication of these microorgan-

isms in AMD functioning. This is the first molecular

analysis of the archaeal community present in the Carnoules

mine system.

Materials and methods

Description of the study site

The lead and zinc mine of Carnoules, which has been

abandoned since 1963, produced 1.2 MT of spoil material

containing sand, sulfide minerals, heavy metals (Pb, Zn, Tl)

and metalloids (As, Sb). The material is deposited in the

middle of and across the upstream part of a creek (the

Reigous) at the site of its natural spring. The Reigous

collects downstream seepage waters from the surroundings

before joining, at 1.5 km, the relatively pristine Amous

river.

The source of the Reigous Creek, now located at the foot

of the dike retaining the mining spoil, is acid (pH 2.7–3.4)

and very rich in dissolved arsenic and iron (80–350 and

750–2,700 mg l-1 respectively, Leblanc et al. 2002) pre-

dominantly in their reduced forms: As(III) and Fe(II). The

water discharge is comprised between 0.8 and 1.7 l s-1.

In the Reigous Creek, As(III) is the dominant As species

whereas Fe occurs as Fe(II). Along the first 30 m of the

creek (about 1 h residence time), the microbial mediated

oxidation of Fe(II) leads to the coprecipitation of 20–60%

of the dissolved As. As-rich (up to 20%) yellow sediments

cover the bottom of the creek. The precipitate is mainly

composed of amorphous Fe(III)–As(III) associated with

tooeleite, a rare nanocrystal mineral of Fe(III)–As(III)

during the winter period and with amorphous Fe(III)–

As(V) the rest of the year (Casiot et al. 2003b; Morin et al.

2003). Bacteria play an essential role in the oxidation of Fe

and As (Casiot et al 2003b). Bacterial diversity is lower

than in unpolluted water. Sequences related to G. ferrugi-

nea, a neutrophilic Fe-oxidizing bacterium, are dominant

(Bruneel et al. 2006).

The biogeochemical processes that occur in the Car-

noules spoil heaps are more complex than those in the

creek. The general hydrochemistry and aquifer hydrody-

namics have already been broadly characterized (Koffi

et al. 2003; Casiot et al. 2003a). The spoil heaps are cov-

ered by an impermeable layer of clay which prevents

rainwater percolation from the surface towards the unsat-

urated zone. The aquifer originates from former natural

springs that were buried under the tailings (Koffi et al.

2003). Therefore, the primary region of oxidation is located

at the base of the tailing, where the oxygen rich rainwater

can penetrate directly. The dominant organisms (27–65%)

are related to Desulfosarcina variabilis a sulfate-reducing

bacterium. Acidithiobacillus ferrooxidans represent the

second most important group (8–14%).

Cultivable bacterial strains of A. ferrooxidans and

Thiomonas (shown to be very active in the oxidation of As)

were identified both in the tailing stock and in the Reigous

Creek (Bruneel et al. 2003).

Sampling and analysis

Three surveys were carried out in November 2004, April

2005, and September 2005 in the tailing stock. Ground-

waters were collected in a borehole (S5, between 10 and

12 m deep) located in the center of the tailings. Samples

were also taken along the Reigous Creek, (collecting

downstream seepage waters from the surroundings) in

November 2005, at the spring (S1), 30 m downstream from

the spring (station COWG), 150 m downstream (COWS),

and 1,500 m (CONF) upstream from the confluence

between the Reigous and the Amous river. Water samples

Extremophiles

123

(300 ml) were filtered through sterile 0.22 lm Nuclepore

filters that were then transferred to cryotubes, frozen in

liquid nitrogen, and stored at -80�C until further analysis.

The main physicochemical parameters [pH, T�C, dis-

solved oxygen (DO), etc.] were measured at the sampling

points. Measurements of pH and water temperature were

made in the field with an Ultrameter Model 6P (Myron L

125 Company, Camlab, Cambridge). Water samples were

immediately filtered through 0.22 lm Millipore mem-

branes fitted on Sartorius polycarbonate filter holders.

Samples for total Fe and As determination were acidified to

pH = 1 with HNO3 (14.5 M), and stored at 4�C in poly-

ethylene bottles until analysis. The samples for Fe and As

speciation and sulfate determination were stored in the dark

and analyzed within 24 h.

DNA isolation

Genomic DNA was extracted from filtered water using the

UltraClean Soil DNA Isolation Kit according to the rec-

ommendations of the manufacturer (MoBio Laboratories

Inc., USA). All the extracted genomic DNA samples were

stored at -20�C until further processing.

PCR amplification

Amplification of archaeal 16S rRNA genes was obtained

using primers Arch21F (50-TTCCGGTTGATCCYGCCG

GA-30) and Arch958R (50-YCCGGCGTTGAMTCCAA

TT-30) (Delong 1992). The PCR amplifications were per-

formed as previously described (Bruneel et al. 2006). The

amount of PCR product was determined by comparison to

known concentrations after migration on agarose gel.

Archaeal 16S rRNA gene library analysis

Archaeal 16S rRNA gene libraries were constructed to

characterize the archaeal populations. Archaeal 16S rRNA

genes were amplified with Arch21F and Arch958R prim-

ers. These PCR products were cloned in E. coli TOP 10

using the pCR2.1 Topo TA cloning kit (Invitrogen, Inc.),

according to the manufacturer’s instructions. Cloned 16S

rRNA gene fragments were reamplified using the primers

TOP1 (50-GTGTGCTGGAATTCGCCCTT-30) and TOP2

(50-TATCTGCAGAATTCGCCCTT-30) located on the

vector and surrounding the inserted PCR fragment, and

then digested with the enzymes HaeIII or HinfI. Restriction

profiles were analyzed using 2.5% agarose gel electro-

phoresis (small-fragment resolution agarose; QA agarose,

QBiogene, Inc.). Around 60–70 clones from each library

were analyzed and grouped according to their RFLP pat-

terns (HaeIII and HinfI digestion). The sequences of clones

from dominant groups were determined.

16S rRNA gene sequencing

Partial sequences of the 16S rRNA gene were determined

by the dideoxy nucleotide chain-termination method using

the BigDye 3.1 kit (Applied Biosystems) on an ABI

PRISM 3730XL Genetic analyzer (Applied Biosystems).

Sequences were checked for chimeras using the CHI-

MERA CHECK function of the Ribosomal Database

Project II (Maidak et al. 2001). DNA sequence analyses

were performed using the BLAST, ALIGNN, and CLU-

STALW programs (Altschul et al. 1990; Felsenstein 1993;

Thompson et al. 1994). A phylogenetic tree was con-

structed using the PHYLIP computer package (Felsenstein

1993). The confidence level of the phylogenetic tree

topology was evaluated by performing 100 bootstrap rep-

lications with the SEQBOOK program. All the sequences

obtained were submitted to the EMBL databases under

accession numbers AM765808 to AM765809 and

AM778965 to AM778977.

Chemical analysis

The determination of total dissolved As was performed by

hydride generation atomic fluorescence spectrometry (HG-

AFS). Analyses of As species were carried out using

coupled anion-exchange chromatography–HG-AFS. This

method, described by Bohari et al. (2001), has a detection

limit of 2.3 nM for As(III) and 6.1 nM for As(V). The

precision is better than 5%. Total dissolved Fe was deter-

mined by flame atomic absorption spectrometry. Fe(II) was

determined using colorimetry at 510 nm after complexa-

tion with 1,10-phenanthrolinium chloride solution in

buffered samples (pH 4.5) (Rodier et al. 1996). The

detection limit is 0.2 lM and the precision better than 5%.

The sulfate concentration was determined after precipita-

tion of BaSO4 with BaCl2 and spectrophotometric

measurement at 650 nm.

Rarefaction analysis, diversity index, and coverage

values

PAST (PAleontological STatistics v 1.19) software from

the website http://folk.uio.no/ohammer/past/ was used for

different diversity indices (Rarefaction analysis, Taxa,

Total clones, Singletons, Dominance, Coverage, Shannon,

Equitability, and Simpson) for each clone library. To per-

form rarefaction analysis, the total number of clones

obtained compared with the number of clones representing

each unique phylotype was used to produce the rarefaction

curves. Coverage values were calculated to determine how

efficiently the libraries described the complexity of a

theoretical community like an original archaeal commu-

nity. The coverage (Good 1953) value is given as

Extremophiles

123

http://folk.uio.no/ohammer/past/

C = 1 - (n1/N) where n1 is the number of clones that

occurred only once in the library.

Results

Aqueous chemistry

The physicochemical composition of the water is presented

in Table 1. The pH inside the piezometer was between 3.73

and 5.78. The temperature varied from 15.5 to 20.6�C and

was relatively stable throughout the year (Koffi et al.

2003). The DO was quite low particularly in April 2005

(between 0.1 and 0.2 mg l-1). The concentration of As

inside the tailing stock varied greatly. As(III) was pre-

dominant, comprised between 78 and 277 mg l-1, and

As(V) varied between 42 and 66 mg l-1. The concentra-

tion of Fe(II) (Fe(III) not detected, data not shown) varied

greatly, i.e. between 778 and 1,299, and sulfate between

3,264 and 4,195 mg l-1. The concentrations of As(III), Fe

and SO42- were highest in November 2004.

In the Reigous creek, the 2.5 pH at the spring increased

along the creek to reach 3.43 at COWS and 3.25 just before

the confluence with the Amous (CONF), 1.5 km away. The

DO content was 1 mg l-1 in the spring but it increased

along the creek to reach 5–6 at COWS and 3–4 mg l-1 at

CONF. Dissolved As and Fe concentrations decreased at

varying degrees along the course of the creek, (30 mg l-1

for As(III), 39 mg l-1 for As(V), 879 mg l-1 for Fe(II) and

4,388 mg l-1 for sulfate at the spring station (S1) but only

0.53 for As(III and V), 25 mg l-1 for Fe(II) and 749 for

sulfate at the CONF station. These elements are removed

by coprecipitation with Fe(III). This process results from

bacterially mediated As- and Fe-oxidation (Casiot et al.

2003b). Furthermore, the increase in pH as a result of

dilution by unpolluted tributaries after COWG also con-

tributes to an increase in As and Fe precipitation. During

this sampling period, the concentrations of As, Fe and

SO42- were not particularly high in comparison to the

concentrations usually found in these waters (up to 10,000

and 20,000 mg l-1 for As and Fe, respectively) in the

tailing stock (Casiot et al. 2003a) and from 80 to

350 mg l-1 for As, 750 to 2,700 mg l-1 for Fe, and 2,000

to 7,500 mg l-1 for sulfate in the head waters of the

Reigous creek (Leblanc et al. 2002).

Composition of archaeal communities

16S rRNA gene library analyses were performed to identify

the dominant groups of archaeal populations. The most

representative sequences of the dominant clones are sum-

marized in Table 2 and the phylogenetic filiations of the

sequences obtained are presented in Fig. 1. DNA could be

extracted from all sampling sites except the S5 borehole in

November 2005. In the Carnoules mine drainage, numer-

ous sequences in the libraries are related to sequences

previously found in AMD, showing that the clone libraries

were not contaminated.

Clones analysis revealed that all the sequences were

affiliated to the phylum Euryarchaeota, while Cre-

narchaeota were not represented. The most abundant

sequence types present in the water of the tailing (S5)

displayed from 99 to 100% homology with Ferroplasma

acidiphilum strain DR1, that was detected in microbial

consortia from AMD and in industrial bioleaching envi-

ronments (Dopson et al. 2004, AY22042). They were

recovered in the groundwater in November 2004 and April

2005, representing a large majority of the clones (65–72%).

The second most abundant group (9% in November 2004

but 65% in September 2005) was similar (99–100%) to the

uncultured archaeon clone ant h4 (Table 2, Fig. 1) found in

two anaerobic sludges (DQ462728, unpublished). The

sequences representing the second most abundant type in

April 2005 (15%) were similar (91%) to clones of the

uncultured archaeon clone YAC1 (Table 2, Fig. 1) found

in communities of different hot springs (DQ237924,

unpublished). In September 2005, the second most

important group (20%), (Table 2), was related to the

uncultured archaeon clone ASL1 found in AMD (Baker

and Banfield 2003; AF544224).

Table 1 Physico-chemical characteristics of the water (mg l-1) during the sampling in S5, S1, COWG, COWS and CONF

Sampling station Sampling period pH (±SD) T (�C) DO (±SD) As(III) (±SD) As(V) (±SD) Fe (II) (±SD) SO42- (±SD)

Tailing

stock

S5 November 2004 5.78 (±0.05) 15.5 2 277 (±14) 42 (±2) 1299 (±104) 4195 (±420)

April 2005 4.05 (±0.05) 17.3 0.1–0.2 128 (±6) 66 (±3) 784 (±62) 3264 (±326)

September 2005 3.73 (±0.05) 20.6 4–5 78 (±4) 53 (±3) 778 (±62) 3629 (±363)

Reigous

Creek

S1 November 2005 2.5 (±0.05) 14.6 1 30.0 (±0.8) 39 (±2) 879 (±70) 4388 (±441)

COWG 2.74 (±0.05) 10.6 5–6 22.0 (±0.8) 22.0 (±0.8) 501 (±40) 1785 (±182)

COWS 3.43 (±0.05) 7.2 5–6 4.5 (±0.2) 1.50 (±0.08) 95 (±8) 902 (±90)

CONF 3.25 (±0.05) 6.7 3–4 0.53 (±0.02) 0.53 (±0.02) 25 (±2) 749 (±75)

SD Standard deviation

Extremophiles

123

In the Reigous creek during the sampling campaign in

November 2005, the most abundant group (21% at the

spring S1, 59% at COWG, 93% at COWS and 74% at

CONF) was related (92–94%) to the uncultured archaeon

clone YAC1. These clones were found in low abundance

(15%) in the groundwater and only in April 2005. The

second most abundant group in the creek was similar (99–

100%) to F. acidiphilum, also numerically significant

members in Carnoules tailing stock. The abundance of this

group decreased along the creek, representing 54% of the

clones at the spring S1, but only 4% at COWG and was

undetected at COWS and CONF. The least abundant

sequences (6%) found only at the COWS station was

related (99% similarity) to the uncultured archaeon clone

ant g10 isolated in macroscopic filaments from an extre-

mely acidic environment, Tinto River (DQ303253,

unpublished). Phylogenetic analyses (Fig. 1) did not enable

affiliation of the clone sequences with any representative of

the subdivision. The closest relative (91%) was Thermo-

plasma sp. SO2 (AB262009, unpublished).

Rarefaction analysis, diversity index and coverage

values of the clone libraries analyzed

Table 3 shows Dominance, Shannon, Equitability, Simp-

son index and Coverage values calculated for each library.

Table 2 Archaeal clones found in Carnoules mine drainage with closest match organism or clone name, percent similarity, phylogenetic group,closest relative and percent number of each group compared to the total number of clones

Sampling station Sampling period Clones Phylum Closest relative(accession number)

Number of bpidentical and% similarity

Relativeabundance ofclones (%)a

Tailing stock S5 November 2004 S5Nov04 73 Euryarchaeota F. acidiphilum strainDR1 (AY222042)

100 72

S5Nov04 82 Euryarchaeota Uncultured archaeonclone ant h4(DQ303256)

100 9

April 2005 S5Apr05 12 Euryarchaeota F. acidiphilum strainDR1 (AY222042)

99 65

S5Apr05 47 99

S5Apr05 45 Euryarchaeota Uncultured archaeonclone YAC1(DQ237924)

91 15

September 2005 S5Sep05 53 Euryarchaeota Uncultured archaeonclone ant h4(DQ303256)

99 65

S5Sep05 56 Euryarchaeota Uncultured archaeonclone ASL1(AF544224)

97 20

Reigous Creek S1 November 2005 S1Nov05 90 Euryarchaeota F. acidiphilum strainDR1 (AY222042)

99 54

S1Nov05 58 Euryarchaeota Uncultured archaeonclone YAC1(DQ237924)

93 21

COWG CGNov05 19 Euryarchaeota Uncultured archaeonclone YAC1(DQ237924)

93 59

CGNov05 94 93

CGNov05 32 Euryarchaeota F. acidiphilum strainDR1 (AY222042)

100 4

COWS CSNov05 10 Euryarchaeota Uncultured archaeonclone YAC1(DQ237924)

92 93

CSNov05 20 Euryarchaeota Uncultured archaeonclone ant g10(DQ303253)

99 6

CONF CFNov05 6 Euryarchaeota Uncultured archaeonclone YAC1(DQ237924)

94 74

a The abundance of clones was calculated for each library

Extremophiles

123

To estimate diversity coverage and to determine whether a

sufficient number of clones from each library had been

sequenced, rarefaction analysis was performed. The gen-

erated curves were near saturation (data not shown),

consistent with the high coverage values (between 0.82 and

0.93). In November 2005, the COWS library showed lower

diversity indices (Shannon: 0.5704; Simpson: 0.2397) than

the other libraries (Shannon ranging from 1.151 to 1.604;

Simpson from 0.4488 to 0.6545). Inversely, in November

2005, the COWS library presented a higher Dominance

S5Apr05 12 (AM778965)

S1Nov05 90 (AM778970)

CGNov05 32 (AM778974)

Ferroplasma acidiphilum strain DR1 (AY222042)

Ferroplasma acidiphilum strain YT DSM 12658T (AJ224936)

Uncultured archaeon ASL32 (AF544222)

S5Nov04 73 (AM765808)

Uncultured archaeon ant c8 (DQ303251)

S5Apr05 47 (AM778966)

Ferroplasma acidarmanus (AF145441)

Uncultured archaeon ant c7 (DQ303250)

Ferroplasma sp. MT17 (AF513710)

Uncultured archaeon ant h10 (DQ303255)

S5Sep05 53 (AM778968)

S5Nov04 82 (AM765809)

Uncultured archaeon ant h4 (DQ303256)

Ferroplasma sp. JTC3 (AY830840)

Uncultured archaeon MS14 (AF232925)

Ferroplasma cyprexacervatum (AY907888)

Thermoplasma volcanium (AF339746)

Thermoplasma sp. S02 (AB262009)

Uncultured archaeon ASL1 (AF544224)

Uncultured archaeon ARCP1-28 (AF523940)

Uncultured archaeon ant g4 (DQ303254)

S5Sep05 56 (AM778969)

Uncultured archaeon ant g10 (DQ303253)

CSNov05 20 (AM778976)

Uncultured archaeon AS1 (AF544219)

Uncultured archaeon ant b7 (DQ303249)

Unidentified archaeon pISA42 (AB019742)

Uncultured euryarchaeote pLM14A-1 (AB247822)

Uncultured Thermoplasmatales archaeon OPPD020 (AY861955)

Uncultured archaeon YAC1 (DQ237924)

CFNov05 6 (AM778977)

CGNov05 94 (AM778973)

S1Nov05 58 (AM778971)

CGNov05 19 (AM778972)

S5Apr05 45 (AM778967)

CSNov05 10 (AM778975)

Archaeoglobus fulgidus strain VC-16 (X05567)

Sulfolobus solfataricus (D26490)

Sulfurisphaera ohwakuensis DSM 1242T (D85507)

Metallosphaera hakonensis (D86414)

Acidianus infernus DSM 3191T (D85505)

Acidianus ambivalens DSM 3772T (D85506)

Uncultured archaeon PMA5 (DQ399817)

Uncultured archaeon ZAR100 (AY341269)

Acidithiobacillus caldus (X72851)

61

53

76

9650

37

16

8

95

66 49

50

53

99

65

38

75

17

29

29

76

71

97

63

76

57

0.05

Fig.1 Phylogenetic analysis of

16S rRNA gene sequences

affiliated with Archaea

members from the AMD of

Carnoules (France). Clone

names in bold correspond to

sequences found in the

Carnoules mine drainage

Extremophiles

123

index (0.7603) than the other libraries (from 0.3456 to

0.5512).

Discussion

In the AMD site of Carnoules, more than 65% of the ar-

chaeal sequences could not be closely related to cultured

organisms, suggesting that they may constitute new taxa.

Only sequences close to F. acidiphilum were related to

cultured organisms. Rarefaction data and percent coverage

calculations suggested that the archaeal 16S rRNA gene

libraries reach saturation.

Whatever the sampling period, the water of S5 inside the

tailing stock, where intensive pyrite oxidation takes place,

was numerically dominated by sequences clearly related to

F. acidiphilum, or to the uncultured clone ant h4 which

showed more than 98% similarity with F. acidiphilum.

This isolate was an acidophilic, mesophilic, ferrous-iron

oxidizing, cell-wall lacking microbe that became the basis

of a new archaeal lineage: the new genus Ferroplasma

within the new family Ferroplasmaceae, in the order

Thermoplasmatales, which includes the families Thermo-

plasmaceae and Picrophilaceae (Golyshina and Timmis

2005). These two populations represented 81% of clones in

November 2004, 65% in April 2005, and 65% in

September 2005. Previous analysis of the bacterial com-

munity in the Carnoules tailing showed that the dominant

population was related to the sulfate-reducing bacteria

Desulfosarcina variabilis (Bruneel et al. 2005). This pop-

ulation could not clearly explain the leaching of the

Carnoules tailing as it is well known that it was mostly

acidophilic ferrous iron-oxidizing microorganisms that

were found to be involved in the production of acid mine

drainage (Baker and Bandfield 2003). Iron oxidizing bac-

teria like A. ferrooxidans and Sulfobacillus spp. were also

present in the Carnoules mine tailing but represented a

minor population (Bruneel et al. 2005). Thus, F. acidiph-

ilum could explain the intensive leaching observed in the

Carnoules tailing and the high concentration of As, up to

10,000 mg l-1, one of the highest concentrations reported

in the world. Furthermore, some strains of this genus like

Ferroplasma acidarmanus Fer1 was shown to be an

arsenic-hypertolerant acidophilic archaeon (Gihring et al.

2003; Baker-Austin et al. 2007). This strain, isolated from

the Iron Mountain mine, California, was able to grow with

up to 10 g arsenate per litre but his growth was reduced

with 5 and 10 g of arsenite per litre. This population, which

is more acid-resistant than iron- and sulfur-oxidizing bac-

teria, is in fact known to mobilize metals from sulfide ores,

e.g. pyrite, arsenopyrite and copper-containing sulfides.

According to Golyshina and Timmis (2005) Ferroplasma

spp. are probably the major players in the biogeochemical

cycling of sulfur and sulfide metals in highly acidic envi-

ronments, and may have considerable potential for

biotechnological applications such as biomining and bio-

catalysis under extreme conditions. These results are

consistent with those of Edwards et al. (2000) at the Iron

Mountain acid-generating site (United State), where the

microbial community is dominated (85%) by an archaeon

of the genus Ferroplasma. For these authors, the presence

of this population and other closely related Thermoplas-

matales suggests that these acidophiles are important

contributors to acid mine drainage and may substantially

impact iron and sulfur cycles. The growth of F. acidiphi-

lum occurs between 20 and 45�C with an optimum at 35�C

and at pH 1.3–2.2 with an optimum at pH 1.7 (Golyshina

et al. 2000). Surprisingly, we detected this population in a

less acidic environment (3.73–5.7). Isolation and charac-

terization of members of this population are needed to

determine their physiological capabilities especially at the

pH range found in Carnoules waters.

The clone sequences from the Reigous Creek were

related to the same groups detected in the tailing S5 but the

abundance of each varied. The dominant population in the

Reigous Creek (21% of total clones at the spring S1, 59%

at COWG, 93% at COWS and around 74% CONF) was

related to the uncultured archaeon clone YAC1 found in

communities in different hot springs. Phylogenetic analy-

ses (Fig. 1) did not enable affiliation of the clone sequences

with any cultured representative of the subdivision and this

clone could thus represent a new species. The closest

Table 3 Diversity indices calculated for the seven clone libraries from different stations in Carnoules mine drainage

Clone library Taxa Total clones Singletons Dominance Coverage(C) Shannon (H) Equitability Simpson (1-D)

S5 November 2004 9 66 5 0.4913 92 1.151 0.5241 0.5087

S5 April 2005 11 66 6 0.4564 90 1.277 0.5323 0.5436

S5 September 2005 10 61 6 0.4512 90 1.234 0.5361 0.5488

S1 November 2005 14 57 10 0.3456 82 1.604 0.6077 0.6545

COWG November 2005 13 69 9 0.3989 86 1.424 0.5552 0.6011

COWS November 2005 6 61 4 0.7603 93 0.5704 0.3183 0.2397

CONF November 05 12 61 6 0.5512 90 1.189 0.4785 0.4488

Extremophiles

123

relative (91%) was the uncultured Thermoplasmatales

archaeon found in the Yellowstone geothermal ecosystem

(Spear et al. 2005). The order Thermoplasmatales includes

the families Ferroplasmaceae, Thermoplasmaceae and

Picrophilaceae (Golyshina and Timmis 2005). The known

members of the Thermoplasmales are all acidophilic. Some

groups, like the family Ferroplasmacea within this order,

are capable of iron oxidation (Edwards et al. 2000; Go-

lyshina and Timmis 2005). A previous study of bacterial

populations in the Carnoules creek showed that the domi-

nant bacterial population was related to G. ferruginea, a

neutrophilic bacterium that oxidizes Fe (Bruneel et al.

2006). Consistent with previous observations demonstrat-

ing that G. ferruginea efficiently remove As (III and V) in

water by coprecipitation with Fe (Katsoyiannis and Zou-

boulis 2004), this population may play a key role in the

remediation process observed in the Reigous creek (Casiot

et al. 2003b). If the uncultured archaeon clone YAC1

oxidizes Fe, this population could play a role in the natural

remediation processes occurring in the Reigous Creek in

association with G. ferruginea, but until the archaeal

strains are isolated, their physiological role in the creek

ecology will remain uncertain. Environmental genome data

like those obtain with analysis of assembled random

shotgun sequence data can also provide detailed insight

into the metabolic potential of uncultivated organisms

(Tyson et al. 2005).

Our study demonstrated the existence of a complex

prokaryotic community in the Carnoules AMD where

bacterial and archaeal populations are present. Both phyl-

otype communities were significantly altered in terms of

size and structure with microhabitats varying inside the

AMD particularly in underground water from the tailing

and in the Reigous and the small creek draining the site.

The occurrence of different dominant communities is likely

associated with the formation of environmental gradients

of temperature, pH, oxidation–reduction potential, etc.

Other methods such as fluorescence in situ hybridization

(FISH) will help to clearly assess the relative proportion of

population. However, this method has not been widely

applied to samples of thermophilic archaea and may be

limited by cross-hybridization. Furthermore, methods such

as metagenomic research (study of the entire genetic

composition of communities of an environment) could help

to study the total diversity, physiology, ecology and phy-

logeny of microbial population but all of the approaches

that are available today have advantages and limitations

(Pontes et al. 2007). Only, the isolation of archaeal strains

at the Carnoules mine will extend our understanding of the

ubiquity of archaea in such environments, and help eluci-

date the microbial component driving the biogeochemical

processes present in this and other extreme AMD sites.

Acknowledgments The study was financed by the EC2CO pro-

gramme (Institut National des Sciences de l’Univers, CNRS). We

thank Marjorie Cloez for identification of the archaeal population in

the site, and Marie Ange Cordier for assistance in analysis of

physical–chemical parameters.

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ENVIRONMENTAL MICROBIOLOGY

Characterization of the Active Bacterial CommunityInvolved in Natural Attenuation Processes in Arsenic-RichCreek Sediments

Odile Bruneel & Aurélie Volant & Sébastien Gallien & Bertrand Chaumande &

Corinne Casiot & Christine Carapito & Amélie Bardil & Guillaume Morin &

Gordon E. Brown Jr & Christian J. Personné & Denis Le Paslier & Christine Schaeffer &

Alain Van Dorsselaer & Philippe N. Bertin & Françoise Elbaz-Poulichet &Florence Arsène-Ploetze

Received: 7 August 2010 /Accepted: 20 January 2011 /Published online: 12 February 2011# Springer Science+Business Media, LLC 2011

Abstract Acid mine drainage of the Carnoulès mine(France) is characterized by acid waters containing highconcentrations of arsenic and iron. In the first 30 m alongthe Reigous, a small creek draining the site, more than 38%of the dissolved arsenic was removed by co-precipitation

with Fe(III), in agreement with previous studies, whichsuggest a role of microbial activities in the co-precipitationof As(III) and As(V) with Fe(III) and sulfate. To investigatehow this particular ecosystem functions, the bacterialcommunity was characterized in water and sediments by

Electronic supplementary material The online version of this article(doi:10.1007/s00248-011-9808-9) contains supplementary material,which is available to authorized users.

O. Bruneel (*) :A. Volant : C. Casiot :A. Bardil :C. J. Personné : F. Elbaz-PoulichetLaboratoire HydroSciences Montpellier,UMR5569 (CNRS-IRD-Universités Montpellier I et II),Université Montpellier II, CC MSE,Place Eugène Bataillon,34095 Montpellier Cedex 05, Francee-mail: [email protected]

B. Chaumande : P. N. Bertin : F. Arsène-PloetzeGénétique Moléculaire,Génomique Microbiologie,UMR7156, Université de Strasbourg/CNRS,28 rue Goethe,67083 Strasbourg Cedex, France

S. Gallien : C. Carapito : C. Schaeffer :A. Van DorsselaerLaboratoire de Spectrométrie de Masse Bio-organique,Institut Pluridisciplinaire Hubert Curien,UMR7178 (CNRS-Université de Strasbourg),25 rue Becquerel,67087 Strasbourg, France

G. MorinInstitut de Minéralogie et de Physique des Milieux Condensés(IMPMC), UMR7590 (CNRS - Universités Paris 6&7 - IPGP),140, rue de Lourmel,75015 Paris, France

G. E. Brown JrSurface and Aqueous Geochemistry Group,Department of Geological and Environmental Sciences,Stanford University,Stanford, CA 94305-2115, USA

G. E. Brown JrStanford Synchrotron Radiation Laboratory, SLAC,2575 23 Sand Hill Road, MS 69,Menlo Park, CA 94025, USA

D. Le PaslierGénomique Métabolique, UMR8030, CNRS,2 rue Gaston Crémieux,91057 Evry Cedex, France

D. Le PaslierCommissariat à l’Energie Atomique (CEA),Direction des Sciences du Vivant, Institut de Génomique,Genoscope, Laboratoire de Génomique Comparative,2 rue Gaston Crémieux,91057 Evry Cedex, France

Microb Ecol (2011) 61:793–810DOI 10.1007/s00248-011-9808-9

http://dx.doi.org/10.1007/s00248-011-9808-9

16S rRNA encoding gene library analysis. Based on theresults obtained using a metaproteomic approach on sedi-ments combined with high-sensitivity HPLC-chip spec-trometry, several GroEL orthologs expressed by thecommunity were characterized, and the active members ofthe prokaryotic community inhabiting the creek sedimentswere identified. Many of these bacteria are β-proteobacteriasuch as Gallionella and Thiomonas, but γ-proteobacteriasuch as Acidithiobacillus ferrooxidans and α-proteobacteriasuch as Acidiphilium, Actinobacteria, and Firmicutes werealso detected.

Introduction

Acid Mine Drainage (AMD) is one of the most serious formsof water pollution in industrial and post-industrial areasworldwide [38]. AMD is generated when the wastes fromthe mining and processing of sulfide ores (such as pyrite orarsenopyrite) come into contact with oxygenated water [5].AMD is often characterized by pH values of 2–4. Suchwaters generally contain high levels of iron, toxic metals(such as aluminum, manganese, lead, cadmium, and zinc),and metalloids (arsenic) [5, 32, 48]. AMD can still occurhundreds of years after mine closure and tens of thousands ofkilometers of groundwater, streams, lakes, and estuariesthroughout the world have been directly impacted [40]. Inseveral cases of AMD, natural remediation has beenobserved, as for example at the Carnoulès site in Franceand the Rio Tinto site in Spain [18, 53]. In such AMD, toxiccompounds are accumulated in sediments consisting of avariety of iron (oxyhydr)oxides and hydroxysulfates such asjarosite, schwertmannite, and ferrihydrite [48]. Naturalremediation of metal pollutants is generally due to theoccurrence of abiotic reactions and/or microbial activitiesthat make these toxic compounds insoluble and lead them toaccumulate in sediments [32, 40]. This toxic compoundprecipitation processes mainly involve the oxidation andprecipitation of iron, which is often the main soluble metalpresent in AMD, and the adsorption of other metals andmetalloids by the ferric minerals formed [32, 51]. Indeed,many elements such as Sr, Cs, Pb, U, and As show a strongaffinity for solid iron oxide [18, 27, 48]. Abiotic oxidation ofFe(II) proceeds very slowly in acidic (pH 3.5) waters [51]. Incontrast, iron-oxidizing bacteria catalyze the reaction andthus accelerate the formation of solid iron oxide [32, 41, 51].In addition, several bacteria contribute to the immobilizationof arsenic via their ability to oxidize this metalloid [6, 14, 18,22, 48], arsenate (As(V)) being adsorbed more strongly thanarsenite (As(III)) by Fe(III) oxides and hydroxides at acidicpH levels [10]. Thiomonas strains show a high arseniteoxidation capacity, and these metabolic activities have beenextensively analyzed under laboratory conditions [6, 14, 22].

However, to be able to develop remediation processes and/oroptimize existing processes, further knowledge is requiredabout how these bacteria function in situ. In particular, it isof crucial importance to determine which bacteria are viableand active in such ecosystems.

The AMD of Carnoulès mine in Southern France is ahighly suitable site for analyzing how microorganismscontribute to the transformation of metals and metalloids insitu, since efficient natural remediation processes are knownto occur at this site [12, 18]. This former mine generatedaround 1.2 Mt of tailings containing 0.7% Pb, 10% FeS2,and 0.2% As. Water percolating through the tailings formsthe head of the Reigous creek. This creek is acidic (pHaround 3) and highly contaminated with As (100 to350 mg L−1). The behavior of As and Fe in the Reigouscreek has been intensively studied [18, 23, 48]. In the creekspring, As(III) is the main As species present and Fe occursin the form of Fe(II) [18]. Along the first 30 m of the creek(about 1 h residence time), the oxidation of Fe(II) leads tothe co-precipitation of more than 38% of the dissolved As[18, 23]. Arsenic accounts for up to 22% of the total dryweight of the sediments formed along the first 10 m alongthe creek. In the wet season, approximately 30 m down-stream of the spring, these sediments are mainly composedof As(III)–Fe(III) oxyhydroxysulfates, whereas As(V)–Fe(III) oxyhydroxysulfates compounds predominate during thedry season [48]. Because of the very high molar As/Fe ratio(up to 0.3) existing in the dissolved phase of the Carnoulèscreek, the mineralogical content of the sediments differssignificantly from that classically observed at most AMD[47], especially along the first 50 m of the creek. Severalstrains of Thiomonas and Acidithiobacillus ferrooxidanshave been isolated from Reigous creek waters, and basedon the results of laboratory experiments, it has beensuggested that these bacteria may contribute to the oxidationof Fe(II) and the co-precipitation of As [14, 18, 21–23, 48].Preliminary analyses have shown that the DNAs of bothbacteria are present in the Reigous creek, as well as that ofGallionella sp., Thiobacillus sp., and some sulfate-reducingbacteria [12]. Archaea have also been found to occur in theReigous creek (Ferroplasma acidiphilum and sequencesaffiliated to uncultured Thermoplasmatales archaeon) as wellas a eukaryotic microorganism, Euglena mutabilis [12, 13,15]. However, the bacterial population inhabiting the As-richReigous sediments has never been characterized so far. It,therefore, seemed to be necessary not only to identify thebacteria present in this creek but also to determine whichmembers of this community are viable and, therefore,perform metabolic activities in situ.

The aim of this study was to describe the bacterialpopulations occurring in both the sediments and waters atthe disused Carnoulès site and to identify the bacteria atwork. For this purpose, three complementary approaches

794 O. Bruneel et al.

were used. First, chemical and mineralogical studies wereperformed in order to determine the arsenic species present.A 16S rRNA encoding gene library was then analyzed inorder to identify the bacterial population present in thecreek sediments and waters. Lastly, based on the findingsobtained using a metaproteomic approach combined withhigh-sensitivity mass spectrometry methods, the activespecies inhabiting the sediments were identified.

Methods

Sampling and Analysis

Samples were collected from Reigous creek in April 2006at COWG station located 30 m downstream from thespring. This sampling was part of a long-term monitoring ofthe physicochemistry of the Reigous Creek water [23]. Themain physicochemical parameters (pH, temperature, anddissolved oxygen concentrations) were measured in situ atthis sampling point. The 5-cm deep sediments on thebottom of the creek and a thin column (less than 10 cm) ofrunning water covering the sediments were sampled. Solidsamples were removed with a sterile spatula from thesurface of the sediments. Water samples (300 ml) wereimmediately filtered through 0.22 μm Millipore membranesfitted on Sartorius polycarbonate filter holders (for waterchemical analysis) or through sterile 0.22-μm Nucleoporefilters that were then transferred to a collection tube (Nunc),frozen in liquid nitrogen, and stored at −80°C until DNAextraction (for 16S rRNA encoding gene analysis). Sam-pling was repeated three times. For total Fe and Asdetermination, filtered water was acidified to pH=1 withHNO3 (14.5 M) and stored at 4°C in polyethylene bottlesuntil analysis. For As and Fe speciation, a 10 μl aliquot offiltered sample water was added to either 0.5 ml of 5% (v/v)0.25 M EDTA solution for As speciation [7] or a mixture of0.5 ml acetate buffer (pH 4.5) and 1 ml of 1,10-phenanthrolinium chloride solution for Fe speciation [50].The vials were completed to 10 ml with deionized water.The samples used for arsenic speciation and Fe(II) andsulfate determination were stored in the dark and analyzedwithin 24 h.

Chemical Analysis

The determination of total dissolved As was performed byICP-MS using Thermo X7 series with a conventionalexternal calibration procedure. Indium was used as internalstandard to correct for instrumental drift and possiblematrix effects. It was not necessary to correct interferencewith chloride because of the extremely high As levelspresent. Certified reference material SLRS-4 (freshwater

samples) was used to check analytical accuracy andprecision. The results showed that the recovery rateobtained was within ±5%.

Analyses of inorganic arsenic species (As(III), As(V))were carried out using anion-exchange chromatography(25 cm×4.1 mm i.d. Hamilton PRP-X100 column withVarian ProStar gradient solvent delivery system) coupled toa hydride generation (VGS 200, FISONS, France) with anatomic fluorescence spectrometry detector (Excalibur,PSAnalytical, GB) [17]. The detection limit obtained was172 ng L−1 for As(III) and 458 ng L−1 for As(V), with aprecision better than 5%. Total dissolved Fe was deter-mined by flame atomic absorption spectrometry. Fe(II)concentration was determined using colorimetry at 510 nmafter complexation with 1,10-phenanthrolinium chloridesolution in buffered samples (pH 4.5) [50] (detection limit:11 μg L−1; precision better than 5%). Sulfate concentrationwas determined after precipitation of BaSO4 with BaCl2and spectrophotometric measurement at 650 nm [50].

Solid Sample Characterization

XAFS data were gathered on the laboratory samples andthe sample taken at COWG on April 2006, at 10 K intransmission mode on a bending magnet D44 at the LUREsynchrotron (Orsay, France), and in fluorescence mode onthe 11-2 wiggler beamline at SSRL (Stanford, CA),respectively. Experiments and data reduction were previ-ously reported [48, 49].

DNA Isolation, 16S rRNA Encoding Gene Cloning,Restriction Analysis, and Sequencing

Genomic DNA was extracted in triplicate from filteredwater and sediments using the UltraClean Soil DNAIsolation Kit according to the manufacturer’s recommenda-tions (MoBio Laboratories Inc., USA). These triplicates werepooled before PCR amplification. All the genomic DNAsamples extracted were stored at −20°C until further process-ing. Bacterial diversity was analyzed by cloning PCRamplified 16S rRNA encoding genes. Bacterial 16S rRNAencoding genes were amplified with 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1489R primers (5′-TACCTTGTTACGACTTCA-3′) [43, 57], as previously described [12].These PCR products were cloned into Escherichia coli TOP10 strain using the pCR2.1 Topo TA cloning kit (Invitrogen,Inc.). Cloned 16S rRNA encoding gene fragments were re-amplified using the primers TOP1 (5′-GTGTGCTGGAATTCGCCCTT-3′) and TOP2 (5′-TATCTGCAGAATTCGCCCTT-3′) that anneal to the vector and surroundthe inserted PCR fragment and then digested with HaeIII orHinfI enzymes. Restriction profiles were analyzed using2.5% agarose gel electrophoresis (small fragment resolution

Active Bacteria in Arsenic-Rich Sediments 795

agarose; QA agarose, QBiogène, Inc.). Around 200 clonesfrom each library were analyzed and grouped accordingto the RFLP patterns obtained. PAST (PaleontologicalSTatistics v 1.19) software from the website http://folk.uio.no/ohammer/past/ was used to calculate differentdiversity indices (rarefaction analysis, taxa, total clones,singletons, dominance, coverage, shannon, equitability,and simpson, Table 1) for each clone library. The totalnumber of clones obtained compared with the number ofclones representing each unique phylotype was used toproduce the rarefaction curves. Coverage values werecalculated to determine how efficiently the libraries describedthe complexity of a theoretical community such as an originalbacterial community. The coverage [29] value is given as C=1—(n1/N) where n1 is the number of clones that occurredonly once in the library. Rarefaction analysis showed that thecurves generated were near saturation (data not shown) andconsistent with the high coverage values of the two clonelibraries (97.8 for the sediment and 98.6 for the water). Thisindicated that the clone libraries were sufficiently sampled.

Partial sequences of the clones from dominant groupswere determined by the dideoxy nucleotide chain-termination method using the BigDye 3.1 kit (AppliedBiosystems) on an ABI PRISM 3730XL Genetic analyzer(Applied Biosystems). The PINTAIL program [4] was usedto check the presence of chimerae. Sequences were alsoexamined manually for chimerae, which were excludedfrom further analyses. These sequences were comparedwith known sequences (NCBI database) using the BLAST,ALIGN, and CLUSTALW programs [1, 26, 55]. Allsequence data obtained were submitted to the EMBLdatabases under accession numbers (FR676963-FR677013;AM988784-AM988794; AM988796; AM988798;AM988801-AM988805; AM988807-AM988809).

Preparation of Protein Extracts and Gel ElectrophoresisAnalysis

Prokaryotes were separated from sediments and eukaryotesusing a Nycodenz density gradient. It should be noted thatthe main population determined from the DNA directlyextracted from sediments was similar to that identified afterNycodenz treatment (data not shown), which suggests thatthis treatment did not result in enrichment of the sample byany particular microorganisms. Ten grams of sediments

were washed in 10 mL of saline buffer and agitatedovernight at 4°C. After 10 min of decantation, 7.5 mL ofsupernatant were added without mixing to 17.5 mL ofNycodenz solution (Axis-Shield, Dundee, Scotland), andthen centrifuged for 30 min at 10,000×g. The cellularfraction (nycodenz/sample interface) was removed andwashed by adding two volumes of NaCl 0.9% andcentrifuged for 15 min at 10,000×g at 4°C. Proteins wereextracted from this cellular fraction as previously described[58], further purified using the 2-D Clean-up kit (GEHealthcare), and resuspended in rehydration buffer(364 g L−1 thiourea, 1,000 g L−1 urea; 25 g L−1 CHAPS,0.6% v/v IPG buffer Pharmalyte, 10 g L−1 of DTT, and0.01% bromophenol blue). Protein concentrations werequantified using the 2-D Quant kit (GE Healthcare). Theseproteins were separated by 2-D gel electrophoresis aspreviously described [58] and finally stained with silvernitrate. Gels were analyzed using an Image Scanner,LabScan v 3.0 (GE Healthcare), and the ImageMaster2D platinum software program (v. 6.01, GE Healthcare).The spots selected were cut out of the 2-D gels and stored at−20°C. Eighty-one spots were analyzed by performingnanoLC-Chip-MS/MS.

In-Gel Digestion, Mass Spectrometry Analysis, and ProteinIdentification

Unless otherwise specified, all chemicals were obtained fromSigma (St. Louis, MO, USA). In-gel digestion of gel spotswas performed as previously described [58]. The resultingpeptides were analyzed by performing nanoLC-MS/MS onan Agilent 1100 Series HPLC-Chip/MS system (AgilentTechnologies, Palo Alto, USA) coupled to an HCT Ultra iontrap (Bruker Daltonics, Bremen, Germany). The MS/MSdata were analyzed using the MASCOT 2.2.0 algorithmgiving a maximum of one missed cleavage, with a masstolerance of 0.5 Da for MS and MS/MS data andcarbamidomethylation of cysteines and oxidation of methio-nines were specified as the variable modifications. MS/MSdata searches were performed against two in-house generateddatabases. The first database was composed of the proteinsequences of all the organisms related to the groupsidentified by performing 16S rRNA encoding gene analysison the Reigous creek sediments and water (α-, β-, δ-,and γ-proteobacteria, Bacilli, Clostridia, Actinobacteria,

Table 1 Diversity indices calculated from the two clone libraries in sediment and water at the Carnoulès mine drainage creek

Clone library Taxa Total no. of clones Singletons Dominance (D) Coverage (C) Shannon (H) Equitability Simpson (1-D)

Sediments 15 229 5 0.5503 97.8 1.146 0.4231 0.4497

Water 18 221 3 0.2551 98.6 1.949 0.6744 0.7449




Nitrospira), as well as unclassified bacteria from http://beta.uniprot.org/, Thiomonas sp. from http://www.genoscope.cns.fr/ (FP475956–FP475957), Euglenozoa, and Viridiplantae.The second database included all bacterial and archaealGroEL chaperonins (12501 and 291 sequences, respectively)extracted from the Uniprot database (http://www.uniprot.org/uniprot).

To assess the false positive rate in the protein identifi-cation, a target-decoy database search was performed [25].With this approach, peptides are matched against a databaseconsisting of the native protein sequences detected in thedatabase (target) and the sequence-reversed entries (decoy).Protein identification was confirmed when at least twopeptides with a minimum Mascot ion score of 30 weredetected. In the case of one-peptide hits, the score of theunique peptide had to be greater than the 95% significanceMascot threshold level. All the proteins identified wereadded to the “InPact” proteomic database developed at ourlaboratory (http://inpact.u-strasbg.fr/~db/) [8].

Phylogenetic Analyses

A search for GroEL homologs and 16S rRNA encodingsequences was carried out in the Uniprot and NCBIdatabases, respectively. A total number of 530 reviewedGroEL bacterial sequences were retrieved from the RefSeqdatabase. These sequences were 500–550 amino acids inlength. Only one sequence representative of each genus(259 sequences in all) was kept. GroEL and 16S rRNAencoding sequences were aligned using ClustalW [55].Alignments were checked by hand and positions with morethan 1% of gaps were automatically removed. Neighbor-joining trees were constructed with 185 amino acids in thecase of the GroEL sequences and with 310 nt in that of the16S rRNA encoding sequences. Trees were drawn up usingthe iTOL website (http://itol.embl.de/) [44].

Results and Discussion

Physical and Chemical Characteristics of Samples

The physicochemistry of the Reigous Creek water at thetime of sampling was typical of that revealed during thelong-term monitoring study [23]. The water sample wasacid (pH=3.28) and moderately oxygenated (dissolvedoxygen concentration=3.5±0.5 mg L−1); it containedextremely high concentrations of SO4

2− (2700±300 mg L−1), Fe (620±30 mg L−1), and As (140±4 mg L−1), with a large predominance of Fe(II) (90±10%of total Fe concentration) and equal proportion of As(III)and As(V). The removal of As during the course of theReigous Creek from its source to the sampling station

COWG reached 38%, corroborating the typical removalrates measured during the long-term monitoring study [23].The pale-yellow loosely packed sediments previouslyobserved during the dry season at the sampling pointchosen in this study (COWG ~30 m downstream ofthe spring), consisted of an amorphous Fe(III)–As(V)hydroxysulfate mineral with an As/Fe molar ratio of 0.5to 0.6 [48]. However, various other biominerals may beformed from dissolved Fe(II) and As(III) [47, 48]. Theexact nature and structure of the sediment sample studiedwas, therefore, further investigated. X-ray powder diffrac-tion (data not shown) and X-ray absorption spectroscopydata obtained at the As–K edge (Fig. 1) showed that arsenicwas present in these samples in an amorphous Fe(III)–As(V) hydroxysulfate phase as previously observed [48].These analyses showed that despite the presence of a minorAs(III) impurity, the oxidized arsenic form As(V) predom-inates in this sediment (Fig. 1a). In a previous study, it hasbeen shown that the catalytic oxidation of As(III) byThiomonas sp. strains accelerates such As–Fe precipitationprocess [48]. Therefore, using extended X-ray absorptionfine structure analysis at the As–K edge (Fig. 1b), thestructure observed in our samples was compared with thatof the minerals obtained after As(III) oxidation by theThiomonas sp. strain B2 in bioassays in which sterilizedCarnoulès Creek water was used [48]. These comparisons(Fig. 1b, c) showed that the molecular structure of theamorphous Fe(III)–As(V) hydroxysulfate phase observed inthese sediments was similar to that of the Fe(III)–As(V)hydroxysulfate obtained in the presence of Thiomonas,further supporting its role in situ.

Composition of Bacterial Communities in Reigous CreekWater and Sediments

Two 16S rRNA encoding gene libraries were constructed(Table 2), containing 229 clones in the sediment library and221 in that of the water library. The Shannon index (see“Methods”) and equitability values were greater in thewater library than in the sediment library (Table 1), whichsuggests that the bacterial diversity was lower in thesediment than in the water samples. Eleven differentspecies were identified in the sediments and 13 in thewater (Table 2, Fig. 2).

Several of the bacteria identified in the present study inboth the waters and the sediments have been previouslydetected in the Reigous waters [12, 14, 15, 18, 21, 22].Classified first by abundance order, several sequences wereaffiliated to Thiobacillus sp. ML2-16. This bacteriumhas been frequently reported to occur in AMD [5].The presence of several strains affiliated to thisproteobacteria is in agreement with the results of previousstudies and shows that these bacteria persist in this


http://beta.uniprot.org/

http://beta.uniprot.org/

http://www.genoscope.cns.fr/

http://www.genoscope.cns.fr/

http://www.uniprot.org/uniprot

http://www.uniprot.org/uniprot

http://inpact.u-strasbg.fr/~db/

http://itol.embl.de/

ecosystem [12]. Secondly, bacteria affiliated to Gallionellacapsiferriformans were detected in both the water andsediments. G. capsiferriformans is an oxygen-dependentferrous iron-oxidizing bacterium that grows at circum-neutral pH [59]. Relatives of Gallionella, such as G.ferruginea in particular, have often been detected, some-times as the dominant group in microbial mine watercommunities, including Carnoulès [12, 33–35]. Bacteriarelated to At. ferrooxidans as well as Thiomonas strainshave been previously isolated from this site [14, 18, 21,22]. At. ferrooxidans, which was the first microorganism tobe isolated from an acidic leaching environment, occursubiquitously in AMD, as does Thiomonas [32]. In additionto these groups, other species that have not previously beendescribed at this site were detected in this study. Some ofthese species were found to occur in both sediments andwater. For example, bacteria affiliated to the FirmicutesAlicyclobacillus sp. BRG 73 were identified. This genus,found in AMD [5], is characterized by moderatelythermophilic, acidophilic, strictly aerobic, and endospore-forming bacilli [30]. Likewise, bacteria related to “Ferrovummyxofaciens” PSTR were detected in both the waters andsediments. “Ferrovum myxofaciens” is an autotrophic iron-oxidizer which predominates in some AMD and is able togrow litho-autotrophically, using ferrous iron as an electron

donor [32]. Lastly, bacteria affiliated to Leptospirillumferrooxidans, an iron-oxidizing member of the Nitrospirae[36], were also present. This bacterium has been found tooccur in several acidic environments and in biofilmsoriginating from AMD [5, 9, 32].

Other newly characterized groups were identified only insediments (Table 2). The presence in acidic mine waters ofAcidocella sp., a non-iron-oxidizing heterotrophic acido-phile is quite common in AMD [33, 35]. Ferrimicrobium isan iron-oxidizing heterotroph that can also use iron as anelectron acceptor [20]. Other prokaryotes detected insediments were affiliated to Acidiphilium sp. CCP3, anon-iron-oxidizing heterotrophic acidophile that is alsoquite common in AMD [33] and Dokdonella koreensis, aγ-proteobacteria.

Six newly characterized groups were identified only inwater. Some of these bacteria were related to Sideroxydanslithotrophicus LD-1, an oxygen-dependent ferrous iron-oxidizing bacterium that grows at circumneutral pH [59];Rhodoferax ferrireducens, a psychrotolerant, facultativeanaerobic bacterium which is able to oxidize acetate withthe reduction of Fe(III) [28]; and an Acidobacteriaceaebacterium, CH1. Members of Acidobacteria have previouslybeen reported in AMD [5, 32]. Finally, three sulfate-reducingbacteria were related to strain JHA1, Desulfomonile limimaris,

Figure 1 X-ray absorption spectra at the As–K edge of the sample,(COWG April 11, 2006,) showing similarities with the X-rayamorphous Fe(III)–As(V) hydroxysulfate phases obtained after incu-bating sterilized Carnoulès water with the Thiomonas sp. strain B2isolated at the site [48]. These phases reached a molar As/Fe ratio of0.8, as described in [48]. a Linear least-squares fitting of XANES datashowed that the largest arsenic fraction (90±2%) was in the As(V)oxidation state. A small arsenic fraction (10±2%) was in the As(III)oxidation state, which resulted in a slight decrease in the amplitude inthe EXAFS spectrum. b Shell by shell fit of the EXAFS spectra in k-

space for the COWG sediment sample and the Thiomonas sp. strainB2 precipitate sample. c Corresponding Fourier transforms of theexperimental and fit curves. Dotted lines experimental; solid linesfitting curves. The local structure of both the COWG and thelaboratory Thiomonas sp. samples includes bidentate arsenate–oxygen–iron complexes characterized by ~1.5–2.0 Fe atoms at anAs–Fe distance of 3.31±0.02 Å. A small arsenic fraction (10±2%)was in the As(III) oxidation state, which resulted in a slight decreasein the amplitude in b the EXAFS spectrum and in c the correspondingFourier transform


Tab

le2

Bacterial

clon

esdetected

attheCarno

ulès

minedrainage

with

theirph

ylog

enetic

grou

p,theclosestisolated

relativ

eandtherelativ

eabun

danceof

each

grou

pversus

thetotalnu

mberof

clon

es(100

%)

Sam

pling

Clones

Phy

logenetic

grou

pClosestisolated

relativ

e(accession

number)

Percentage

ofsimilarity

Relative

abun

dance

ofclon

es(%

)

Sedim

ent

CGA6S

d1a,

5a,10

b,13

c,23

c,34

c,59

b,89

c,92

cß-Proteob

acteria

Thiob

acillus

sp.ML2-16

(DQ14

5970

)95

–96

31

CGA6S

d4b,

36c,

48c

ß-Proteob

acteria

Gallio

nella

capsiferrifo

rman

sES-2

(DQ38

6262

)96

21

CGA6S

d13a

γ-Proteob

acteria

Acidithioba

cillu

sferroo

xida

nsDX-1

(EU08

4695

)99

10

CGA6S

d10a,37

aα-Proteob

acteria

Acido

cella

sp.M21

(AY76

5998

)99

–100

8

CGA6S

d31c,36

aß-Proteob

acteria

Thiom

onas

sp.PK44

(AY45

5806

)99

6

CGA6S

d6b,

18a,

27b

γ-Proteob

acteria

Dokdo

nella

koreensisNML01

–023

3(EF58

9679

)92

5

CGA6S

d32a

Actinob

acteria

Ferrimicrobium

sp.BGR49

(GU16

7992

)99

3

CGA6S

d38c

α-Proteob

acteria

Acidiph

ilium

sp.CCP3(AY76

6000

)99

3

CGA6S

d20a,76

cFirmicutes

Alicycloba

cillu

ssp.BGR73

(GU16

7996

)92

–99

3

CGA6S

d58b

Nitrospirae

Leptospirillum

ferroo

xida

nsSy(A

F35

6839

)99

2

CGA6S

d51c

ß-Proteob

acteria

“Ferrovum

myxofaciens”PSTR(EF13

3508

)10

02

Water

CGA6W

t4c,

7a,15

c,20

a,21

b,22

b,23

a,31

b,32

a,33

c,45

b,54

c,63

b,73

cß-Proteob

acteria

Thiob

acillus

sp.ML2-16

(DQ14

5970

)94

–96

26

CGA6W

t7c,

17b,

21a,

23c,

27b,

35b,

36b,

67c,

80c,

86c

ß-Proteob

acteria

Gallio

nella

capsiferrifo

rman

sES-2

(DQ38

6262

)89

–97

18

CGA6W

t25c,56

bγ-Proteob

acteria

Acidithioba

cillu

sferroo

xida

nsBGR:110

(GU16

8011)

100

10

CGA6W

t5a,

48c,

79b

Acidithioba

cillu

sferroo

xida

nsDSM

2392

(AJ459

800)

91–92

CGA6W

t9a,

19a,

27a,

9b,61

cFirmicutes

Alicycloba

cillu

ssp.BGR73

(GU16

7996

)91

–99

8

CGA6W

t11a,29

c,78

cß-Proteob

acteria

Sideroxyda

nslitho

trop

hicusLD-1

(DQ38

6859

)94

–97

8

CGA6W

t3a,

8cß-Proteob

acteria

“Ferrovum

myxofaciens”PSTR(EF13

3508

)98

–99

4

CGA6W

t86b

ß-Proteob

acteria

Thiom

onas

sp.PK44

(AY45

5806

)94

3

CGA6W

t15a

Nitrospirae

Leptospirillum

ferroo

xida

ns(A

B51

0912

)94

3

CGA6W

t51b

ß-Proteob

acteria

Rho

doferaxferrireducensT118(CP00

0267

)99

3

CGA6W

t61b

Acido

bacteria

Acido

bacteriaceae

bacterium

CH1(D

Q35

5184

)96

3

CGA6W

t42c

Sulfate-reducingbacterium

JHA1(EF44

2984

)82

3

CGA6W

t30a

δ-Proteob

acteria

Desulfomon

ilelim

imaris(N

R_0

2507

9)87

3

CGA6W

t10a

δ-Proteob

acteria

Desulfuromon

assvalba

rdensis60

(AY83

5390

)82

1

Sequences

closelyrelatedto

16SrRNA

genesfrom

Eug

lena

spp.

chloroplastwerealso

detected

(datano

tshow

n).The

16SrRNA

encoding

gene

ofchloroplastsiscloselyrelatedto

thebacterial

16SrRNA

encoding

gene

andcanthereforebe

amplifiedby

prim

ers8F

and14

89R


an anaerobic dehalogenating bacterium from marine sediments[54] and Desulfuromonas svalbardensis 60, a psychrophilic,Fe(III)-reducing bacterium isolated from Arctic sediments [56](Table 2).

All in all, 17 species of bacteria were identified in thewater and sediments sampled at the Reigous creek. Onlyseven genera were found to be present in both phases,six were found only in water, and four only in thesediments (Table 2). Most of these species are commonresidents of AMD [5, 32]. This quite low bacterialdiversity was probably due to the high concentration oftoxic compounds in this AMD and was consistent withprevious observations showing that the biodiversity ofacidic, metal-rich mine waters is mainly restricted tospecialized prokaryotes and some eukaryotes such asEuglena [15, 52], which has been detected in this study(data not shown). In the Reigous system, in both sedi-ments and water, the populations observed were mainlyinvolved in the Fe, As, and S cycles. The populationsinvolved in Fe(II) oxidation were related to Gallionella, At.ferrooxidans, Ferrimicrobium, Leptospirillum, Sideroxydanslithotrophicus, or “Ferrovum myxofaciens” [32, 59], whereasferric iron reduction has been described for populations likeAcidiphilium spp., Acidocella, Desulfuromonas svalbardensis,Rhodoferax ferrireducens, or even At. ferrooxidans andFerrimicrobium acidiphilum [20, 28, 56]. Since the Carnoulèscreek spring contains mainly Fe(II) and As(III) in the form ofdissolved species [18], the Fe(III) and As(V) may be formedas the result of microbial oxidation processes via the activityof acidophilic iron- and arsenite-oxidizing bacteria [24, 48]. Inother words, the large amounts of soft pale-yellow As(V)–Fe(III) hydroxysulfate sediments analyzed here (Fig. 1) wereprobably formed by the joint activities of iron-oxidizing (e.g.,At. ferrooxidans or Gallionella) and arsenic-oxidizing (e.g.,Thiomonas sp.) microorganisms. Concerning S cycling, wefound populations able to oxidize the reduced inorganic sulfurcompounds, like Thiobacillus, Thiomonas, or At. ferrooxidans[32, 42, 46]. Sulfate-reducing bacteria such as Desulfomonilelimimaris or Desulfuromonas svalbardensis were also foundto be present in sediments and may be involved in sulfateconsumption [54, 56].

Among these prokaryotes, some bacteria may be presentbut not functionally active, and it was, therefore, crucial todifferentiate between dead or inactive cells and functionalcells. To determine which organisms play a significant rolein the natural remediation processes such as the Fe(II)oxidation processes observed at the study site, a metapro-teomic approach was used to list the bacterial populationexpressing proteins, i.e., those which were active. Themetaproteomic approach was possible with sediments butfailed with the water samples because larger numbers ofbacterial cells were recovered from sediments than fromwater (data not shown).

Characterization of the Main Proteins Expressedby the Sediment Community

Proteins expressed by this community were liable to corre-spond to orthologs originating from diverse prokaryotes andto have similar amino acid sequences. For those reasons and toimprove their characterization, proteins were separated byperforming 2-D gel electrophoresis (supplementary Fig. 1). Atotal number of 89 proteins were identified, 44% of which(39 proteins, Table 3) originated from bacteria and 39% (35proteins) from protists, while 15 proteins originated fromhigher plants, probably from decomposed plant debrispresent at the Reigous creek. One third of the proteinsoriginated from protists, mainly consisting of E. mutabilisdetected in our samples (data not shown) and could not becompletely removed using the Nycodenz Gradient. E.mutabilis is a common inhabitant of AMD [2, 11, 15, 39]and was also present at the surface of sediments. TheseEuglena proteins are involved in various metabolic processes,suggesting that this eukaryote may play a relevant role in thisecosystem, in agreement with recent results (Gouhlen-Cholletand Bertin, unpublished data). Only proteins originating frombacteria were further analyzed in this study. The 2-D gelelectrophoresis approach used here allowed identifying from asingle sample only the most abundant cytoplasmic proteins.Therefore, some relevant proteins that might be expressed inthis environment, such as rusticyanin from At. ferrooxidans,which is known to be involved in Fe(II) oxidation and waspreviously thought to possibly play a functional role at thissite [21, 24, 48], were not detected in this study. Thismembrane protein may not be resolved in the 2-D gel, or itmay not be abundant at the sampling point. In addition, themajority of the bacteria forming the Carnoulès communityhave never been grown and studied in vitro so far. Theirgenome sequences, and hence their protein sequences, whichare required for MS identification purposes, may not beavailable in public databases, except for those of At.ferrooxidans, Thiomonas, and Gallionella strains. All thesehypotheses may explain why so few bacterial proteins wereidentified in this study.

The bacterial proteins identified originated from Aquificaewere Actinobacteria, Deinococcus, Synergistetes, Firmicutes,Bacteroidetes, and α-, β-, and γ-proteobacteria. Among the

Figure 2 Phylogenetic tree based on 16S rRNA-encoding sequences.Sequences were aligned using ClustalW. Alignments were checked byhand and positions with more than 1% of gaps were automaticallyremoved. Neighbor-joining trees were drawn up with 310 nt usingITOL (http://itol.embl.de/) [44]. Accession numbers: see supplemen-tary data. In red: bacteria identified based on the metaproteomic(GroEL identifications) approach; in blue: bacteria identified using the16S rRNA encoding gene library; in black: sequences detected inNCBI databases which are closely related to the bacteria present in theReigous sediment

b


http://dx.doi.org/10.1007/s00248-011-9808-9

-proteobacteria

-proteobacteria

-proteobacteria

-proteobacteria

Nitrospirae

Actinobacteria

Synergistetes

Firmicutes

Acidobacteria

Deinococcus/Thermus


Tab

le3

Bacterial

proteins

identifiedin

theReigo

ussediment’smicrobial

commun

ity

Phylum

Class,Fam

ily,Genus

Organism

Level

ofdiscrimination

Protein

name

Protein

accessionnumbers

Spotnumberb

Peptid

esequence

Aquificae

Aquificae(class);Aquificales;

Aquificaceae;Hydrogenobaculum

Hydrogenobaculum

sp.

Y04AAS1

Species

Putativeuncharacterizedprotein

A7W

FJ8

25IG

AAVIG

R

Deinococcus-

Therm

usDeinococci;Deinococcales;

Deinococcaceae;

Deinococcus

Deinococcus

deserti

Species

60kD

achaperoninsa

C1C

ZP1

4,6

APGFGDR

QLV

FDEAAR

Deinococci;Deinococcales;

Deinococcaceae;

Deinococcus

Deinococcus

sp.A62

Species

60kD

achaperoninsa

A1Y

UK7

2AVLV

AIEEIK

Synergistetes

Synergistia

;Synergistales;

Synergistaceae;Therm

anaerovibrio

Therm

anaerovibrio

acidam

inovorans

Genus

orbelow

60kD

achaperoninsa

D1B

621

4IA

QVASISANDK

FGSPTITNDGVTIA

K

Firmicutes

Bacilli;Lactobacilla

les;

Lactobacilla

ceae

Lactobacillu

sdelbrueckiisubsp.

Indicusor

delbrueckii

Subspecies

60kD

achaperoninsa

Q70BV2,

Q70BV5

4,5

APGFGDR

YGAPTITNDGVTIA

K

Bacilli;Bacillales;Bacillaceae

Virgibacillus

pantothenticus

Fam

ilyor

below

60kD

achaperoninsa

A9L

HR1

4,5,

6AVEVAVK

Bacilli;Bacillales;Bacillaceae;

Bacillus

Bacillus

halodurans,

pseudofirmus

orselenitireducens

Species

60kD

achaperoninsa

O50305,

A8V

UQ6,

D3F

SF9

2NVTSGANPMVIR

Clostridia;

Clostridiales;

Clostridiaceae;

Alkaliphilus

Alkaliphilus

metalliredigensor

ormlandii

Genus

orbelow

60kD

achaperoninsa

A6T

LJ1,A8H

J57

6LSGGVAVIQ

VGAATETELK

Clostridia;

Clostridiales;

Clostridiaceae;

Clostridium

Clostridium

botulin

umSpecies

60kD

achaperoninsa

A5I723,

A7F

YP3,

A7G

IN3,

B1IFD4,

B1L

1K0,

C1F

LV5,

C3K

UC8,

B1Q

9U6,

B1Q

I57

4APGFGDR

LGID

IIR

Clostridia;

Clostridiales;

Clostridiaceae;

Clostridium

Clostridium

hiranonis

Species

60kD

achaperoninsa

B6F

W06

4,5,

6APGFGDR

KALEEPLR

VGAATEVEMK

TNDIA

GDGTTTATVLAQAIIR

Clostridia;

Clostridiales;

Clostridiaceae;

Clostridium

Clostridium

papyrosolvens

Species

orsubspecies

60kD

achaperoninsa

C7IKN8

4,6

APGFGDR

FGSPTITNDGVTIA

K

Bacteroidetes

Flavobacteria;Flavobacteriales;

Flavobacteriaceae;Gramella

Gramella

forsetii(strain

KT0803)

Species

ATPsynthase;beta

subunit

A0M

791

11MPSAVGYQPTLATEMGAMQER

Phosphoglyceratekinase

A0M

6J2

13,20

LGDIY

VNDAFGTAHR

Actinobacteria

Actinobacteria(class);

Actinobacteridae;

Actinom

ycetales;

Propionibacterineae;

Propionibacteriaceae

Propionibacterium

freudenreichii

Species

60kD

achaperoninsa

A5JUG8

4,5,

6NVTA

GANPIELK


Actinobacteridae;

Actinom

ycetales;

Glycomycineae;

Glycomycetaceae;

Stackebrandtia

Stackebrandtia

nassauensis

Suborderor

below

60kD

achaperoninsa

C4D

UC7

6GMNALADAVK


Actinobacteridae;

Actinom

ycetales;

Streptosporangineae;

Streptosporangiaceae;

Streptosporangium

Streptosporangium

roseum

Fam

ily60

kDachaperoninsa

D2B

BD1

4APGFGDR

GTFTSVAVK


Actinobacteridae;

Actinom

ycetales

Arthrobacter,

Janibacter,

Clavibacter

orKineococcus

Order

ATPsynthase;beta

subunit

A0JY64,A1R

7V3,

A3T

GD9,

A5C

Q60,A6W

7G9

10,31,32,33,38

DVQNQDVLLFID

NIFR

VALSALT

MAEYFR

IGLFGGAGVGK

Proteobacteria

α-proteobacteria,

Rhizobiales;

Sinorhizobium

medicae

Genus

or60

kDachaperoninsa

A6U

H06

2,4

LVAAGMNPMDLK


Tab

le3

(con

tinued)

Phylum

Class,Fam

ily,Genus

Organism

Level

ofdiscrimination

Protein

name

Protein

accessionnumbers

Spotnumberb

Peptid

esequence

Rhizobiaceae;

Sinorhizobium/

Ensifergroup;Sinorhizobium

below

AAVEEGIVAGGGVALLR

α-proteobacteria,

Rhodospirillales;

Acetobacteraceae;

Acidiphilium

Acidiphilium

cryptum

Genus

orbelow

60kD

achaperoninsa

A5G

1G2

5,6

APGFGDR

AAVEEGIV

PGGGVALAR

AVAAGMNPMDLK

AGIIDPTK

ENTTIV

EGAGK

α-proteobacteria,

Rhodobacterales;

unclassified

Rhodobacterales

Rhodobacterales

bacterium

HTCC2083

Fam

ilyor

below

60kD

achaperoninsa

B6A

WC8

4,5

APGFGDR

EIELADPFENMGAQLVK

SVAAGMNPMDLK

β-proteobacteria,

Burkholderiales;

Com

amonadaceae;

Acidovorax

Acidovoraxavenae

Species

60kD

achaperoninsa

A1T

KQ5,

D1S

TJ1

4APGFGDR

VGAATEVEMK

AVTA

LVAELKK

VTLADLGQAK

AAVEEGIVAGGGVALLR

β-proteobacteria,

Burkholderiales;

Alcaligenaceae;

Bordetella

Bordetella

petrii

Species

60kD

achaperoninsa

A9I685

2,4,

5APGFGDR

AVEEPLR

VGAATEVEMK

EGVITVEDGK

VQIEEATSDYDREK

DLLPVLEQVAK

VEDALHATR

VQIEEATSDYDR

β-proteobacteria

Bordetella

avium

(strain197N)

Species

50Sribosomal

protein

L7/L12

Q2L

2M6

NC14

AEILDAIA

GMTVLELSELIK

DLV

DGAPKPVK

β-proteobacteria;

Burkholderiales;

Oxalobacteraceae;

Herminiim

onas

Herminiim

onas

arsenicoxydans

Species

Glutathione-dependent

form

aldehyde

dehydrogenase

(alcohol

dehydrogenaseclass

III),HEAR2039

A4G

6P6

NC12

IIAID

TNPA

K

TNLCVAVR

Glutathione-independent

form

aldehyde

dehydrogenase,

HEAR2048

A4G

6Q5

NC3,

NC11

LEDAPA

AYK

VID

YVGVDCR

GMTMGHEMTGEVIEVGSDVEVVK

FPELITPQGK

β-proteobacteria,

Burkholderiales;

unclassified

Burkholderiales;

Burkholderiales

Generaincertaesedis

Leptothrixcholodniior

Thiom

onas

interm

edia

Genus

60kD

achaperoninsa

B1X

XY9,

C7H

ZY6

2,3,

4,5,

36,60

SFGAPTVTK

YVAAGMNPMDLKR

APGFGDR

LQNMGAQMVK

EGVITVEDGK

VGAATEVEMK

VIA

EEVGLT

LEK

YVAAGMNPMDLK

VTLADLGQAK

IQIEEATSDYDREK

VEDALHATR

IQIEEATSDYDR


Tab

le3

(con

tinued)

Phylum

Class,Fam

ily,Genus

Organism

Level

ofdiscrimination

Protein

name

Protein

accessionnumbers

Spotnumberb

Peptid

esequence

AMLEDIA

ILTGGK

AAVEEGIVAGGGVALLR

Thiom

onas

3As

Genus

50Sribosomal

proteinL1;

THI3722

FP475956

38VAVSSTMGIG

VR

VDTA

TVNAAVAGQ

β-proteobacteria,

Burkholderiales;

Burkholderiaceae;

Limnobacter

Limnobacter

sp.

MED105

Genus

orbelow

60kD

achaperoninsa

A6G

TE5

4APGFGDR

VGAATEVEMK

GVNILANAVK

β-proteobacteria,

Burkholderiales;

unclassified

Burkholderiales;

Burkholderiales

Genera

incertaesedis;

Methylib

ium

Methylib

ium

petroleiphilu

mGenus

orbelow

60kD

achaperoninsa

A2S

CV1

2,4

SFGAPTVTK

YVAAGMNPMDLKR

APGFGDR

VQIEEATSDYDREK

LQNMGAQMVK

VGAATEVEMK

EGVITVEDGK

VIA

EEVGLT

LEK

YVAAGMNPMDLK

VTLADLGQAK

VEDALHATR

AAVEEGIVAGGGVALLR

VQIEEATSDYDR

VQIEEATSDYDR

AMLEDIA

ILTGGK

β-proteobacteria,

Burkholderiales;

Burkholderiaceae;

Ralstonia

Ralstonia

pickettii

Subspecies

60kD

achaperoninsa

B2U

6M6

4APGFGDR

VGAATEVEMK

DLLPILEQVAK

AAVEEGIVAGGGVALLR

β-proteobacteria,

Burkholderiales;

Com

amonadaceae;

Verm

inephrobacter

Verm

inephrobacter

eiseniae

Genus

60kD

achaperoninsa

A1W

L05

2,4

APGFGDR

EVVFGGEAR

VGAATEVEMK

EGVITVEDGK

VIA

EEVGLT

LEK

VQIEEATSDYDREK

AVTA

LVAELKK

VTLADLGQAK

VEDALHATR

VQIEEATSDYDR

AMLEDIA

ILTGGK

β-proteobacteria,

Methylophila

les;

Methylophila

ceae;Methylotenera

Methylotenera

mobilis

Genus

orbelow

60kD

achaperoninsa

C6W

TL6

5,6

SVAAGMNPMDLK


Tab

le3

(con

tinued)

Phylum

Class,Fam

ily,Genus

Organism

Level

ofdiscrimination

Protein

name

Protein

accessionnumbers

Spotnumberb

Peptid

esequence

TNDIA

GDGTTTATVLAQAIIR

β-proteobacteria,

Gallio

nella

les;

Gallio

nella

ceae;Gallio

nella

Gallio

nella

ferruginea

Genus

orbelow

60kD

achaperoninsa

C5V

7N1

2VGAATEVEMK

GYLSPYFIN

NQDR

DLLPVLEQVAK

VEDALHATR

γ-proteobacteria,Acidithiobacilla

les;

Acidithiobacilla

ceae;

Acidithiobacillu

s

Acidithiobacillu

sferrooxidans

Species

60kD

achaperoninsa

B5E

N19,B7J561

2,3

APGFGDR

HALEGFK

AVIA

GMNPMDLK

GVNVLADAVK

VVSEEIG

MK

VEDALHATR

AMLEDMAILTGGR

LESTTLADLGQAK

γ-proteobacteria,Methylococcales;

Methylococcaceae;

Methylococcus

Methylococcus

capsulatus

Order

orbelow

60kD

achaperoninsa

Q60AY0

2,3,

4APGFGDR

VGAATEVEMK

VEDALHATR

QIVANAGDEPSVVLNK

γ-proteobacteria;Pseudom

onadales;

Pseudom

onadaceae

Pseudom

onas

Genus

Outer

mem

branelip

oprotein

OprI

A2V

C34,A4X

VE5,

O85409-

085430-O

85432-O8543

7,O85439-O85444,

Q3K

906,

Q48K14,Q883S

8

4,15,17,19,22,26–

27,40,44,49,55,

60,62,64

–65,

NC7-NC10

LTATEDAAAR

KADEALAAAQK

ADEALAAAQK

ITATEDAAAR

γ-proteobacteria,unclassified

Gam

maproteobacteria;

OMG

group;

SAR92

clade

Gam

maproteobacterium

HTCC2207

Group

orclade

60kD

achaperoninsa

Q1Y

SA6

5SVAAGMNPMDLK

AQIEDTSSDYDR

δ-proteobacteria,Myxococcales;

Nannocystineae;

Haliangiaceae;

Haliangium

Haliangium

ochraceum

Order

orbelow

60kD

achaperoninsa

D0L

RR3

4,6

APGFGDR

VGAATEVEMK

GYLSPYFVTDSER

Proteobacteria

Several

Proteobacteria

Phylum

Succinyl-CoA

synthetase;beta

subunit

Q3K

FU6c

NC1,

NC3,

NC11

LEGNNAELGAK

aMS/M

Sdata

weresearched

againstan

in-hou

sedatabase

includ

ingallthebacterialandarchaeal

GroELsequ

encesob

tained

from

Uniprot

bSpo

tsfrom

1to

65originated

from

apH

4to

7gradient

gel(Sup

plem

entary

Fig.1),spotsfrom

NC1to

NC14

originated

from

apH

3to

10gradient

gel(datano

tshow

n)cSeveral

proteins

may

correspo

ndto

thisidentification:

Q3K

FU6,

A1A

8Y0,

A1F

GM7,

A1JRB6,

A1K

TM6,

A2U

EB0,

A3H

HN5,

A3M

887,

A4T

NT8,

A4W

879,

A4X

V90

,A5W

114,

A5W

C33

,A6B

TC9,

A6T

6F6,

A6V

7K5,

A7F

KR4,

A7M

QX5,

A7Z

JA8,

A7Z

XY8,

A8A

J84,

A8G

B83

,P0A

836,

P0A

837,

P0A

838,

P0A

839,

P53

593,

P66

869,

P66

870,

Q02

K73

,Q0T

6W6,

Q0T

JW6,

Q1C

AG1,

Q1C

FM0,

Q1I7L

3,Q1R

EJ8,Q21

IW6,

Q2N

UM2,

Q2S

D35

,Q32

4I4,

Q32

IK3,

Q3Z

476,

Q48

K68

,Q4F

VH9,

Q4K

FY6,

Q4Z

UW7,

Q57

RL3,

Q5F

878,

Q5P

CM7,

Q66

DA0,

Q6D

7G2,

Q6F

8L4,

Q7N

6V5,

Q7N

Z47

,Q88

3Z4,

Q88

FB2,

Q8Z

H00

,Q9JUT0,

Q9JZP4


39 bacterial proteins detected, there were two distinct ATPsynthases, two distinct 50 S ribosomal proteins, onephosphoglycerate kinase, and one succinyl-CoA synthetase,which are involved in energy metabolism, translation,glycolysis, and TCA cycle, respectively (Table 3). Theseproteins may not play a specific role in this environmentsince they are known to be housekeeping proteins in bacteria.Among the other proteins, one outer protein OrpI, oneuncharacterized protein with an unknown function, oneglutathione-dependent, and one glutathione-independentformaldehyde dehydrogenases were identified. The lattertwo proteins belong to the formaldehyde detoxificationpathway. Although no correlation with the environmentalconditions might explain the functional specificities of theseproteins, it has been reported that the arsenite-oxidizingbacteria H. arsenicoxydans synthesizes alcohol dehydroge-nase and glutathione-dependent formaldehyde dehydrogenasewhen grown in the presence of arsenic [58].

It is worth noting that more than half of the identifiedbacterial proteins were similar to the 60-kDa GroEL chaper-onin. These data suggest that multiple chaperonins of variousgenetic origins are expressed by the Reigous creek commu-nity. GroEL is known to be ubiquitously present in Bacteriaand Archaea. These proteins are generally abundantlyexpressed in bacterial cells, especially under stress conditionssuch as those occurring in this particularly toxic environment[3]. The groEL gene is conserved in prokaryotes, and hasbeen found to be present in one copy in the majority ofsequenced genomes, except in the case of some pathogens[45, 60]. Because of its conservation properties (supplemen-tary Fig. 2), this gene is often used as a phylogenetic marker[31]. In addition, it has been previously established that someof the amino acids stretch occurring in GroEL are specific toone genus or family of bacteria. These peptide sequences cantherefore be used as the signature of a specific phylogeneticgroup. These GroEL identifications (Table 3, Fig. 3) were,therefore, considered for use as a possible taxonomic tool inaddition to the 16S rRNA-based taxonomic approach. Todetermine which bacteria in the whole community wereactive, i.e., able to express proteins, the organisms identifiedusing the 16S rRNA encoding gene library- and GroEL-based approaches (Fig. 2) were compared.

Most of the bacteria identified based onGroEL belonged tofive phyla divisions. Bacteria belonging to Deinococcus andSynergistetes did not feature among those identified based onthe 16S rRNA encoding gene. One possible explanation forthis discrepancy may be that a PCR or cloning bias mayhave prevented those bacteria from being detected with thismethod. These findings suggest that metaproteomic methodsused as taxonomic tools can provide a useful complementarytool in addition to the 16S rRNA encoding gene approach.Interestingly, Thiomonas, At. ferrooxidans, Acidiphilium, andGallionella, expressed proteins and were, therefore, active.

16S rRNA-encoding gene analysis showed that thesebacteria abound in this ecosystem. Bacteria affiliated to At.ferrooxidans and Gallionella are able to oxidize iron. Inaddition, many strains of the Thiomonas genus are able tooxidize As(III) into As(V) under laboratory conditions[6, 14, 22]. The fact that their proteins were detected showsthat these bacteria were viable and metabolically active. Thisfinding supports the hypothesis that the oxidation of Fe(II) toFe(III) catalyzed by iron-oxidizing microorganism such asAt. ferrooxidans and Gallionella and oxidation of As(III) intoAs(V) by As(III) oxidizers such as Thiomonas, probablyleads to the precipitation of the more or less ordered ironoxy-hydroxides (Fe(III)–As(V) hydroxysulfate) detected inthis study (Fig. 1) [48]. This finding is in agreement withprevious data showing that Gallionella ferruginea efficientlyremove Fe, As(III), and As(V) in water [41]. The presentdata show for the first time that this bacterium is activeand probably plays a functional role in the sediments ofthe Reigous creek. Some heterotrophic bacteria such asAcidiphilium were also found to be active in this AMD,suggesting that they could cope with the low amount oforganic carbon (dissolved organic carbon concentration1.7±0.4 mg/L [16]) of Reigous creek water. It has previouslybeen suggested that these acidophilic heterotrophic bacteriamay be involved in organic carbon turnover processes [32].Interestingly, these four bacteria (At. ferrooxidans, Thiomonas,Gallionella, and Acidiphilium) found to be active members ofthis AMD community have been previously identified inAMD, but some of them were thought to have differentoptimum pH levels. Indeed, G. ferruginea is a neutrophilicbacterium which oxidizes Fe, but relatives of Gallionella,have often been detected in AMD [12, 33–35]. The strainoccurring at Carnoulès showed less than 97% homology withG. capsiferriformans and its physiological characteristics areprobably different. It seems probable that an acid-tolerantrelative of this bacterium is able to oxidize iron under acid pHconditions.

In addition to the bacteria belonging to these four genera,other bacteria were also found to be active, but adiscrepancy was again observed between the bacteriaidentified based on the results of 16S rRNA encoding geneanalysis and the metaproteomic approach. The GroELprotein sequences of some bacteria identified using the16S rRNA encoding gene library were not available in theUniprot database, which might explain this discrepancy.However, phylogenetic comparisons between the 16SrRNA and metaproteomic data obtained (Fig. 2) suggestedwhich of the bacteria present in this ecosystem may expressa GroEL identified in the metaproteomic study. Based onthese comparisons, it seems likely that in addition toThiomonas, At. ferrooxidans, Acidiphilium, and Gallionella,clones related to β-, γ-, and δ-proteobacteria, such asLimnobacter or Methylococcus (At. ferrooxidans DSM


A. avenae subsp. avenae A. avenae subsp. citrulli A. ferrooxidans ATCC53993 A. ferrooxidans ATCC23270T A. cryptum A. metalliredigens A. oremlandii B. halodurans B. pseudofirmus B. selenitireducens B. petrii C. botulinum C. hiranonis C. papyrosolvens D. desertii D. sp. A62 G. ferruginea Gamma-proteobacterium HTCC2207 H. ochraceum L. delbrueckii subsp. delbrueckii L. delbrueckii subsp. indicus L. cholodnii Limnobacter sp. MED105 M. petroleiphilum M. capsulatus M. mobilis P. freudenreichii R. picketii Rhodobacterales bacterium HTCC2083 S. medicae S. nassauensis S. roseum T. acidaminovorans Thiomonas 3As T. intermedia V. eiseniae V. pantothenticus


Figure 3 Alignment of the amino acid sequences of 60 kDa chaper-onins. Thirty-six sequences matched the MS results: 29 Proteins wereidentified in Table 3: 22 of them unambiguously matched one protein(the bacterial name of which is given in orange), whereas fifteenidentifications matched at least two proteins (22 bacterial names givenin black). Thiomonas sp. 3As data were added for the sake ofcomparison (in blue). Thirty-seven sequences in all were aligned. Thesesequences were compared with ClustalW2 using the default parameters(http://www.ebi.ac.uk/Tools/clustalw2/index.html). Alignment was per-formed using the Jalview software program [19]. The blue highlightedletters correspond to identical residues among the 37 orthologs(BLOSUM62 score option). Highlighted letters corresponding to thepeptide sequences identified by MS allowed distinguishing severalorthologs. First, 23 proteins were identified because at least oneidentified peptide matched only this GroEL: these peptides (labeled inred) corresponding to signature sequences specific to one species,genus, or family of bacteria are located in the same homology regionsbut differed from other peptides by at least one amino acid substitution.Secondly, 13 of the proteins identified were confirmed, since severalidentified peptides (labeled in green) matched this GroEL. Each of theseindividual peptides matched several proteins; however, only one proteindetected in Uniprot contained all these amino-acid sequences, whichsuggests that a relative of this protein was probably present in thisextract. Since the full amino-acid sequences of almost all the

chaperonins expressed by the bacteria present at the study site areunknown, the possibility cannot be ruled out that two identified peptideswere erroneously assigned to two distinct chaperonins, whereas thesepeptides may in fact have originated from one protein, the amino acidsequence of which has not yet been included in the databases.Nevertheless, because of the high level of conservation observed inGroEL proteins (Supplementary Fig. 2), the proteins identified may beexpressed by a close relative of the organism identified using theUniprot database. GroEL originated from Acidiphilium cryptum; Acid-ithiobacillus ferrooxidans ATCC23270T; Acidithiobacillus ferrooxidansATCC53993; Acidovorax avenae subsp. avenae; Acidovorax avenaesubsp. citrulli; Alkaliphilus metalliredigens; Alkaliphilus oremlandii;Bacillus halodurans; Bacillus pseudofirmus; Bacillus selenitireducens;Bordetella petrii; Clostridium botulinum; Clostridium hiranonis; Clos-tridium papyrosolvens; Deinococcus desertii; Deinococcus sp. A62;Gallionella ferruginea; Haliangium ochraceum; Lactobacillus del-brueckii subsp. delbrueckii; Lactobacillus delbrueckii subsp. indicus;Leptothrix cholodnii; Limnobacter sp. MED105; Methylibium petrolei-philum; Methylococcus capsulatus; Methylotenera mobilis; Propioni-bacterium freudenreichii; Ralstonia picketii; Rhodobacterales bacteriumHTCC2083; Sinorhizobium medicae; Stackebrandtia nassauensis;Streptosporangium roseum; Thermanaerovibrio acidaminovorans; Thi-omonas 3As; Thiomonas intermedia; Verminephrobacter eiseniae;Virgibacillus pantothenticus; and Gammaproteobacterium HTCC2207


http://www.ebi.ac.uk/Tools/clustalw2/index.html

2392 affiliated bacteria), Methylotenera (Thiobacillus-related clones), D. koreensis (which is affiliated to theγ-proteobacteria HTCC2207), Ferrimicrobium-like bacteria(which are related to the Actinobacteria S. nassauensis andS. roseum), and Haliangium (clones affiliated to sulfate-reducing bacteria JHA1, D. limimaris and D. svalbardensis)may play a role in this ecosystem. All in all, the dataobtained here show that Firmicutes, which could be affiliatedto Alicyclobacillus ferrooxidans, are also active. Thesebacteria are able to oxidize ferrous iron [37] and may,therefore, participate in the transformation of the iron presentin high concentrations in these waters. The active popula-tion as a whole was not only composed of several iron-oxidizers in addition to At. ferrooxidans but alsocontained iron reducers, one known arsenite-oxidizer,sulfate-reducing, and sulfur compound oxidizers, and bothautotrophic and heterotrophic bacteria. All these bacteria

may contribute importantly to the remediation processobserved in situ.

In Conclusion

The active bacterial species inhabiting Carnoulès AMDecosystem were identified in this study using high-sensitivity nanoLC-chip-MS/MS methods combined witha 16S rRNA based phylogenetic approach. The meta-proteomic data obtained here show for the first time thatGallionella, Thiomonas, At. ferrooxidans, and Acidiphi-lium actively express proteins in situ. Previous hypothesesbased on experiments performed under laboratory con-ditions [14, 18, 21, 22, 24, 48] suggest that microbialactivity may contribute to the arsenite oxidation and Asimmobilization occurring in the heavily contaminatedAMD at the Carnoulès mine via iron oxidation processes.



Figure 3 (continued)


Since these bacteria were found to be active and to expressproteins which are among the most abundant proteinsencountered at this site, it seems likely that the largeamounts of pale-yellow As(V)–Fe(III) hydroxysulfatesediments forming at Carnoulès, which were characterizedhere, may result from the conjugate activities of iron-oxidizing microorganisms (such as At. ferrooxidans, Alicyclo-bacillus ferrooxydans, Ferrimicrobium, or Gallionella) andarsenic-oxidizing microorganisms (such as Thiomonas sp.).Several bacteria may be responsible in situ for changing theratio between the oxidized and reduced forms of iron, arsenic,and sulfur compounds, promoting the formation of the Fe(III)–As(V) hydroxysulfate precipitates detected in this study.These bacteria are therefore of prime importance in the partialbut efficient natural process of remediation undergone by thecontaminated Carnoulès ecosystem. In addition, autotrophiciron, arsenic, and sulfur oxidizers may provide the organiccarbon sources required by the functional heterotrophs suchas Acidiphilium present in this ecosystem. All in all, thepresent findings provide evidence that the functionalgenomics approach provides a useful means of describingbacterial communities such as those inhabiting the Reigouscreek and determining their contribution to natural attenuationprocesses. It is now proposed to use approaches of this kindin future studies to complete this picture of the functionalprocesses at work in this ecosystem, as well as to investigatethe role played by the less abundant active bacteria identifiedin this study.

Acknowledgements The study was financed by the EC2CO program(“Institut National des Sciences de l’Univers,” CNRS), the “Observatoirede Recherche Méditerranéen en Environnement” (OSU-OREME), and bythe ANR 07-BLANC-0118 project (“Agence Nationale de la Recherche”).Sébastien Gallien and Aurélie Volant were supported by a grant from theFrench Ministry of Education and Research. This work was performed inthe framework of the “Groupement de recherche: Métabolisme del’Arsenic chez les Microorganismes” (GDR2909-CNRS).

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ORIGINAL PAPER

Archaeal diversity: temporal variation in the arsenic-rich creeksediments of Carnoules Mine, France

A. Volant • A. Desoeuvre • C. Casiot • B. Lauga • S. Delpoux •

G. Morin • J. C. Personne • M. Hery • F. Elbaz-Poulichet •

P. N. Bertin • O. Bruneel

Received: 16 February 2012 / Accepted: 3 May 2012 / Published online: 20 June 2012

� Springer 2012

Abstract The Carnoules mine is an extreme environment

located in the South of France. It is an unusual ecosystem due

to its acidic pH (2–3), high concentration of heavy metals,

iron, and sulfate, but mainly due to its very high concentration

of arsenic (up to 10 g L-1 in the tailing stock pore water, and

100–350 mg L-1 in Reigous Creek, which collects the acid

mine drainage). Here, we present a survey of the archaeal

community in the sediment and its temporal variation using a

culture-independent approach by cloning of 16S rRNA

encoding genes. The taxonomic affiliation of Archaea showed

a low degree of biodiversity with two different phyla: Eur-

yarchaeota and Thaumarchaeota. The archaeal community

varied in composition and richness throughout the sampling

campaigns. Many sequences were phylogenetically related

to the order Thermoplasmatales represented by aerobic or

facultatively anaerobic, thermoacidophilic autotrophic or

heterotrophic organisms like the organotrophic genus Ther-

mogymnomonas. Some members of Thermoplasmatales can

also derive energy from sulfur/iron oxidation or reduction. We

also found microorganisms affiliated with methanogenic

Archaea (Methanomassiliicoccus luminyensis), which are

involved in the carbon cycle. Some sequences affiliated with

ammonia oxidizers, involved in the first and rate-limiting step

in nitrification, a key process in the nitrogen cycle were also

observed, including Candidatus Nitrososphaera viennensis

and Candidatus nitrosopumilus sp. These results suggest that

Archaea may be important players in the Reigous sediments

through their participation in the biochemical cycles of ele-

ments, including those of carbon and nitrogen.

Keywords Archaea � Diversity � Arsenic �Acid mine drainage � Lead and zinc mine

Introduction

Acid mine drainage (AMD) water is a worldwide environ-

mental problem caused by active and abandoned mines

(Johnson and Hallberg 2003). Mining and processing of sul-

fide-rich ores produce large amounts of pyrite-rich waste. In

contact with meteoric water, oxidation of this material gen-

erates AMD. These effluents are generally characterized by a

low pH and contain significant quantities of sulfates, metals

and metalloids including arsenic. AMD generation is mainly

mediated by acidophilic iron-oxidizing microorganisms

Communicated by F. Robb.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00792-012-0466-8) contains supplementarymaterial, which is available to authorized users.

A. Volant � A. Desoeuvre � C. Casiot � S. Delpoux �J. C. Personne � M. Hery � F. Elbaz-Poulichet � O. Bruneel (&)

Laboratoire HydroSciences Montpellier, HSM, UMR 5569

(IRD, CNRS, Universites Montpellier 1 et 2),

Universite Montpellier 2, Place E. Bataillon, CC MSE,

34095 Montpellier, France


B. Lauga

Equipe Environnement et Microbiologie, EEM, UMR 5254

(IPREM, CNRS), Universite de Pau et des Pays de l’Adour,

BP 1155, 64013 Pau, France

G. Morin

Institut de Mineralogie et de Physique des Milieux Condenses,

IMPMC, UMR 7590 (CNRS, Universite Pierre et marie

curie/Paris 6), 4 place Jussieu, 75252 Paris, France

P. N. Bertin

Laboratoire de Genetique Moleculaire, Genomique,

Microbiologie, GMGM, UMR 7156 (Universite de Strasbourg,

CNRS), Departement Microorganismes, Genomes,

Environnement, 28 Rue Goethe, 67083 Strasbourg, France

123

Extremophiles (2012) 16:645–657

DOI 10.1007/s00792-012-0466-8

Author's personal copy

http://dx.doi.org/10.1007/s00792-012-0466-8

(Edwards et al. 1999). Natural remediation of AMD can be

observed at the Carnoules site (France) or at Rio Tinto (Spain)

(Casiot et al. 2003; Sanchez Espana et al. 2005). This natural

remediation of metal pollutants is generally due to the

occurrence of abiotic reactions and/or microbial activities that

make these toxic compounds insoluble and lead to their

accumulation in sediments (Hallberg 2010). These precipi-

tations mainly involve the oxidation and precipitation of iron

and the adsorption of other metals and metalloids by the

resulting ferric minerals. Sulfate-reducing bacteria also have

the ability to reduce sulfate to sulfide, which then reacts with

certain dissolved metals to form insoluble precipitates

(Hallberg 2010). In addition, several bacteria contribute to the

immobilization of arsenic, thanks to their ability to oxidize

this metalloid, arsenate As(V) being less soluble than arsenite

As(III) (Bowell 1994).

The microbiology of AMD streams has been the subject of

numerous studies. While a large amount of information is

available on acidophilic bacteria indigenous to AMD, little is

known about Archaea (Hallberg 2010). Several studies evi-

denced the presence of archaeal communities in acidic

waters (Edwards et al. 2000; Dopson et al. 2004). The

Archaea reported in AMD systems include groups of sulfur

and/or iron-oxidizers, such as Sulfolobus, Acidianus, Met-

allosphaera, Sulfurisphaera, and Ferroplasma (Edwards

et al. 2000; Golyshina et al. 2000; Baker and Banfield 2003).

It has consequently been suggested that Archaea could also

play a major role in the generation and remediation of AMD

via oxidation of iron (Baker and Banfield 2003). Archaea

may also play a role in the biogeochemical cycling of arsenic,

for example, through the presence of Archaea that respire

As(V) like Pyrobaculum aerophilum and Pyrobaculum

arsenaticum (Huber et al. 2000; Oremland and Stolz 2003).

In a previous study, Bruneel et al. (2008) investigated the

archaeal community in water samples from Carnoules, an

AMD very rich in metallic elements and especially arsenic

compared to many others AMD. This study reported the

presence of Ferroplasma acidiphilum and sequences affili-

ated to uncultured Thermoplasmatales archaeon. However,

the archaeal population that inhabits the arsenic-rich Reigous

sediments has never been characterized. Thus, to improve

our understanding of AMD functioning, we characterized the

archaeal communities present in sediment samples from the

arsenic-rich AMD of the Carnoules mine (France) and their

temporal variations using a 16S rRNA encoding gene library.



The Pb-Zn Carnoules mine, located in southern France, was

closed in 1962. The mining extraction left about 1.2 Mt of

solid sulfidic wastes containing 0.7 wt% lead, 10 wt% iron

and 0.2 wt% arsenic, which are stored behind a 6 m high

dam on the uppermost course of Reigous Creek. The

seepage water, which percolates through the wastes,

emerges at the base of the tailings dam, and is the initial

source of Reigous Creek. The water is acidic (2 \ pH \ 3)

and rich in dissolved sulfate, iron and arsenic (2000–7700,

500–1000 and 50–350 mg L-1, respectively) the later being

predominantly in reduced forms: Fe(II) and As(III) (Casiot

et al. 2003). The arsenic concentration decreases within the

first 30 m of the creek mainly due to bacterial iron oxidation

which leads to the coprecipitation of 20–60 % of dissolved

arsenic (Casiot et al. 2003). Although the arsenic level

remains high, its concentration subsequently decreases by

around 95 % between the source of Reigous Creek and its

confluence with the Amous River, 1.5 km downstream. The

precipitates, which form around stromatolitic-like bacterial

structures, are mainly composed of Fe(III)–As(III) in winter

in the first 10 m and of amorphous Fe(III)–As(V) during the

rest of the year (Casiot et al. 2003; Morin et al. 2003). Many

studies (including culture-dependent and independent) have

been conducted on the bacterial communities inhabiting the

Carnoules mine. Two of them focused specifically on sed-

iment. The active bacterial species were identified in the

sediments in the April 2006 library using high sensitivity

nanoLC-chip-MS/MS methods combined with a 16S rRNA

based phylogenetic approach (Bruneel et al. 2011). This

study showed that Gallionella, Thiomonas, Acidithiobacil-

lus ferrooxidans, and Acidiphilium actively expressed pro-

teins in situ. Meta- and proteo-genomics approaches were

also used on sediments in the May 2007 library and allowed

reconstruction of seven bacterial strains (Bertin et al. 2011).

These studies and previous results (Casiot et al. 2003;

Morin et al. 2003) suggest that the large amounts of As(V)–

Fe(III) hydroxysulfate sediments forming at Carnoules may

result from the combined activities of iron-oxidizing

microorganisms (such as At. ferrooxidans, Alicyclobacillus

ferrooxidans, Ferrimicrobium, or Gallionella) and arsenic-

oxidizing microorganisms (such as Thiomonas sp.).

Sampling procedure and measurement

of physicochemical properties

Four sampling campaigns were carried out in April 2006,

October 2008, January 2009 and November 2009. Samples

were collected at the station called COWG (Carnoules

Oxidizing Wetland, point G) located 30 m downstream of

the spring (Bruneel et al. 2003). 5 cm deep pale yellow

loosely packed sediments were collected at the bottom of

the creek using a sterile spatula and pooled [global posi-

tioning system (GPS) coordinates: 44107001.8000N/

4100006.9000E]. This sampling was done in three repli-

cates. Solid phases were harvested by centrifugation and

646 Extremophiles (2012) 16:645–657

123


dried under vacuum before mineralogical and spectro-

scopic analyses. The main physicochemical parameters

(pH, T �C, and dissolved oxygen concentration) of the

running water at the sampling point were measured in the

field. pH and water temperature were measured with an

Ultrameter Model 6P (Myron L 125 Company, Camlab,

Cambridge). Water samples (500 ml) were immediately

filtered through 0.22 lm Millipore membranes fitted on

Sartorius polycarbonate filter holders. For total iron and

arsenic determination, the filtered water was acidified to pH

1 with HNO3 (14.5 M) and stored at 4 �C in polyethylene

bottles until analysis. For arsenic and iron speciation, a

20 ll aliquot of filtered water sample was added to either a

mixture of acetic acid and EDTA (Samanta and Clifford

2005) for arsenic speciation or to a mixture of 0.5 ml

acetate buffer (pH 4.5) and 1 ml of 1,10-phenanthrolinium

chloride solution (Rodier et al. 1996) for Fe(II) determi-

nation. The vials were completed to 10 ml with deionized

water. The samples for iron and arsenic speciation and

sulfate determination were stored in the dark and analyzed

within 24 h. Chemical analysis were carried out as previ-

ously described (Bruneel et al. 2011).

Solid sample characterization

The mineralogical composition of the solid samples col-

lected at COWG was qualitatively determined using powder

X-ray diffraction analysis (XRD). Data were collected with

Co K-alpha radiation on an X’Pert PRO P analytical dif-

fractometer equipped with an X’Celerator detector, in con-

tinuous mode and a counting time of 4 h per sample. X-ray

absorption spectroscopy data were collected on the solid

phases sampled at COWG in October 2008, January 2009,

and November 2009. X-ray absorption near edge structure

(XANES) and extended X-ray absorption fine structure

(EXAFS) spectra were recorded at a temperature 10–15 K

in fluorescence mode on the FAME BM30B bending mag-

net beamline at ESRF (Grenoble, France). Data for the April

2006 COWG sample were previously collected at the 11-2

wiggler beamline at SSRL (Stanford, CA) and analyzed in

Bruneel et al. (2011). Experimental details and data reduc-

tion procedures are reported in previous studies (Morin et al.

2003; Ona-Nguema et al. 2005; Hohmann et al. 2011).

XANES and EXAFS data were interpreted by linear com-

bination fitting using a set of model compound spectra. This

set includes As(V)- and As(III)–Fe(III) oxyhydroxides and

oxyhydroxysulfates synthesized via biotic and abiotic

pathways (Morin et al. 2003; Maillot 2011).

DNA isolation

Triplicate genomic DNA was extracted from sediments

using the UltraClean Soil DNA Isolation Kit according to

the manufacturer’s recommendations (MoBio Laboratories

Inc., Carlsbad, CA, USA). These triplicates were pooled

before PCR amplification. All extracted genomic DNA

samples were stored at -20 �C until further analysis.

PCR amplification

Amplification of archaeal 16S rRNA genes was obtained

by PCR using primers Arch21F (50-TTCCGGTTGATCC

YGCCGGA-30) and Arch958R (50-YCCGGCGTTGAMTC

CAATT-30) (Delong 1992). Two PCR protocols were used

due to major amplification difficulties. The first PCR

amplification mixture contained 2 ll of DNA template,

2 ll of both primers (20 lM), 25 ll of PCR Master Mix

Ampli Taq Gold 360 (Applied Biosystems, Foster City,

CA, USA). Sterile distilled water was added to reach a final

volume of 50 ll. The PCR conditions were as follows, an

initial denaturation step of 95 �C for 7 min followed by 35

denaturation cycles at 95 �C for 1 min, an annealing cycle

at 55 �C for 45 s and an extension cycle at 72 �C for

1 min. Final extension was at 72 �C for 10 min. As

amplification of the January 2009 sample failed with this

protocol, another enzyme was used, the PCR Extender

Polymerase Mix (5Prime, Hamburg, Deutschland) as well

as for a part of the November 2009 sample, which was also

very difficult to amplify. The second PCR amplification

mixture contained 2 ll of DNA template, 2 ll of both

primers (20 lM), 2.5 ll of dNTPs 10 mM, 5 ll reaction

Tunning buffer 910 and 0.5 ll of PCR Extender Poly-

merase Mix (5Prime, Hamburg, Deutschland). Sterile dis-

tilled water was added to reach a final volume of 50 ll. The

PCR conditions were as follows: initial denaturation step at

94 �C for 3 min followed by 35 denaturation cycles at

94 �C for 1 min, an annealing cycle at 55 �C for 1 min,

and an extension cycle at 72 �C for 1.5 min. Final exten-

sion was at 72 �C for 10 min. PCR products were purified

with the GFX PCR DNA purification kit (Amersham-

Pharmacia). The PCR Extender polymerase mix creates

blunt ended products. For TA Cloning�, 30 A-overhangs

are needed on these PCR products, which are obtained with

a different Taq polymerase. To 25 ll of purified PCR

product, 2.5 ll of buffer 109, 0.5 ll of dATPs 10 mM, and

0.5 ll of Taq DNA polymerase (Eurobiotaq�, Eurobio,

France) were added. The PCR amplification mixture was

then incubated at 72 �C for 20 min.

Cloning and 16S rRNA gene sequencing

The PCR products were cloned in E. coli TOP 10 strain

using the pCR2.1 Topo TA cloning kit (Invitrogen, Inc.,

Carlsbad, CA, USA). Cloned 16S rRNA gene fragments

were re-amplified using the primers TOP1 (50-GTGTGCT

GGAATTCGCCCTT-30) and TOP2 (50-TATCTGCAGAA

Extremophiles (2012) 16:645–657 647

123


TTCGCCCTT-30) that anneal to the vector and flank the

inserted PCR fragment. A total of 340 clones from the four

libraries were sequenced. Partial sequences of the clones

were determined by the dideoxy nucleotide chain-termi-

nation method using the BigDye 3.1 kit (Applied Bio-

systems) on an ABI PRISM 3730XL Genetic analyzer

(Applied Biosystems). The MALLARD program (Ashelford

et al. 2006) was used to detect and then remove chimera.

Sequences were also examined manually for chimera, which

were excluded from further analyses. Sequences were then

aligned in Mothur (http://www.mothur.org) (Schloss et al.

2009) using the SILVA archaeal database as reference

alignment. The same program was used to calculate a

neighbor-joining (NJ) (Saitou and Nei 1987) distance

matrix using the Jukes-Cantor (JC) correction. The matrix

was then used to assign sequences to operational taxo-

nomic units (OTUs) defined at 97 (species level) and 85 %

(class level) cutoff using the furthest-neighbor algorithm.

Sequences were compared with the available databases

NCBI and Greengenes (http://greengenes.lbl.gov) by

BLAST online searches (Altschul et al. 1990) and Mothur

to identify their taxonomic identities. Representative

sequences for each OTU defined at 97 % cutoff were

identified using the tool implemented in Mothur and were

submitted to the EMBL databases under accession numbers

(HE653775–HE653816).

Phylogenetic analysis

Archaeal 16S rRNA gene homologs were collected from

the database at NCBI using the BLAST program with

default parameters; one representative of each OTU was

selected, giving a dataset of 99 sequences for final analy-

sis. Multiple sequence alignment of partial prokaryotic

sequences was performed using Clustal W (Thompson

et al. 2000). A maximum likelihood phylogenetic recon-

struction was obtained using the PhyML program (Guindon

and Gascuel 2003) with the GTR model, four evolutionary

rates, a calculated proportion of invariant sites and calcu-

lated nucleotide frequencies (default parameters). Statisti-

cal likelihood at nodes was calculated via a likelihood-ratio

test (Anisimova and Gascuel 2006).

Statistical analysis of diversity and comparison

of archaeal libraries

The Mothur software package was also used to generate

diversity indices and statistics (OTUs, total clones, single-

tons, Chao1, Shannon, evenness, coverage) for each clone

library as sequence similarity with a 97 % cutoff. The total

number of clones obtained compared with the number of

clones representing each unique phylotype was used to

produce the rarefaction curves at the 85 % level. Coverage

values were calculated to determine how efficiently the

libraries described the complexity of a theoretical com-

munity like an original archaeal community. The coverage

(Good 1953) value is given as C = 1 - (n1/N) where n1 is

the number of clones that occurred only once in the library.

To determine the significance of differences between

archaeal libraries, a LIBSHUFF statistical analysis was

performed in Mothur following Singleton et al.’s (2001)

method. A LIBSHUFF comparison of libraries yielded the

following formula using the Bonferroni correction:

0.05 = 1-(1 - a)k(k - 1), where a is the critical P value

and k is the number of libraries. The critical P value is

0.0042 when four libraries are compared. If any comparison

of two libraries has a P value below or equal to 0.0042, then

there is 95 % confidence to believe that the two libraries

concerned differ significantly in community composition.

Jaccard and Yue & Clayton theta tree clustering analysis

(Yue and Clayton 2005) were also performed in Mothur to

identify community membership and structure relationships

between the libraries.

Results

Physical and chemical characteristics of samples

The physicochemical characteristics of the waters are listed

in Table 1. The physicochemistry of Reigous Creek water

at these sampling periods was typical of that observed

during a previous long-term monitoring study (Egal et al.

2010). The water samples were acid (pH = 2.91–3.28)

and very rich in sulfate (1830–3400 mg L-1), iron

(510–1735 mg L-1), and arsenic (70–194 mg L-1), with

predominance of the reduced forms Fe(II) and As(III).

Dissolved oxygen concentrations ranged from 3.5 to

7.86 mg L-1. The January 2009 sample showed the lowest

iron, arsenic, and sulfate concentrations.

The nature and structure of the sediment samples were

investigated using mineralogical and spectroscopic meth-

ods. XANES analyses at the arsenic K-edge showed that,

despite the presence of an As(III) component equal to

12–34 ± 5 % of total arsenic, the oxidized arsenic form

As(V) predominated in all the sediments (Fig. S1). EXAFS

data (Fig. S2) showed that As(V) was mainly present in the

samples in an amorphous Fe(III)–As(V) hydroxysulfate

phase, as previously observed (Morin et al. 2003; Bruneel

et al. 2011), As(III) being likely sorbed to poorly ordered

schwertmannite. For January 2009, there was not enough

time exposure to X-ray beam in EXAFS analysis to record

this sample, however, based on the XANES data we can

assume that this sample should be similar to the others.

XRD analyses (Fig. S3) showed that these arsenic-bearing

phases were mixed with sandy components (quartz,

648 Extremophiles (2012) 16:645–657

123


http://www.mothur.org

http://greengenes.lbl.gov

K-feldspar and micas) and that pyrite was only detected

after October 2008.

Diversity analysis

A total of 340 clones obtained from the four independent

16S rRNA gene libraries were fully sequenced and phy-

logenetically analyzed. Thirteen sequences were identified

as likely chimeras and excluded from further analyses.

Sequencing and phylogenetic analysis of the 327 remaining

cloned sequences led to the identification of 9 and 42 OTUs

defined at two different levels of identity (85 and 97 %,

respectively). Rarefaction curves calculated at the class

level (85 % identity, the rank usually used for representing

the microbial community) were near saturation (Fig. 1).

Table 2 shows the Shannon, evenness, and Chao1 indices

and the coverage values calculated for each library at 97 %

identity. The coverage values of the four clone libraries

(90, 88, 96 and 92, respectively, for April 2006, October

2008, January 2009, and November 2009) indicate that the

clone libraries were sufficiently sampled. The estimations

of the diversity indices show that the structure and mem-

bership composition of the archaeal community changed

over the sampling period. The Shannon diversity (H) and

Chao1 richness indices ranged from 1.37 to 2.57 and 6.5 to

30.5, respectively. The diversity (H = 1.37) and richness

(Chao1 = 6.5) were significantly lower in January 2009

whereas November 2009 displayed the highest values

(H = 2.57; Chao1 = 30.5), which is consistent with the

rarefaction curves.

Comparison of archaeal community

The overall community structure was analyzed for each

sample using the Mothur software package. LIBSHUFF

analysis was performed to compare the OTU compositions

of each clone library revealing a high degree of variation

between individuals and showing that with Bonferroni

correction, each library differed significantly from all

others (Table 3). The resulting dendrograms of Jaccard and

Yue & Clayton theta similarity coefficient analysis (Fig. 2)

identified one major cluster and one outlier (January 2009).

The similarity in community membership (Jaccard index,

Fig. 2a) showed that April 2006 and November 2009 were

more related to each other in this respect, whereas April

2006 and October 2008 were more related to each other in

terms of community structure (Yue & Clayton index,

Fig. 2b).

Phylogenetic analysis of archaeal community

Four 16S rRNA encoding gene libraries were constructed

each containing a distinct archaeal community, which

varied in composition and richness throughout the sam-

pling campaigns.

In April 2006, the 16S rRNA phylogenetic reconstruc-

tion (Fig. 3) showed that all the sequences corresponding

to 17 OTUs (OTUs 1–17) were affiliated to the phylum

Euryarchaeota, as previously observed in the water samples

from Carnoules (Bruneel et al. 2008). The most abundant

OTU (OTU 1, 53 clones representing around 61 % of the

sample) was affiliated to the order Thermoplasmatales

which contained 97 % of the sequences grouped in 15

OTUs. Within this order, the majority of the OTUs were

closely related to uncultured clones from an acidic envi-

ronment such as acidic mine water and sediments (Fig. 3).

Table 1 Physico-chemical characteristics of the water (mg L-1) during sampling at COWG

Sampling period pH (±SD) T (�C) DO (±SD) As(III) (±SD) As(V) (±SD) Fe (total) (±SD) Fe(II) (±SD) SO42- (±SD)

April 2006 3.28 (±0.05) 12.9 3.5 (±0.5) 69 (±3) 71 (±4) 620 (±30) 560 (±28) 2700 (±300)

October 2008 3.13 (±0.05) 14.3 5.7 (±0.1) 133 (±7) 20 (±1) 1250 (±62) 1220 (±61) 3400 (±340)

January 2009 2.91 (±0.05) 9.4 5.5 (±0.5) 43 (±2) 27 (±1) 510 (±25) 540 (±27) 1830 (±183)

November 2009 3.26 (±0.05) 13.1 7.9 (±0.1) 161 (±8) 33 (±2) 1735 (±87) 1440 (±72) 3300 (±330)

SD standard deviation

Fig. 1 Rarefaction curves of the archaeal 16S rRNA sequences from

Carnoules mine sediments at 85 % identity. The total number of

sequenced clones is plotted against the number of OTUs observed in

the same library

Extremophiles (2012) 16:645–657 649

123


The BLAST affiliation (Table 4) showed that some of

these OTUs displayed 89–94 % similarity with Thermo-

gymnomonas acidicola, a moderately thermophilic, acido-

philic, strictly aerobic heterotroph that uses yeast extract,

as well as glucose and mannose (in the presence of yeast

extract) as carbon and energy sources (Itoh et al. 2007).

Additionally, OTU 15 related to the uncultured clone

ORCMO 26 retrieved from a copper mine drainage (Rowe

et al. 2007, Fig. 3) was found. This OTU was assigned

to methanogenic lineage (Methanomicrobia, Fig. 3) with

the closest relative Methanomassiliicoccus luminyensis, a

methanogenic Archaea recently isolated from human

faeces (Dridi et al. 2011). However, the Greengenes clas-

sification (Table 4) assigned this OTU to the order Ther-

moplasmatales. Lastly, an unknown group belonging

to the Euryarchaeota and represented by OTU 8 was

detected. This group formed an independent branch that

was distantly related to the identified groups and showed

low similarity with the uncultured archaeon clone hfm29

isolated from an iron-rich microbial mat (Kato et al. in

press).

Twenty OTUs were retrieved from the October 2008

library, 18 of which belonged to the Euryarchaeota and two

to the Thaumarchaeota (Fig. 3). Like in April 2006, most

of the Euryarchaeota sequences were affiliated with the

Thermoplasmatales (OTUs 1, 6, 9, 11, 12, 17, 19, 20,

22–27, and 31) which accounted for 86 % of the total

archaeal clones including the same most abundant OTU

(OTU1; around 54 %, Fig. 3). The BLAST affiliation

(Table 4) also revealed similarity of some OTUs with

Thermogymnomonas acidicola. Three OTUs (18, 21, and

28) affiliated with uncultured clones isolated from acidic

environments (clone SALE1B1 and clone anta6) and from

a forested wetland impacted by reject coal (clone ARCP2-

12) (Brofft et al. 2002; Garcıa-Moyano et al. 2007, Fig. 3),

respectively, were assigned to Methanomicrobia. The

remaining OTUs (OTUs 29 and 30) were affiliated with

environmental sequences originating from acidic soil and

acidic hot springs, which likely represent uncultured lin-

eages of Thaumarchaeota.

A significant change in the archaeal community

appeared in the January 2009 library, when diversity

decreased and no cultured species were identified. Indeed,

almost all the sequences (96 %) clustered in five OTUs

(OTUs 8, 32, 33, 34, and 35, Fig. 3) were related to the

uncultured archaeon clone hfmA029 previously found in

April 2006. This group formed an independent branch that

was far away from the remaining groups. This clone dis-

played around 97 % similarity with Methanothermobacter

thermautotrophicus, an autotrophic thermophilic methan-

ogen recovered from an anaerobic sewage sludge digester

(Zeikus and Wolee 1972). Remaining sequences grouped

Table 2 Diversity indices and statistics calculated for the four clone libraries from COWG station at different sampling periods

Clone library No. of sequences No. of OTUsa Singletons Good’s coverageb Shannon diversityc Chao1 richness

April 2006 87 17 9 90 1.63 24.2

October 2008 80 20 10 88 1.96 27.5

January 2009 47 6 2 96 1.37 6.5

November 2009 113 25 9 92 2.57 30.5

a OTUs were defined at 97 % cutoffb Coverage: sum of probabilities of observed classes calculated as (1 - (n/N)), where n is the number of singleton sequences and N is the total

number of sequencesc Takes into account the number and evenness of species

Table 3 Community comparison using LIBSHUFF

Y library

Apr-06 Oct-08 Jan-09 Nov-09

X library

Apr-06 – 0.0260 \0.0001* 0.1529

Oct-08 0.0001* – \0.0001* 0.0001*

Jan-09 \0.0001* \0.0001* – 0.2222

Nov-09 \0.0001* \0.0001* \0.0001* –

* Significant difference. Bonferroni correction P value = 0.0042

– Not compared

Fig. 2 Similarities in archaeal community membership (Jaccard a)

and in community structure (Yue & Clayton b) between samples.

Values are based on 0.03 distances

650 Extremophiles (2012) 16:645–657

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Fig. 3 Maximum likelihood tree of 16S rRNA gene homologs from

the archaeal clones (in bold) along with a selection of representatives

of archaeal diversity. Numbers at nodes indicate a LTR (approximate

likelihood ratio test) branch support as computed by PhyML. The

scale bar gives the average number of substitutions per site. The

number in parenthesis indicates the number of clones for the sampling

period which is represented by a symbol (star April 2006, squareOctober 2008, circle January 2009 and diamond November 2009)

Extremophiles (2012) 16:645–657 651

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Table 4 Identification number of the OTUs retrieved from the Reigous Creek sediment of Carnoules mine, taxonomic affiliation and repre-

sentative sequence for each OTU

OTU

ID

Number of

sequences

Representative

sequence

Taxonomic affiliation Closest relative (% of identity)

Phylum Class Order

1 132 ArCMSdO8D35 Euryarchaeota Thermoplasmata Thermoplasmatales Aciduliprofundum sp. EPR07-39

(85 %)

2 15 ArCMSdA6A12 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (89 %)

3 2 ArCMSdA6A86 Euryarchaeota Thermoplasmata Thermoplasmatales Aciduliprofundum sp. EPR07-39

(85 %)


13583 (90 %)


13583 (91 %)


13583 (92 %)

7 3 ArCoSdN9H63 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (93 %)

8 24 ArCMSdJ9B78 Euryarchaeota – – Clone hfmA029 (86 %)


13583 (88 %)

10 3 ArCoSdN9D80 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (94 %)

11 7 ArCMSdO8B50 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (94 %)

12 4 ArCMSdO8B53 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (90 %)

13 1 ArCMSdA6A52 Euryarchaeota Thermoplasmata Thermoplasmatales Thermoplasma volcanium (84 %)


13583 (90 %)

15 3 ArCoSdN9H35 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (80 %)


13583 (91 %)


13583 (88 %)

18 5 ArCMSdO8A3 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (82 %)

19 1 ArCMSdO8A13 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (89 %)


13583 (87 %)

21 2 ArCoSdN9H67 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (83 %)


13583 (88 %)


13583 (89 %)

24 4 ArCMSdO8A49 Euryarchaeota Thermoplasmata Thermoplasmatales Thermoplasma volcanium GSS1

(89 %)


13583 (90 %)


13583 (99 %)


13583 (93 %)

652 Extremophiles (2012) 16:645–657

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in a single OTU (OTU 29) belonged to an unknown group

of Thaumarchaeota previously found in October 2008 and

were affiliated with clone GBX-A-COQ1-158 isolated

from an acidic hot spring (Fig. 3).

An increase in archaeal diversity was observed in the

November 2009 library, with 25 OTUs belonging to

the Euryarchaeota (69 clones corresponding to 61 % of the

sample) and to the Thaumarchaeota (22 clones corre-

sponding to 19 % of the sample, Fig. 3). The sequences

from the Euryarchaeota were distributed in 22 OTUs.

Fifteen were related to the order Thermoplasmatales, 11 of

which (OTUs 1, 2, 4, 6, 7, 9, 10, 11, 17, 24, 26) were

previously found in the April 2006 and October 2008

libraries (Fig. 3). As in the results observed in these two

sampling periods, OTU 1 was also the most abundant

group in the sample in November 2009 (32 %). Addi-

tionally, OTUs 15 and 21, also found in the two first

libraries, were assigned to the order Methanomicrobia.

The remaining five OTUs (8, 32, 33, 35, and 38), were not

shown to be related to any known species and formed

unknown groups of the Euryarchaeota (Fig. 3). Among

these, the most abundant sequences belonging to OTU 38,

displayed a strong similarity (99 %) with an uncultured

archaeon clone LC15_L00B08 isolated from the monim-

olimnion of a stratified lake (Gregersen et al. 2009). The

four other OTUs (8, 32, 33, and 35) were affiliated with

the uncultured archaeon clones hfmA029 mainly present

in the January 2009 library. The Thaumarchaeota detected

in this study fell into different lineages clustered in three

OTUs. The first (OTU 36) belonged to Thaumarchaeota

group I.1b and the 15 sequences within this OTU showed

from 95 to 96 % similarity with Candidatus Nitrososph-

aera viennensis a chemolithoautotrophic ammonia-oxi-

dizing archaeon (Tourna et al. 2011). The second (OTU

37) was assigned to Thaumarchaeota group I.1a and the

sequences displayed 92–93 % similarity with Candidatus

Nitrosopumilus sp., another ammonia-oxidizing prokaryote

(Matsutani et al. 2011). The last OTU (OTU 29), previ-

ously found in October 2008 could not be related to any

known species.

Table 4 continued

OTU

ID

Number of

sequences

Representative

sequence

Taxonomic affiliation Closest relative (% of identity)

Phylum Class Order

28 1 ArCMSdO8A89 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (85 %)

29 5 ArCMSdJ9A29 – – – Candidatus Nitrosocaldusyellowstonii HL72 (84 %)

30 2 ArCMSdO8C25 – – – Candidatus Nitrososphaeragargensis (83 %)

31 1 ArCMSdO8E23 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (92 %)

32 15 ArCMSdJ9C75 Euryarchaeota – – Clone SVB_Fis_02_pl37c09 (86 %)

33 16 ArCMSdJ9C68 Euryarchaeota – – Clone hfmA029 (85 %)

34 1 ArCMSdJ9C55 Euryarchaeota Methanomicrobia Methanomicrobiales Clone hfmA029 (84 %)

35 2 ArCoSdN9A45 Euryarchaeota – – Clone hfmA029 (85 %)

36 15 ArCoSdN9B9 Thaumarchaeota No class Nitrososphaerales Candidatus Nitrososphaera sp.

EN76 (96 %)

37 6 ArCoSdN9F14 Thaumarchaeota No class Cenarchaeales Candidatus Nitrosopumilus sp.

NM25 (93 %)

38 11 ArCoSdN9D53 Euryarchaeota – – Clone TG_FD0.2_SA043 (100 %)


13583 (90 %)

40 1 ArCoSdN9E14 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (88 %)

41 1 ArCoSdN9G7 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM

13583 (93 %)


13583 (92 %)

OTU definition and taxonomic identification of representative sequences were done using mothur (Schloss et al. 2009; see ‘‘Materials and

methods’’ for details). Only taxonomic affiliations with 100 % similarity are shown. The closest relative was obtained by BLAST search on

NCBI nr database

Extremophiles (2012) 16:645–657 653

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Discussion

Archaeal 16S rRNA gene analysis of the sediment sampled

at the Reigous Creek showed that the Carnoules archaeal

community includes the phylum Euryarchaeota and

Thaumarchaeota. The relatively low archaeal diversity

revealed by molecular-based methods is consistent with the

results of studies in similar environments (Bond et al. 2000;

Baker and Banfield 2003; Bruneel et al. 2008; Sanchez-

Andrea et al. 2011). This may reflect the limited number of

different electron donors and acceptors available in this

AMD and the high concentration of toxic compounds along

with the low pH. Most of the phylotypes identified in this

study were related to genera and species usually found in

extreme environments (hot springs, acidic springs, hydro-

thermal vents, etc.) and showed similarities with sequences

obtained in previous studies of Tinto River and other AMD

(Sanchez-Andrea et al. 2011; Garcıa-Moyano et al. 2007;

Rowe et al. 2007, Fig. 3).

Regarding the dynamics of the archaeal community, our

study showed that significant modifications in this com-

munity occurred throughout the sampling period. All the

sampling periods showed differences in community struc-

ture and membership although April 2006 and October

2008 were more similar in terms of community structure.

Similarity coefficient analysis showed that January 2009

was very different from all the other sampling periods.

In January 2009, the archaeal community changed and

diversity decreased. Almost all the sequences were related

to an uncultured archaeon clone hfmA029 affiliated with

methanogenic lineage (Methanothermobacter thermautot-

rophicus). This clone, hfmA029, previously found in April

2006 (OTU 8) in only 2 % of the sample became the

dominant population in January 2009. The differences in

the archaeal community observed in January 2009 may

result from a modification in the composition of the sedi-

ment, although the physicochemical analysis of the sedi-

ments appeared to be similar throughout the sampling

period, and consisted mainly of an amorphous Fe(III)–

As(V) hydroxysulfate mineral. Indeed, XRD analyses

revealed that pyrite first appeared in October 2008. Like-

wise, since late 2007, a leakage of fine grey sulfide-reach

sands out of the tailings pile has been observed after the

rainfall events that generally occur in September and

October. This is probably due to the corrosion of the drains

at the bottom of the tailing stock that are responsible for the

water discharge inside the mine tailing. In January 2009,

the sulfide sands, originated from the tailings stock, formed

a very thick layer (around 3 cm deep) in the bottom of the

creek which could explain the change in the archaeal

community.

In the Reigous sediment, most of the sequences were

phylogenetically related to the order Thermoplasmatales,

although none of the clones could be identified with high

similarity ([97 %) as belonging to any cultured species.

This order is represented by thermoacidophilic organisms

(Reysenbach 2001), which often derive energy from sulfur

oxidation or reduction. So far, the order contains three

families, each represented by one genus: the Thermoplas-

mataecae, the Picrophilaceae and the Ferroplasmaceae

(Itoh et al. 2007). The Thermoplasmataecae comprises

species like Tp. acidophilum that couple the oxidation of

organic carbon with reduction of elemental sulfur, whereas

members of the Ferroplasmaceae are strict iron-oxidizing

chemolithotrophs such as Ferroplasma acidiphilum (Itoh

et al. 2007). Microorganisms affiliated with methanogenic

Archaea such as Methanomassiliicoccus luminyensis were

also identified. Methanogenic communities play an

important role in the global carbon cycle, completing the

conversion of organic carbon into methane gas by utilizing

the metabolic products of bacteria (CO2, H2, acetate, and

formate) and other simple methyl compounds available in

the environment (Sanz et al. 2011). Lastly, we found

microorganisms involved in ammonia oxidation, a key step

in the nitrogen cycle (Brochier-Armanet et al. 2011), with

presence of sequences affiliated to Candidatus Nitro-

sosphaera viennensis and Candidatus Nitrosopumilus sp.

Until recently, ammonia oxidation, the first nitrification

step of the nitrogen cycle was thought to be carried out

only by autotrophic ammonia-oxidizing bacteria (AOB)

belonging to the Beta- and Gammaproteobacteria lineages

(Purkhold et al. 2000) occasionally supported by hetero-

trophic nitrifiers in soil environments (De Boer and

Kowalchuk 2001). Ammonia-oxidizing Archaea (AOA) are

members of the proposed novel Phylum Thaumarchaeota,

and are currently being indentified in almost all environ-

ments (Brochier-Armanet et al. 2008). These Archaea may

thus play a major role in the nitrogen cycle in the Carnoules

sediments.

Previous studies focused on the bacterial communities

inhabiting the Carnoules AMD sediment. These studies

showed that the active population of bacteria also con-

tained iron reducers, sulfate-reducing, and sulfur com-

pound oxidizers, and both autotrophic and heterotrophic

bacteria (Bruneel et al. 2011). Statistical analysis of

genomic and proteomic data demonstrated that both met-

abolic specificity and partnerships can co-exist in this

arsenic-rich sediment (Bertin et al. 2011). These processes

include the fixation of inorganic carbon and nitrogen by

several strains, in particular those belonging to the Thio-

monas, Acidithiobacillus, and Gallionella related genera.

However, this study did not find evidence for the presence

of archaeal species among the dominant organisms, sug-

gesting that they may represent a small proportion of the

microbial community in the sediment. Despite the fact that

we cannot really infer the implication of the Archaea

654 Extremophiles (2012) 16:645–657

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detected in most of these metabolic pathways because

most of them could not be affiliated to cultured species,

we can point to their probable implication in a specific

metabolism currently unknown in bacteria (Forterre et al.

2002), methanogenesis. Archaea are also involved in the

nitrogen cycle (Candidatus Nitrososphaera viennensis and

Candidatus Nitrosopumilus sp.) and some of them may

also be involved in the sulfur and iron cycles (Ther-

moplasmatales). All these microorganisms may contribute

to the remediation process observed in situ and could also

be involved in the stability of this sediment by changing

the ratio between oxidized and reduced forms of iron,

arsenic, and sulfur compounds, promoting the formation

and/or dissolution of the Fe(III)–As(V) hydroxysulfate

precipitates.

Because isolation and phenotypic characterization of

many environmental Archaea are currently not possible,

the physiological features and ecological significance of

some Archaea detected in this AMD remain difficult to

assess. Moreover, the fact that most of the archaeal

sequences were only distantly related (\94 % similarity) to

known archaeal species suggests that other taxa may exist.

Additionally, the contradictions observed in the taxonomic

affiliation resulting from the 16S rRNA phylogenetic

reconstruction (Fig. 3) and the Greengenes classification

(Table 4) suggest that there is still a lack of information

making the taxonomic identification difficult to assess.

Indeed, almost half of the 16S rRNA gene sequences

archived in GenBank database lacks clear taxonomic

information (DeSantis et al. 2006). As a consequence,

different authors use different names for uncultured clus-

ters which lead to conflicting nomenclatures. Recently

developed high-throughput techniques (metagenomics,

metaproteomics, and microarrays) may help link the

identity of AMD-promoting prokaryotes to their function in

mining environments (Mohapatra et al. 2011; Bertin et al.

2011) in the absence of laboratory culture. In the future,

these new genomic tools should provide a more precise

assessment of the archaeal diversity that will probably lead

to substantial changes in current archaeal phylogeny and

taxonomy (Brochier-Armanet et al. 2008; Schleper et al.

2005) and to a better understanding of the evolution and

metabolic capacities of uncultured Archaea. In conclusion,

to increase our insight into the functioning of these highly

acidic environments and to elucidate the role of these

microorganisms, both improving culture strategies for

further physiological and metabolic characterization of

newly detected species and a greater sequencing effort are

still needed.

Acknowledgments The French CRG is gratefully acknowledged

for provision of beamtime on the FAME BM30B beamline. This work

was supported by EC2CO CNRS/INSU program, by ACI/FNS Grant

#3033 and by SESAME IdF Grant #1775. Part of the field chemical

data was acquired through the OSU OREME.

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R E S EA RCH AR T I C L E

Diversity and spatiotemporal dynamics of bacterialcommunities: physicochemical and other drivers along an acid

mine drainage

Aur�elie Volant1, Odile Bruneel1, Ang�elique Desoeuvre1, Marina H�ery1, Corinne Casiot1, No€elle Bru2,Sophie Delpoux1, Anne Fahy3, Fabien Javerliat3, Olivier Bouchez4, Robert Duran3, Philippe N.Bertin5, Franc�oise Elbaz-Poulichet1 & B�eatrice Lauga3

1Laboratoire HydroSciences Montpellier, HSM, UMR 5569 (IRD, CNRS, Universit�es Montpellier 1 et 2), Universit�e Montpellier 2, Montpellier,

France; 2Laboratoire de Math�ematiques et de leurs Applications, UMR 5142 (CNRS), Universit�e de Pau et des Pays de l’Adour, Pau, France;3�Equipe Environnement et Microbiologie, EEM, UMR 5254 (IPREM, CNRS), Universit�e de Pau et des Pays de l’Adour, Pau, France; 4INRA Auzeville,

Plateforme G�enomique Chemin de Borde Rouge, Castanet-Tolosan, France; and 5D�epartement Microorganismes, G�enomes, Environnement,

Laboratoire de G�en�etique Mol�eculaire, G�enomique, Microbiologie, GMGM, UMR 7156 (Universit�e de Strasbourg, CNRS), Strasbourg, France

Correspondence: Odile Bruneel, Laboratoire

HydroSciences Montpellier, UMR5569,

Universit�e Montpellier 2, Place E. Bataillon,

CC MSE, 34095 Montpellier, France.

Tel.: (+33)4 67 14 36 59;

fax: (+33)4 67 14 47 74;


Received 16 April 2014; revised 10 July

2014; accepted 16 July 2014.

DOI: 10.1111/1574-6941.12394

Editor: Tillmann Lueders

Keywords

spatial and temporal dynamics; bacterial

diversity; acid mine drainage; arsenic.

Abstract

Deciphering the biotic and abiotic factors that control microbial community

structure over time and along an environmental gradient is a pivotal question

in microbial ecology. Carnoul�es mine (France), which is characterized by acid

waters and very high concentrations of arsenic, iron, and sulfate, provides an

excellent opportunity to study these factors along the pollution gradient of

Reigous Creek. To this end, biodiversity and spatiotemporal distribution of

bacterial communities were characterized using T-RFLP fingerprinting and

high-throughput sequencing. Patterns of spatial and temporal variations in bac-

terial community composition linked to changes in the physicochemical condi-

tions suggested that species-sorting processes were at work in the acid mine

drainage. Arsenic, temperature, and sulfate appeared to be the most important

factors that drove the composition of bacterial communities along this contin-

uum. Time series investigation along the pollution gradient also highlighted

habitat specialization for some major members of the community (Acidithioba-

cillus and Thiomonas), dispersal for Acidithiobacillus, and evidence of extinction/

re-thriving processes for Gallionella. Finally, pyrosequencing revealed a broader

phylogenetic range of taxa than previous clone library-based diversity. Overall,

our findings suggest that in addition to environmental filtering processes, addi-

tional forces (dispersal, birth/death events) could operate in AMD community.

Introduction

Acid mine drainage (AMD) is one of the most pernicious

forms of pollution in the world and is widely recognized

as having costly environmental and socioeconomic

impacts (Hallberg, 2010). AMD occurs when waste from

the extraction and processing of sulfide ore comes into

contact with oxygenated water. Drainages are typically

acidic and usually contain high concentrations of sulfate,

metals and metalloids including arsenic. Although per-

ceived as extreme environments hostile to life, a variety

of microorganisms are able to thrive in it. For some of

them, their role in the oxidation of sulfide minerals,

which leads to bioleaching, is well known, as is their role

in natural attenuation of such polluted waters (Edwards

et al., 2000; Hallberg, 2010; Johnson, 2012). Despite the

central role of microorganisms in such ecosystem func-

tioning, our understanding of the mechanisms shaping

microbial community structure and diversity in AMD

remains limited. As pointed out by Miller et al. (2009),

deciphering how microbial communities are patterned

along environmental gradients is a pivotal question in

microbial ecology. Although AMD is characterized by

changing conditions over time and space, few studies

were interested in comparing the microbial communities

along such environmental gradients. The AMD of

FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

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EC

OLO

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Carnoul�es in southern France provides an excellent

opportunity to investigate these fundamental questions of

microbial ecology. This site is characterized by an acid

pH (2–4) and high levels of metal and metalloids, in par-

ticular As (up to 10 g L�1 in the tailings stock pore

water, and 100–350 mg L�1 at the source of Reigous

Creek), and natural attenuation processes result in a

strong spatial pollution gradient along the drainage.

Indeed, nearly 95% of the arsenic in solution is removed

between the source of Reigous Creek, which emerges

from the mine tailings, and its confluence with the

Amous River, 1.5 km downstream. To our knowledge,

this AMD is one of the most As-rich AMDs reported to

date (Morin & Calas, 2006). It is an outstanding example

of adaptation to life in an extreme environment.

In this study, we used a combination of molecular

approaches to investigate the spatial and temporal dynam-

ics of bacterial communities in relation to the physico-

chemical parameters in Carnoul�es acid mine drainage.

Using 16S rRNA gene pyrosequencing and terminal

restriction fragment length polymorphism (T-RFLP), our

aim was (1) to characterize the spatial dynamics of the

structure and composition of the bacterial communities

along an environmental gradient, (2) to evaluate the tem-

poral changes in the composition of the communities,

and (3) to determine whether their dynamics could be

linked to variations of environmental conditions.



The Pb–Zn Carnoul�es mine, located in southern France,

produced 1.2 Mt of solid wastes that are stored behind a

dam and contain 0.7% Pb, 10% Fe, and 0.2% As. The

aquifer is not fed by vertical percolation of rainwater

through the tailings, but rather originates from natural

springs that were buried under the tailings (Koffi et al.,

2003). The water table is 1–10 m below the surface of the

tailings stock, depending on the season and location.

With the exception of temperature, which is almost con-

stant with average values around 15 °C, the physicochem-

ical parameters of the groundwater vary as a function of

the hydrological conditions (Casiot et al., 2003b). In

2001, the groundwater below the tailings contained extre-

mely high levels of arsenic: up to 10 000 mg L�1 (Casiot

et al., 2003b). The water emerges at the bottom of the

dam, forming the source of the Reigous Creek. This

AMD is acid (pH ≤ 3), with high concentrations of

sulfate (2000–7700 mg L�1), iron (500–1000 mg L�1),

and arsenic (50–350 mg L�1). Iron and arsenic are

mainly present in their reduced forms Fe(II) and As(III)

(Casiot et al., 2003a). The natural attenuation of As is the

result of microbiologically mediated As–Fe coprecipitation(Morin et al., 2003; Bruneel et al., 2006). 10–47% of Fe,

and 20–60% of As are removed from the aqueous phase

within the first 30 m of the creek. Beyond this point

(COWG sampling site, located 30 m downstream from

the spring, Fig. 1), the Reigous receives water from quar-

ries and mine galleries, especially after rainfall events,

which strongly influence its acidity and metal content

(Egal et al., 2010).

Sampling procedure and measurement of

physicochemical properties

Six sampling campaigns were carried out in November

2007, February 2008, October 2008, March 2009, Novem-

ber 2009, and January 2010 at five sampling sites, result-

ing in a set of 30 samples. Groundwater was collected

from a borehole (S5, between 10 and 12 m deep) located

within the tailings. Water samples were also taken at four

sites along Reigous Creek (collecting downstream seepage

water from the surroundings) at the spring (S1), 30 m

downstream from the spring (COWG), 150 m down-

stream (GAL), and 1500 m downstream (CONF), just

before the confluence between Reigous Creek and the

Amous River (Fig. 1). Water samples (300 mL) were

immediately filtered through sterile 0.22 lm Nuclepore

filters, which were transferred to a collection tube

(Nunc), frozen in liquid nitrogen, and stored at �80 °Cuntil DNA extraction. This sampling was carried out in

triplicate. Measurements of water conductivity, tempera-

ture, redox potential, pH, and dissolved oxygen concen-

tration were carried out as previously described (Bruneel

et al., 2011). For chemical analyses, 500 mL water sam-

ples were immediately filtered through 0.22 lm Millipore

membranes fitted on Sartorius polycarbonate filter hold-

ers. For total Fe and As determination, the filtered water

was acidified to pH 1 with HNO3 (14.5 M) and stored at

4 °C in polyethylene bottles until analysis. A 20 lL ali-

quot of the filtered water sample was added either to a

mixture of acetic acid and EDTA (Samanta & Clifford,

2005) for As speciation or to a mixture of 0.5 mL acetate

buffer (pH 4.5) and 1 mL of 1,10-phenanthrolinium

chloride solution (Rodier et al., 1996) for Fe(II) determi-

nation. The vials were filled to 10 mL with deionized

water. Samples destined for Fe and As speciation and sul-

fate determination were stored in the dark and analyzed

within 24 h. Chemical analyses were carried out as previ-

ously described (Bruneel et al., 2011).

DNA isolation

Genomic DNA was extracted in triplicate from filtered

water using the UltraClean Soil DNA Isolation Kit

FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 A. Volant et al.

(MoBio Laboratories Inc., Carlsbad, CA) according to

the manufacturer’s recommendations. These triplicate

extractions were pooled before PCR amplification. All

genomic DNA extracts were stored at �20 °C until further

analysis.

Terminal restriction fragment length

polymorphism

The 16S rRNA genes were amplified by PCR, and the

bacterial community structure was identified by T-RFLP.

The fluorescent labeled primers HEX 357F (50-hexa-

chloro-fluorescein-phosphoramidite-CCTACGGGAGGCA

GCAG-30) (Lane, 1991) and 926R (50-CCGTCAATTCMT

TTRAGT-30) (Muyzer & Ramsing, 1995), described as

universal within the bacterial domain, were used. Tripli-

cate PCR amplifications were performed on each sample.

The reaction mixture contained 1 lL of DNA template,

1 lL of both primers (10 lM), and 12.5 lL of PCR Mas-

ter Mix Ampli Taq Gold 360 (Applied Biosystems, Foster

City, CA). Sterile distilled water was added to obtain a

final volume of 25 lL. PCR conditions were as follows:

one cycle at 95 °C for 10 min, 35 cycles at 95 °C for

45 s, 55 °C for 45 s, and 72 °C for 45 s, followed by

10 min at 72 °C. The 90 PCR products were purified

with Illustra GFXTM PCR DNA and the Gel Band Purifica-

tion Kit (GE Healthcare, Munich, Germany). The concen-

tration of PCR product was determined by comparison

with molecular markers (Smartlader, Eurogentec) after

migration on agarose gel. Approximately 100 ng of puri-

fied amplicon was digested in 10 lL reaction with 0.3 U

of enzyme HpaII or AluI (New England Biolabs Inc., Ips-

wich, MA) at 37 °C for 3 h. Terminal restriction frag-

ment (T-RF) profiles were obtained from the digested

amplicons by suspending 1 lL aliquots in 8.75 lL form-

amide with 0.25 lL of Genescan ROX 500 size standard

(Applied Biosystems). T-RFs were separated on an ABI

PRISM 3130xl Genetic Analyser (Applied Biosystems).

Data were collected and analyzed using GENEMAPPER soft-

ware (version 1.4, Applied Biosystem). To increase strin-

gency for the T-RF profiles of 16S rRNA genes, T-RFs

outside the range of the size standard (35–500 bp) were

discarded, and the background noise level was set at 30

fluorescence units. T-ALIGN software (Smith et al., 2005)

was used to compare replicate profiles and to generate

consensus profiles containing only T-RFs that occurred in

replicate reactions. Consensus profiles were then aligned

on the basis of the length of the T-RFs and individual peak

areas as previously described by Smith et al. (2005) with

the confidence interval set at 0.5, resulting in the genera-

tion of data sets of aligned T-RFs that gave individual rela-

tive peak areas as a percentage of the overall profile. T-RFs

were included in the subsequent analysis if they represented

> 1% of the cumulative peak height for the sample.

Construction of the libraries, 454-

pyrosequencing, and sequence quality control

The 16S rRNA genes were also amplified by PCR for

multiplex pyrosequencing using barcoded primers. The

primer pairs used, targeting the V3 to V5 variable regions

of the 16S rRNA gene, were 357F (50-AxxxCCTA

CGGGAGGCAGCAG-30) and 926R (50-BxxxCCGTCAAT

TCMTTTRAGT-30). A and B represent the two FLX Tita-

nium adapters (A adapter sequence: 50-CGTATCGCCTC

CCTCGCGCCATCAG-30; B adapter sequence: 50-CTAT

Fig. 1. Map of Carnoul�es mine and location of sampling sites.


Spatiotemporal dynamics of bacterial communities 3

GCGCCTTGCCAGCCCGCTCAG-30), and xxx represent

the sample-specific barcode sequence. PCR was performed

using 30–35 cycles under conditions identical to those

described above for T-RFLP. The number of cycles was

varied with the samples to obtain a strong band with a

minimum number of cycles to respect the initial abun-

dances of bacterial communities. The 90 PCR products

with a proximal length of 569 bp were excised from 1%

agarose gel and purified with the QIAquick Gel Extrac-

tion Kit (QIAGEN Inc., Valencia, CA). To minimize ran-

dom PCR bias, triplicates were pooled in equimolar

ratios prior to pyrosequencing. Pyrosequencing of the 30

amplicon libraries was performed on a GS-FLX-Titanium

sequencer (Roche 454 Life Sciences) at the GenoToul

genomic platform in Toulouse (France) using four sepa-

rate 1/8 region of a plate.

Processing of pyrosequencing data and

taxonomic classification

Preliminary quality checks, sorting, and trimming of the

454 reads were carried out using the NG6 pipeline

(http://vm-bioinfo.toulouse.inra.fr/ng6/). Tags were

extracted from the 454 reads using the sff file (Roche

software), and three kinds of analysis were performed as

described by Ueno et al. (2010): (1) BLAST search for

E. coli, phage, and yeast contaminants, (2) read quality

analysis, and (3) removal of sequences that were too long

or too short (sequences with more or less than two stan-

dard deviations from the mean), sequences containing

more than 4% of N, low-complexity sequences and

duplicated reads, using Pyrocleaner. The sequences were

then analyzed with the software Mothur version 1.30

(Schloss et al., 2009). Preprocessing of unaligned

sequences included removing sequences < 450 bp, all

sequences containing ambiguous characters, and

sequences with more than eight homopolymers. We also

removed sequences that did not align over the same span

of nucleotide positions. Identical sequences were

grouped, and representative sequences were aligned

against the SILVA bacterial and archaeal reference data-

base using the Needleman–Wunsch algorithm (Needle-

man & Wunsch, 1970). Chimeric sequences were

detected and removed using the implementation of Chi-

mera Uchime. A further screening step (precluster) was

carried out to reduce sequencing noise by clustering

reads differing by only one base every 100 bases (Huse

et al., 2010). The remaining high-quality reads were used

to generate a distance matrix and were clustered into

operational taxonomic units (OTUs) defined at 97% cut-

off using the average neighbor algorithm. Next, the

OTUs were phylogenetically classified to genus level

using the naive Bayesian classifier (80% confidence

threshold) trained on the RDP taxonomic outline imple-

mented in Mothur and a modified bacterial database. In

silico T-RF prediction of the 16S rRNA gene sequences

obtained in this study was performed using the program

TRiFLE (Junier et al., 2008), and predicted T-RFs were

linked to measured T-RFs from the microbial commu-

nity profiles.

Estimation of diversity and statistical analysis

Diversity indices

Nonparametric Chao1 and Shannon alpha diversity esti-

mates, as well as coverage and rarefaction curves, were

calculated with MOTHUR v.1.30 for each sample. Analysis

of variance (ANOVA) was performed with Tukey’s tests to

identify differences between sampling sites.

Cluster analysis

To compare community composition based on T-RFLP

and 454-pyrosequencing data, normalized OTUs abun-

dances were square-root-transformed and pairwise dis-

similarities among samples were calculated using the

relative abundance-based Bray–Curtis index (BC). Non-

metric multidimensional scaling (nMDS) analysis was

performed on the dissimilarity matrices to visualize pat-

terns of community composition. Using the 454-pyrose-

quencing data, we carried out a random sampling

procedure to make equal the number of sequences per

sample (486 sequences) and we removed singleton OTUs

(sequences that only occurred once) to reduce the influ-

ence of rare OTUs. One-way analysis of similarity

(ANOSIM) and multiple pairwise comparisons were used to

test whether there were significant differences in commu-

nity composition in space. R-values > 0.75 are commonly

interpreted as well-separated bacterial compositions;

R > 0.5 as overlapping, but clearly different; and R < 0.25

as practically not separable.

CCA

Canonical correspondence analyses (CCAs) were used to

explore variations in the bacterial communities under the

constraint of our set of environmental variables. Explana-

tory variables were log(x + 1)-transformed where neces-

sary to approximate normal distribution. This model was

tested with Monte Carlo permutation tests (499 random-

ized runs) to determine significance, and each environ-

mental parameter was tested by stepwise analysis to

detect significant predictors. All statistical analyses were

performed with R 3.0.1 (R Development Core Team,

2012) including the VEGAN package.


4 A. Volant et al.

Results

Spatial and temporal analyses of the

environmental data set

The physicochemical characteristics of the water samples

were determined for six sampling dates and at five differ-

ent sites in a borehole and along Reigous Creek (Fig. 1;

Supporting Information, Table S1). With the exception of

pH, the environmental variables measured differed signifi-

cantly between sites (ANOVA, P < 0.05). A significant

decrease (Tukey’s test, P < 0.01) in temperature was

observed in Reigous Creek, where the influence of air

temperature causes a larger range of values (Fig. 2a) in

contrast to the temperature of the water in the borehole

(S5) and at the source of the Reigous (S1), which did not

differ significantly (13.3 � 1.4 °C and 13.6 � 1 °C,respectively, and Tukey’s test, P = 0.99). All samples were

characterized by low pH (≤ 3.7). No significant variations

in pH (ANOVA, P = 0.49) were observed between the sam-

pling sites located downstream from the source (COWG,

GAL and CONF; Fig. 2b). Dissolved oxygen (DO) con-

centrations in the water at the upstream sites presented a

mean of 1.0 � 1.0 mg L�1 for S5 and 0.8 � 0.6 mg L�1

for S1, denoting generally suboxic conditions at these

sites. DO increased sharply between S1 and COWG

(mean of 6.1 � 1.8 mg L�1) (Fig. 2c) and continued to

increase slightly all along the creek, to reach a mean of

10.5 � 1.3 mg L�1 at CONF. The redox potential (Eh)

showed average values of 558 � 85 mV at S5. Eh

increased along Reigous Creek from 512 � 39 mV at S1

to 635 � 99 mV at CONF (Fig. 2d). In contrast, average

conductivity decreased from 7588 � 5203 lS cm�1 at S5

to 5121 � 631 lS cm�1 at S1 and reached minimum

(1612 � 97 lS cm�1) at CONF (Fig. 2e). Sulfate (SO42�)

concentrations were maximum in the groundwater at S5

with a mean of 14 080 � 10 630 mg L�1. After a sharp

decrease at S1 (average values of 2682 � 1180 mg L�1),

concentrations gradually decreased along Reigous Creek

(Fig. 2f). Dissolved Fe concentrations in the groundwater

at S5 exhibited average values of 4474 � 2855 mg L�1.

Fe concentrations decreased from the source (S1, average

values of 1317 � 383 mg L�1) to CONF, where Fe

remained below 82 mg L�1 (Fig. 2g). The proportion of

Fe(III) (difference between Fe(total) and Fe(II)) was gen-

erally negligible except at some sampling dates at S5 and

CONF (Table S1). At S5, concentrations of dissolved

arsenic (As) exhibited an average value of 440 �184 mg L�1 (Fig. 2h). Along Reigous Creek, As concen-

trations decreased with increasing distance from the

source (average value of 175 � 71 mg L�1), to values

below 6 mg L�1 at CONF (Fig. 2h), with predominance

of the reduced form As(III). An average of 65% of sulfate,

96% of iron, and 99% of arsenic were removed from the

aqueous phase between S1 and CONF sampling sites.

Diversity and species richness estimators of

bacterial communities

Hex-labeled PCR products were digested separately with

two restriction enzymes. HpaII that produced the largest

numbers of T-RFs (data not shown) was used to assess

the differences in the microbial communities. T-RFLP

profiles generated showed a total of 43 different T-RFs

for the five sites, and the number of T-RFs detected in

each sample varied from 2 to 17 (Fig. 3). Average T-RF

richness (number of T-RFs) and average Shannon diver-

sity indices calculated from relative peak intensity data

were highest at S1 and COWG (H = 2.03 � 0.3 and

H = 1.96 � 0.3, respectively), while the lowest values

were observed at GAL (H = 1.26 � 0.4; Fig. 4a). Values

at CONF were intermediate (H = 1.67 � 0.7). Although

bacterial community diversity varied among the sampling

sites, the differences were not significant (ANOVA,

F = 2.46, P = 0.071). For each site, the bacterial commu-

nity showed variations over time, but no particular trend

could be identified. Some T-RFs were found in the

majority of the profiles (e.g. T-RF 150), where they usu-

ally accounted for a high proportion of the total T-RFs

(Fig. 3). Between one and three site-specific T-RFs were

identified in all the sites (in red in Fig. 3).

A total of 99 441 sequence reads were generated in a

single run of 454-pyrosequencing from 30 independent

16S rRNA gene libraries. Note that pyrosequencing of

two samples taken in February 2008 (S5 and S1) failed

and were thus excluded from analysis. After trimming

and processing with Mothur, 63 442 reads remained with

an average length of 530 bp. Clustering of the remaining

sequences led to the identification of 6613 OTUs (includ-

ing 4510 singletons) defined at 97% identity. Although

singletons represented 68% of the total number of OTUs,

they only accounted for 7% of the total DNA sequences.

The results of rarefaction analysis along with the Chao1

and the Shannon indexes and coverage values are listed

in Table 1. In the resampled data set, Good’s coverage

ranged from 69% to 97% with an average value of 85%,

indicating that the majority of bacterial phylotypes were

recovered. Species richness (Chao1 index) of the bacterial

communities presented significant variations along Rei-

gous Creek (ANOVA, P = 0.001, F = 6.66) (Fig. 4b). The

nonparametric estimators Chao1 ranged between 52 and

495 estimated OTUs for all the sites considered (Table 1).

The highest average OTU richness was found at CONF

and S1 (Chao1 = 364 � 145 and 296 � 32, respectively),

suggesting that an important number of OTUs were not

revealed by the analysis of these two sites. Situated



(a)

(b)

(c)

(d)

Fig. 2. Variations in the main physicochemical parameters over the course of the study and boxplot of each variable per sampling site. Arrows

indicate sampling dates for T-RFLP and pyrosequencing analysis. Note that some data are missing, as shown by the gaps in the curves. DO,

dissolved oxygen; Eh, redox potential.


6 A. Volant et al.

(e)

(f)

(g)

(h)

Fig. 2. Continued



Fig. 3. Relative abundance of terminal restriction fragments (T-RFs) derived from bacterial communities. Single T-RFs per sampling site are in red.

Dominant T-RFs are in bold. Taxonomic affiliation of T-RFs was carried out by in silico T-RFLP analysis. T-RF 91 and 100 could not be assigned to

any phylogenetic group; T-RF 125 represented Armatimonadetes gp4 and Chlorobi; T-RF 150 was mainly related to Deinococcus-Thermus,

Spirochaetes and Actinobacteria; T-RF 162 was mainly related to A. ferrooxidans but could be assigned to other proteobacterial phylotypes

detected in the AMD. N7: November 2007; O8: October 2008; M9: March 2009; N9: November 2009; J10: January 2010.

(a) (b)

Fig. 4. Average diversity and species richness index per group � standard deviation calculated based on (a) T-RFLP profiles and (b) 454

pyrosequencing reads of the reduced data set based on the 16S rRNA genes.


8 A. Volant et al.

Table

1.Estimated

OTU

richness,

diversity

indices,an

destimated

sample

coverageforeach

16SrRNA

gen

elibrary.Resultsarepresentedforfulldatasetread

s(fulldataset)

andforreduced

datasets

withoutsingletonsan

drandomly

resampledto

makethesample

size

equal

(red

uceddataset)

Sampling

sites

Red

uceddataset

Fulldataset

No.of

read

s

Obs.

OTU

s*Chao

1Sh

annon†

Coverage‡

No.of

read

s

Obs.

OTU

s*Chao

1Sh

annon†

Coverage‡

S5

S5N7

486

79

116(95;163)

3.18(3.04;3.32)

92

2089

216

576(435;810)

3.16(3.07;3.24)

93

S5O8

486

32

52(38;98)

1.66(1.52;1.80)

97

2523

87

258(165;465)

1.49(1.42;1.56)

98

S5M9

486

52

89(67;146)

2.14(1.99;2.29)

94

2838

177

381(298;520)

2.20(2.13;2.28)

96

S5N9

486

92

142(115;199)

3.31(3.17;3.46)

91

1086

159

481(334;751)

3.22(3.11;3.34)

91

S5J10

486

70

102(82;150)

3.40(3.29;3.51)

94

2354

195

598(423;908)

3.41(3.35;3.48)

95

S1

S1N7

486

138

266(209;368)

3.86(3.71;4.01)

83

2719

436

1329(1051;1773)

3.88(3.79;3.96)

90

S1O8

486

131

289(217;422)

3.93(3.80;4.05)

83

3422

727

2396(1994;2925)

4.59(4.52;4.66)

85

S1M9

486

127

270(204;392)

3.82(3.68;3.95)

84

2057

365

1068(844;1397)

3.97(3.89;4.06)

88

S1N9

486

137

315(237;455)

3.48(3.30;3.65)

81

2021

392

1040(848;1313)

3.53(3.41;3.64)

86

S1J10

486

181

342(277;449)

4.48(4.35;4.61)

78

4573

845

2158(1859;2544)

4.81(4.75;4.88)

88

COWG

CGN7

486

121

193(159;256)

3.75(3.61;3.89)

87

688

194

367(301;474)

4.06(3.93;4.19)

82

CGF8

486

105

201(154;295)

3.16(2.99;3.33)

87

2160

305

835(659;1099)

3.21(3.11;3.30)

90

CGO8

486

98

206(152;312)

2.90(2.72;3.07)

87

2119

255

1025(715;1544)

2.82(2.72;2.92)

92

CGM9

486

146

382(279;565)

3.87(3.72;4.01)

79

2756

511

1710(1364;2196)

4.11(4.02;4.19)

87

CGN9

486

112

349(232;578)

3.00(2.81;3.18)

84

1317

201

624(455;907)

2.80(2.67;2.93)

89

CGJ10

486

50

88(65;148)

1.44(1.26,1.62)

94

1827

136

472(311;781)

1.51(1.41;1.62)

95

GAL

GLN

7486

91

144(117;200)

2.28(2.08;2.49)

90

1638

223

543(423;734)

2.23(2.10;2.35)

91

GLF8

486

65

224(135;430)

1.33(1.14;1.52)

90

1679

177

665(456;1030)

1.56(1.44;1.68)

92

GLO

8486

93

253(172;418)

2.57(2.39;2.75)

87

2093

236

614(473;839)

2.64(2.54;2.74)

93

GLM

9486

107

239(178;354)

2.36(2.14;2.57)

85

2489

356

1070(843;1403)

2.49(2.37;2.60)

90

GLN

9486

110

227(171;334)

2.60(2.39;2.82)

86

1682

285

715(570;934)

2.65(2.51;2.78)

88

GLJ10

486

83

201(140;331)

2.05(1.85;2.25)

88

2246

271

862(654;1183)

2.12(2.01;2.23)

91

CONF

CFN

7486

239

492(400;635)

5.07(4.97;5.18)

70

3256

933

1995(1764;2290)

5.92(5.87;5.98)

84

CFF8

486

62

186(115;352)

1.46(1.27;1.65)

91

1768

240

972(692;1428)

1.94(1.82;2.06)

89

CFO

8486

191

335(279;426)

4.40(4.25;4.55)

77

1731

548

1163(1004;1377)

5.02(4.93;5.12)

80

CFM

9486

84

202(142;323)

1.87(1.66;2.07)

88

527

125

462(306;751)

2.28(2.07;2.50)

81

CFN

9486

233

477(390;612)

4.82(4.69;4.95)

69

1994

699

1708(1467;2026)

5.45(5.37;5.53)

77

CFJ10

486

180

495(367;711)

4.06(3.89;4.23)

74

5718

1343

3763(3330;4289)

4.79(4.71;4.86)

84

*OTU

sweredefi

ned

at97%

cutoff.

†Takesinto

accountthenumber

andeven

nessofspecies.

‡Coverage:

sum

ofprobab

ilities

ofobserved

classescalculatedas

(1�

(n/N)),wherenisthenumber

ofsingletonsequen

cesan

dN

isthetotalnumber

ofsequen

ces.

Values

inbracketsare95%

confiden

ceintervals.



between these two sites, COWG and GAL exhibited inter-

mediate richness estimates (Chao1 = 236 � 110 and

215 � 39, respectively). The lowest richness was observed

in the tailing groundwater at S5 (Chao1 = 100 � 33).

Bacterial OTU diversity, estimated by the Shannon index,

also differed significantly between sites (ANOVA, F = 3.01,

P = 0.039), with values ranging from 1.33 to 5.07

(Table 1). In agreement with T-RFLP data analysis

(Fig. 4a), the highest average diversity value was found at

S1 (H = 3.91 � 0.36) and the lowest value at GAL

(H = 2.20 � 0.47) (Fig. 4b). As predictable, average T-RF

diversity is lower than OTU diversity (c. 50%); indeed,

taxon-specific resolution of pyrosequencing is much

higher than fingerprinting (Pilloni et al., 2012). Again, no

seasonal trend was observed. The same richness and

diversity patterns were observed in both the full and

resampled data sets, although the richness estimator

and Shannon index were higher in the full data set, due

to the larger number of sequences (data not shown).

Taxonomic assignment of bacterial

pyrosequencing reads and T-RFs

At a confidence threshold of 80%, we were able to assign

56 426 of 63 442 qualified reads (that is, 89%) to a

known phylum (Table S2) and 76% to a known order

(Supporting Information, Fig. S1). Most of the unclassi-

fied reads (55% representing 9.6% to 37% of the qualified

reads of each sample) were associated with samples col-

lected at S1. Altogether, 23 bacterial phyla were recovered

from our samples, with 4–8 different phyla found in sam-

ples collected at S5, 12–13 at S1, 9–14 at COWG, 10–13at GAL, and 9–20 at CONF (Table S2). Most of the bac-

terial sequences (86%) belonged to phyla that are most

often encountered in acid mine drainages worldwide

(Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria,

Bacteroidetes, and Nitrospirae). In addition, microorgan-

isms representing 0.5% of the total sequences were related

to CARN1, ‘Candidatus Fodinabacter communificans’.

Proteobacteria was the most abundant phylum in all the

samples, accounting for 69.6% of all sequences retrieved.

This phylum was represented by bacteria belonging to the

Alphaproteobacteria, Betaproteobacteria, Gammaproteobac-

teria, Deltaproteobacteria, and Epsilonproteobacteria. The

most abundant classes in nearly all the samples were

Betaproteobacteria and Gammaproteobacteria (average val-

ues of 63.4% and 30.4% of the pyrosequencing reads,

respectively). There were dominated by Gallionellales and

Acidithiobacillales, respectively (Fig. S1a). Three other

phyla, Actinobacteria represented mainly by Acidimicrobi-

ales and Actinomycetales, Firmicutes (principally Clostridi-

ales and Bacilliales), and Acidobacteria, were also

abundant but their proportion varied depending on the

sample analyzed (Fig. S1b–d). Most of the sequences

associated with Acidobacteria could not be classified to

the order level except for Acidobacteriales and Holopha-

gales. As can be seen in Fig. S2, a relatively small number

of OTUs dominated at all sites (> 1% in total abundance

per sample). The most abundant OTUs were phylogeneti-

cally related to Gallionella ferruginea (Gallionellales),

Acidithiobacillus ferrooxidans (Acidithiobacillales), and

Thiobacillus sp. (Hydrogenophilales), collectively account-

ing for 41% of all the sequences.

When possible, T-RFs were assigned to a taxon or a

group of taxon by in silico restriction of 16S rRNA gene

sequences. Among the five dominant T-RFs (91, 100, 125,

150, and 162 bp in size), T-RFs 91 and 100 could not be

assigned to any specific phylogenetic group. Armatimona-

detes gp4 and Chlorobi were represented by T-RF 125,

and T-RF 150 was mainly related to Deinococcus-Thermus,

Spirochaetes, and Actinobacteria. While T-RF 162 was

mainly related to A. ferrooxidans, it could be assigned to

other proteobacterial phylotype detected in this AMD.

Spatial and temporal variations in bacterial

community structure

Spatiotemporal dynamics of bacterial populations were

identified by T-RFLP analysis and 454-pyrosequencing of

16S rRNA genes (Fig. S3).

Although samples formed overlapping clusters on the

nMDS plot of the T-RFLP profiles, weak but significantly

different bacterial communities at the five sites were

revealed (ANOSIM Global R = 0.2819, P < 0.001). S5 dif-

fered significantly from the other sites, with some over-

lapping communities (pairwise tests: r-values ranging

from 0.45 to 0.61, P < 0.05). These results highlighted

changes in the structure of the bacterial communities

between the tailing groundwater (S5) and the water in

Reigous Creek. The high dissimilarity observed within

each site revealed variations in community structure over

time, especially at S5, GAL, and CONF (Fig. S3a). These

variations may have masked a spatial pattern.

nMDS analyses of 454-pyrosequencing data also

showed that the composition of the bacterial communi-

ties differed significantly along the spatial gradient from

the sterile (S5) to the confluence (CONF) (Fig. S3b). Fur-

thermore, an ANOSIM test corroborated the nMDS plot

data, revealing significantly different bacterial composi-

tions in water as a function of the spatial location (Global

R = 0.6192, P < 0.001), except at GAL and COWG which

did not differ significantly (ANOSIM pairwise comparison

r = 0.206, P = 0.37). Higher temporal variation at CONF

was highlighted by the large cluster on the nMDS plot.

The marked temporal variations in the bacterial commu-

nity at S5 and CONF highlighted by the two data sets


10 A. Volant et al.

may be linked to the stronger seasonal fluctuation of

some physicochemical parameters at these sites, particu-

larly temperature at CONF (Fig. 2a) and pH, Eh, sulfate,

Fe, and As at S5 (Fig. 2b, d, f, g and h).

We investigated the four most abundant phyla to get a

global view of the variations of bacterial communities

along the creek (Fig. S1). Proteobacteria distribution var-

ied between samples, with a relative predominance of

Acidithiobacillales in samples from S5 and S1, followed by

the dominance of Gallionellales in the majority of other

samples (Fig. S1a). A general increase in Betaproteobacte-

ria was observed in the downstream direction of Reigous

Creek. Among Actinobacteria, the Acidimicrobiales were

present in all samples except those collected at CONF

where almost equal proportions of Acidimicrobiales and

Actinomycetales were retrieved (Fig. S1b). The Firmicutes

phylum revealed the dominance of Clostridiales in sam-

ples from S5, whereas Bacillales were dominant at the

other sites, again except CONF. Different orders were

dominant at CONF over time, including Lactobacillales

and Selenomonadales (Fig. S1c). No Acidobacteria were

retrieved at S5 in October 2008 (Fig. S1d).

We also assessed the dynamics of the dominant genera

(> 5% in total abundance per sample) (Fig. 5). The rela-

tive abundance of genera at each site varied over the sam-

pling period. While the relative abundance of Gallionella

was almost constant in COWG and GAL, there was an

important temporal variation in the other sites. This was

evident in S5 where this genus was extinct and re-thrived

over time (Fig. 5a). Although Gallionella was present in

almost all sites for a sampling date, there was no clear rela-

tionship between sites. Indeed, GAL and COWG exhibited

a relatively high proportion of Gallionella at almost all the

sampling dates (as much as 85% of all pyrosequencing

reads at GAL) without any link with the upstream sites S5

and S1 (Fig. 5b). In contrast, Acidithiobacillus represented

a minor fraction of the bacterial community, except at S5

where this OTU was dominant (24–72% of the pyrose-

quencing reads). The relative abundance of Acidithiobacil-

lus showed a decreasing trend along the continuum for

each sampling date (Fig. 5b). This genus also exhibited an

increase in October 2008 and March 2009 for S5 and S1

(Fig. 5a). Members of the Thiobacillus genus showed

higher proportion in COWG at each sampling date inde-

pendently of the other sites (Fig. 5b). The temporal varia-

tion of this genus was minor except an important increase

at GAL in October 2008 (Fig. 5a).

T-RFLP profiles from the 30 samples were investigated

to assess the dynamics of T-RFs (Fig. 3). Among the five

dominant T-RFs, T-RFs 91 and 100 represented a large

proportion of the T-RFLP profiles in almost all samples

except those from S5. T-RF 125 related to Armatimonade-

tes gp4 and Chlorobi was more abundant in samples from

S5 and S1 than in downstream samples. T-RF 150 (Dei-

nococcus-Thermus, Spirochaetes, and Actinobacteria –related) was the most abundant phylotype in the tailings

groundwater (S5), accounting for up to 70% of the T-

RFLP profiles. It was relatively abundant along the creek

especially at GAL. T-RF 162 (related to A. ferrooxidans

but also to other proteobacterial phylotype) was not

dominant at S5 and represented a minor fraction of the

bacterial community along the creek.

Linking bacterial community structure to

environmental variables

Canonical correspondence analysis (CCA) was performed

to elucidate the main relationships between physicochemi-

cal variables and bacterial community structure and com-

position (Fig. 6). Samples were plotted in different areas

of the diagram depending on their environmental charac-

teristics. The resolution of 454-pyrosequencing allowed to

account for more variation than T-RFLP (36.4% and

20.5%, respectively) in the species–environment relation-

ship across the first two canonical axes. CCA axis 1 based

on T-RFLP data only separated the samples into two clus-

ters, one containing the tailings site (S5) and the other

grouping the sites along the creek (S1, COWG, GAL, and

CONF), with sulfate, DO, and pH being the strongest

determinants of bacterial community structure (Fig. 6a).

In contrast, a higher resolution was observed with CCA

axis 1 based on 454-pyrosequencing data, which was most

closely correlated with iron, arsenic, and conductivity and

separated the sampling sites into three clusters (CONF,

GAL+COWG, and S1+S5) as a function of the pollution

gradient (Fig. 6b). The upstream site S5 was highly pol-

luted and little oxygenated, whereas the downstream site

CONF was less polluted and characterized by a higher

redox potential (Eh). CCA axis 2 separated samples

according to water temperature. After the Monte Carlo

permutation test, the environmental variables significantly

correlated with the canonical axes based on 454-pyrose-

quencing data were arsenic (F-ratio = 1.9, P = 0.01), tem-

perature (F-ratio = 1.4, P = 0.01), and sulfate (F-

ratio = 1.4, P = 0.01). The differences between the two

data sets were probably due to the power of 454-pyrose-

quencing over T-RFLP for taxon resolution. Focusing on

454-pyrosequencing data, the influence of environmental

variables on dominant OTUs (> 5% of total abundance

per sample) was also investigated (Fig. S4). Nineteen of

the 23 dominant OTUs showed a strong correlation with

the physicochemical parameters. Five OTUs (15, 16, 28,

32, and 52) were strongly correlated with elevated DO and

Eh and 14 with high temperatures and high concentrations

of As, Fe, and sulfate. As indicated by the position of

OTUs 1, 3, 4, and 11 on the graph, near the origin of the



axes, none of the environmental variables measured in the

study could explain their distribution and thus their niche.

At the least polluted site (CONF), Gallionella, Ferrovum,

and Acidiferrobacter were the main genera detected,

whereas, at the most polluted sites (S5 and S1), a higher

number of genera were codominant (Acidithiobacillus,

Ignavibacterium, Ralstonia, Leptospirillum, Gallionella,

Ferrovum, etc.).

Discussion

This study combined a classical fingerprinting method

(T-RFLP) and a high-throughput barcoded pyrosequenc-

ing of 16S rRNA genes to investigate the diversity, spatial

distribution, and seasonal variation of bacterial communi-

ties in Carnoul�es AMD (France), which is heavily con-

taminated with As.

(a)

(b)

Fig. 5. Relative abundance of the dominant genera (> 5% in total abundance per sample) presented by (a) sampling sites and (b) sampling date.

N7: November 2007; O8: October 2008; M9: March 2009; N9: November 2009; J10: January 2010.


12 A. Volant et al.

Spatial and temporal variations in the

environmental data set and in the bacterial

community

Monitoring the physicochemical parameters of Reigous

Creek confirmed previous results (Casiot et al., 2003a;

Egal et al., 2010), showing a significant decrease in

concentrations of dissolved As, Fe, and sulfate with

increasing distance from the source: 72% of sulfate, 96%

of iron, and 99% of arsenic had been removed by the

time Reigous Creek flowed into the River Amous (Table

S1). In addition, the concentrations of As and Fe in the

water from the tailings stock were much lower in 2007–2010 (average values of 440 � 184 mg L�1 and 4474 �2855 mg L�1, respectively), than those measured in 2001

(up to 10 g L�1 for As and around 20 g L�1 for Fe, Casi-

ot et al., 2003b), although these concentrations are still

very high compared to other AMDs.

Both molecular methods highlighted a higher bacterial

diversity than expected in this extreme habitat. T-RFLP

profiles showed for the five sites a total of 43 T-RFs rang-

ing from 2 to 17 T-RFs per sampling site (Fig. 3). For py-

rosequencing data, a total of 63 442 reads led to the

identification of 6613 OTUs, including 4510 singletons

representing 68% of the total number of OTUs. As

expected, a larger number of phylotypes were identified

using the pyrosequencing method leading to a significant

increase in resolution. Average Good’s coverage was over

89%, suggesting that the 16S rRNA gene sequences into

each sample represented the majority of the bacterial

phylotypes present. Nonetheless, additional sequencing

effort would be required to exhaustively characterize the

bacterial community, particularly for samples from the

least polluted site CONF, as shown by the lower coverage

values and the lack of asymptote in the rarefaction curves

(data not shown).

nMDS analyses revealed significant differences in the

composition of the bacterial communities in the five sites

along the AMD (Fig. S3). However, different clustering

patterns were obtained based on T-RFLP or pyrosequenc-

ing data. With pyrosequencing, individual sequences can

be classified at the genus level. In contrast, one T-RF can

correspond to several different bacterial phylotypes

(belonging to different genera or even different higher

taxonomic levels). Such differences in the resolution of

the two methods may explain the differences obtained in

the cluster analyses (Hwang et al., 2012). Nevertheless,

changes in bacterial community structure between the

tailings groundwater (S5) and the Reigous Creek were

revealed by the two sets of data, reflecting important dif-

ferences in ecological conditions between the two habi-

tats. According to both methods, S1 was the most diverse

bacterial community, while GAL was the least diverse.

Therefore, bacterial diversity varied independently of the

sampling site, suggesting that globally upstream commu-

nities do not influence downstream communities. The

apparent minimum effect of immigration suggests that

species-sorting processes best describe bacterial commu-

nity structure in these connected environments, with local

environmental factors driving the composition of the

(a) (b)

Fig. 6. Canonical correspondence analysis (CCA) correlating the bacterial community structure at each sampling site with arsenic (As), iron (Fe),

conductivity (Cond), temperature (T), dissolved oxygen (DO), redox (Eh), pH and sulfate. The bacterial community structures correspond to OTU

abundances from (a) T-RFLP data and (b) pyrosequencing data. The main clusters are highlighted.



bacterial community at each site. Temporal variations of

the bacterial communities could also be observed at each

site although no particular trend could be identified.

However, the important temporal variation of the bacte-

rial community observed at S5 and CONF may be due to

a higher seasonal fluctuation in physicochemical parame-

ters at these two sites, especially temperature at CONF,

and pH, Eh, sulfate, Fe, and As at S5.

However, our fine-scale investigation at the genus level

of the bacterial communities along the Reigous Creek

over time provided some important data and allowed to

establish some hypothesis about community composition.

Indeed, Gallionella in contrast to Acidithiobacillus do not

seem to benefit from the seed bank provided by the most

upstream sites (S5 and S1). This suggests that Gallionella,

under a process that still need to be elucidated, extinct/

re-thrived at each site over time. In contrast, Acidithioba-

cillus that is preferentially encountered upstream of the

Reigous Creek or Thiobacillus that thrived at COWG

could be found at these sites, under conditions that

reflect their preferential habitats. The presence of these

organisms downstream of the sites would instead reflect

dispersal from upstream sites.

Physicochemical parameters shape the

composition of the bacterial community

This work highlighted a spatial gradient of physicochemi-

cal conditions linked to a significant shift in bacterial

community composition along the continuum. Indeed,

canonical correspondence analysis of the whole pyrose-

quencing data set indicated that arsenic, temperature, and

sulfate were the factors that most influence the composi-

tion of the bacterial communities (Fig. 6b). The level of

pollution affects also some dominant bacterial popula-

tions (> 5% of relative abundance). Gallionella, Ferrovum,

and Acidiferrobacter were the dominant genera in water

sampled at the least polluted site (CONF) and were cor-

related with high DO and Eh, whereas in water from the

most polluted sites (S5 and S1), a larger number of dom-

inant genera were detected (Acidithiobacillus, Ignavibacte-

rium, Ralstonia, Leptospirillum, Gallionella, and Ferrovum)

whose relative abundance was correlated with higher tem-

perature and high concentrations of As, Fe, and sulfate

(Fig. S4). Thus, different members of a given genus such

as Gallionella (OTUs 19, 15 and 1) or Ferrovum (OTUs

25 and 28) were correlated with different environmental

parameters, suggesting that these OTUs correspond to

bacterial phylotypes with some specificity explaining these

different behaviors. Furthermore, the high abundance of

Gallionella-related sequences in these acidic ecosystems

characterized by contrasted levels of pollution is consis-

tent with results of a previous study suggesting that

Gallionella-like organisms may be more tolerant to acid

and metal than currently thought (Fabisch et al., 2013).

In accordance with our results, temperature has been pre-

viously suggested as a primary factor controlling the

structure and dynamics of microbial communities in

AMD (Edwards et al., 1999) and in various natural envi-

ronments like hot springs (Ward et al., 1998; Miller et al.,

2009) or marine environments (Fuhrman et al., 2008).

Nevertheless, sulfate and arsenic concentrations have not

previously been shown to be significantly correlated with

bacterial diversity in AMDs. Earlier studies identified dif-

ferent environmental predictors of microbial populations

in AMD including conductivity and rainfall (Edwards

et al., 1999), pH (Lear et al., 2009), oxygen gradient

(Gonz�alez-Toril et al., 2011), and season (Streten-Joyce

et al., 2013), which may result in site-specific physico-

chemical and geochemical characteristics (Kuang et al.,

2012). Furthermore, while many studies highlighted pH

as the most important factor structuring AMD communi-

ties (Kuang et al., 2012; Chen et al., 2013), our study

produced no evidence of the influence of this parameter,

probably due to the limited variation in pH among our

samples (average values of 2.5 � 0.8–3.2 � 0.3).

Composition of the bacterial communities

In this study, we were able to identify a wider phyloge-

netic range of taxa than in any previous clone library-

based diversity survey of the Carnoul�es AMD, including

sequences of several previously undetected taxa. These

new taxa include members of the Bacteroidetes, Chlorobi,

Chloroflexi, Elusimicrobia, Chlamydiae, Cyanobacteria, Dei-

nococcus-Thermus, Spirochaetes, Fibrobacteres, Fusobacteria,

Gemmatimonadetes, Plantctomycetes, Verrumicrobia, and

of the uncultured OD1-PO11-TM7 clade. The majority of

phyla that were not previously detected on clone libraries

accounted only for < 1% of the pyrosequencing data,

explaining why they were missed with the clone library

approach. The high rate of low-abundance populations

(68% of singletons) increased the phylogenetic bacterial

diversity. However, despite the preponderance of this rare

biosphere in most studies, its ecological and functional

roles remain largely unexplored (Galand et al., 2009).

Recent studies indicated that such organisms may be at a

dormant or a spore stage, but in favorable conditions

they may become active and even dominant (Delavat

et al., 2012). Thus, these taxa may play an important role

in extreme habitats like AMD, buffering the effects of

important environmental shifts (Sogin et al., 2006; Mon-

chy et al., 2011). However, further investigations will be

needed to determine whether they play a role in this eco-

system and/or whether they reflect allochthonous input

from surrounding environments. Moreover, the high-


14 A. Volant et al.

throughput sequencing also questions the accuracy of

OTU richness estimates, as sequencing errors and inade-

quate clustering algorithms can lead to overestimates of

community richness (Huse et al., 2010). The majority of

the most abundant taxa detected in this study were

related to orders commonly encountered in AMD, most

of which are known to be involved in Fe, As, and S

cycles: namely Gallionellales (Betaproteobacteria), Acidi-

thiobacilliales (Gammaproteobacteria), Acidimicrobiales

(Actinobacteria), Hydrogenophilales (Betaproteobacteria),

Burkholderiales (Betaproteobacteria), Nitrospiralles (Nitros-

pirae), Desulforomonadales (Deltaproteobacteria), and Des-

ulfobacterales (Deltaproteobacteria). The ecological role of

previously detected taxa has been widely characterized

(Bruneel et al., 2005, 2006, 2011; Bertin et al., 2011) in

this ecosystem. A relatively small number of OTUs domi-

nated at each sampling site (Fig. S2) and the majority of

them were phylogenetically related to taxa previously

found in AMD (Gallionella ferruginea, Acidithiobacillus

ferrooxidans, and Thiobacillus sp.), as well in Carnoul�es

revealing their persistence in such ecosystems (Baker &

Banfield, 2003; Bruneel et al., 2006, 2011; Hallberg et al.,

2006; Heinzel et al., 2009; Hallberg, 2010). These three

genera varied in their relative abundance over the sam-

pling period. Gallionella was present in high proportions

in almost all samples, mainly at GAL and COWG. In

contrast, except at S5 where this genus was dominant,

Acidithiobacillus accounted for a minor fraction of the

bacterial community (Fig. 5). Furthermore, our study

confirmed the presence of relatives of a novel bacterial

phylum, ‘Candidatus Fodinabacter communificans’

detected by a recent metagenomic investigation of Car-

noul�es AMD and prominent in the active COWG com-

munity (Bertin et al., 2011; Fahy et al., unpublished

data). The relatively high number of unclassified bacteria

per sample (0.3–37%) supports the fact that many bacte-

ria remain to be cultured. These results thus corroborate

the main observations made in previous studies, except

for the predominance of organisms related to sulfate

reducing bacteria (SRB) identified in water from the tail-

ings by Bruneel et al. (2005) using a cloning–sequencingapproach. Instead, our results revealed the dominance of

Acidithiobacillales over SRB in these samples. The very

low proportion of SRB populations in our study (on

average 0.1% of total abundance per sample) could be

partly due to differences in the physicochemical variables

of the water, to the choice of a stringent similarity cutoff

but also to the different primers used for PCR amplifica-

tion. Furthermore, relatives of Thiomonas belonging to

the Burkholderiales order were retrieved and accounted

for < 1% of the total sequences (Fig. S1a). Despite their

low abundance, several strains of Thiomonas sp. have

been previously isolated and shown to be active in the

oxidation of As (Bruneel et al. 2003). A metaproteomic

approach also showed that Gallionella, Thiomonas, and A.

ferrooxidans actively express proteins in situ, thus proba-

bly playing a functional role in this AMD (Bruneel et al.,

2011). These populations could play an important role in

the efficient remediation process observed along this creek

by favoring the oxidation of Fe(II) and the co-precipita-

tion of As (Casiot et al., 2003a; Bruneel et al., 2011).

This work has increased our knowledge of bacterial

diversity and dynamics in acid mine drainage. Bacterial

diversity in Carnoul�es AMD was revealed to be much

higher than previously evidenced using clone library

techniques (Bruneel et al., 2011), as suggested by cul-

ture-dependent methods (Delavat et al., 2012). Our study

revealed complex patterns of spatial and temporal varia-

tions in bacterial community composition, suggesting

that community composition reflects changes in physico-

chemical conditions. This investigation provided a first

step to the study of spatial and temporal structure of

bacterial communities and the factors that control it. To

improve our understanding of the functioning of this

ecosystem, future efforts should be oriented toward

active communities and how they fluctuate in response

to environmental changes. Such knowledge will help to

determine their roles in the functioning of the AMD

ecosystems and explain important assembly processes in

microbial ecology.

Acknowledgements

This study was financed by the FRB (Fondation pour la

recherche sur la Biodiversit�e) program blanc AAP-IN-2009-

039, the « Observatoire de Recherche M�editerran�een de l’

Environnement » (OSU-OREME). A.V. was supported by

a grant from the French Ministry of Education and

Research and F.J. by a grant from the Direction G�en�erale

de l’Armement (DGA). This work was performed within

the framework of the Groupement de recherche: M�etabol-

isme de l’Arsenic chez les Microorganismes (GDR2909-

CNRS).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Composition of different phyla based on classifi-

cation of 16S rRNA gene sequences of bacteria from each

sample using RDP Classifier: (a) Proteobacteria orders,

(b) Actinobacteria orders, (c) Firmicutes orders, and (d)

Acidobacteria orders.

Fig. S2. Histogram of the relative abundance of dominant

OTUs at the Carnoul�es sampling sites (G. ferruginea

subsp. capsiferriformans ES-2: NC_014394; A. ferrooxidans

strain HL1: JF815535; Thiobacillus sp. ML2-16:

DQ145970; Pseudomonas migulae: AY605698; A. ferrivo-

rans SS3: NR_074660; Actinobacterium BGR 105:

GU168008; Acidobacteriaceae bacterium CH1: DQ355184;

Ferrimicrobium sp. Py-F2: KC208496; Metallibacterium sp.

911: HE858262; Alicyclobacillaceae bacterium Feo-D4-16-

CH: FN870323; Acidisphaera sp. nju-AMDS1: FJ915153;

Betaproteobacterium OYT1: AB720115.

Fig. S3. Nonmetric multidimensional scaling analysis of

the composition of the bacterial community estimated by

(a) T-RFLP and (b) 454 pyrosequencing based on 16S

rRNA genes.

Fig. S4. (a) Ordination plot of CCA based on pyrose-

quencing data showing OTUs with relative abundance

>5%. (b) Abundant OTUs and their correlation with

environmental variables and phylogenetic affiliation deter-

mined by BLAST search.

Table S1. Physicochemical characteristics of the water at

each sampling site and sampling date.

Table S2. Relative abundance (in %) of total sequences of

bacterial 16S rRNA genes from each sample assigned to

different phyla.



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