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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
année DUT Génie Biologique option Analyses Biologiques et
Biochimiques. UM2, Montpellier
• 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
année DUT Génie Biologique option Analyses Biologiques et
Biochimiques. UM2, Montpellier
• Coencadrement L. Rubini (2011) 2ème
année DUT Génie Biologique, option Analyses
Biologiques et Biochimiques. UM2, Montpellier
• Co-encadrement M. Dufour (2012) 2ème
année DUT Génie Biologique, option Analyses
Biologiques et Biochimiques. UM2, Montpellier
• Co-encadrement L. Causse (2013) 2ème
année DUT Génie Biologique, option Analyses
Biologiques et Biochimiques. UM2, Montpellier
• 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,
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. 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.,
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. 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
sur la croissance des plantes dans des environnements contaminés par des métaux et
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
sur la croissance des plantes dans des environnements contaminés par des métaux et
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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67
VIII ANNEXES : SELECTION DE 5 PUBLICATIONS
Bruneel O, Duran R, Casiot C, Elbaz-Poulichet F, Personné JC (200) Diversity of
microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France. Applied and
Environmental Microbiology. 72, 551-556
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
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,
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
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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554 BRUNEEL ET AL. APPL. ENVIRON. MICROBIOL.
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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556 BRUNEEL ET AL. APPL. ENVIRON. MICROBIOL.
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
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
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
796 O. Bruneel et al.
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
Active Bacteria in Arsenic-Rich Sediments 797
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
798 O. Bruneel et al.
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
Active Bacteria in Arsenic-Rich Sediments 799
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
800 O. Bruneel et al.
-proteobacteria
-proteobacteria
-proteobacteria
-proteobacteria
Nitrospirae
Actinobacteria
Synergistetes
Firmicutes
Acidobacteria
Deinococcus/Thermus
Active Bacteria in Arsenic-Rich Sediments 801
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
Actinobacteria(class);
Actinobacteridae;
Actinom
ycetales;
Glycomycineae;
Glycomycetaceae;
Stackebrandtia
Stackebrandtia
nassauensis
Suborderor
below
60kD
achaperoninsa
C4D
UC7
6GMNALADAVK
Actinobacteria(class);
Actinobacteridae;
Actinom
ycetales;
Streptosporangineae;
Streptosporangiaceae;
Streptosporangium
Streptosporangium
roseum
Fam
ily60
kDachaperoninsa
D2B
BD1
4APGFGDR
GTFTSVAVK
Actinobacteria(class);
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
802 O. Bruneel et al.
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
Active Bacteria in Arsenic-Rich Sediments 803
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
804 O. Bruneel et al.
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
Active Bacteria in Arsenic-Rich Sediments 805
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
806 O. Bruneel et al.
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
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
Active Bacteria in Arsenic-Rich Sediments 807
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.
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
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 (continued)
808 O. Bruneel et al.
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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810 O. Bruneel et al.
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
e-mail: [email protected]
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
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(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.
Materials and methods
Description of the study site
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
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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
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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,
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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
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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
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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)
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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 %)
4 4 ArCMSdA6A46 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (90 %)
5 1 ArCMSdA6A17 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (91 %)
6 16 ArCMSdA6A67 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (92 %)
7 3 ArCoSdN9H63 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (93 %)
8 24 ArCMSdJ9B78 Euryarchaeota – – Clone hfmA029 (86 %)
9 4 ArCMSdA6A30 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
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 %)
14 2 ArCMSdA6A84 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (90 %)
15 3 ArCoSdN9H35 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (80 %)
16 1 ArCMSdA6A92 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (91 %)
17 4 ArCoSdN9H43 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (88 %)
18 5 ArCMSdO8A3 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (82 %)
19 1 ArCMSdO8A13 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (89 %)
20 1 ArCMSdO8A16 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (87 %)
21 2 ArCoSdN9H67 Euryarchaeota Thermoplasmata – Methanomassiliicoccus luminyensisB10 (83 %)
22 1 ArCMSdO8A24 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (88 %)
23 2 ArCMSdO8A56 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (89 %)
24 4 ArCMSdO8A49 Euryarchaeota Thermoplasmata Thermoplasmatales Thermoplasma volcanium GSS1
(89 %)
25 1 ArCMSdO8A54 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (90 %)
26 3 ArCMSdO8A74 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (99 %)
27 1 ArCMSdO8A85 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (93 %)
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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 %)
39 2 ArCoSdN9H79 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (90 %)
40 1 ArCoSdN9E14 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (88 %)
41 1 ArCoSdN9G7 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
13583 (93 %)
42 1 ArCoSdN9H80 Euryarchaeota Thermoplasmata Thermoplasmatales Thermogymnomonas acidicola JCM
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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Author's personal copy
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;
e-mail: [email protected]
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
GY
EC
OLO
GY
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.
Materials and methods
Description of the study site
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.
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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.
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 5
(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.
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
6 A. Volant et al.
(e)
(f)
(g)
(h)
Fig. 2. Continued
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 7
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.
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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.
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 9
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
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 11
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.
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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.
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 13
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-
FEMS Microbiol Ecol && (2014) 1–17ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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.
FEMS Microbiol Ecol && (2014) 1–17 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Spatiotemporal dynamics of bacterial communities 17