Aquatic Sciences

, Volume 76, Supplement 1, pp 103–116 | Cite as

Endospore-forming bacteria as new proxies to assess impact of eutrophication in Lake Geneva (Switzerland–France)

  • Tina Wunderlin
  • Juan Pablo Corella
  • Thomas Junier
  • Matthieu Bueche
  • Jean-Luc Loizeau
  • Stéphanie Girardclos
  • Pilar JunierEmail author
Research Article - Based on MIR Investigations in Lake Geneva


Measurements of chemical composition and biological parameters of sediment cores are used as proxies for changes in past environmental conditions and more recently the human impact on ecosystem health. In this study, endospore-forming bacteria are proposed as a new biological proxy for such paleoecological reconstructions. A sediment core providing a record for the past 90 years (137Cs and magnetic susceptibility dating) was retrieved from the Rhone Delta of Lake Geneva. X-ray fluorescence was analyzed at a 0.2-cm resolution, while DNA extracts, elemental geochemistry and grain size were obtained at 4-cm intervals. The total number of bacteria and endospore-forming bacteria were quantified by qPCR using the 16S rRNA gene and the endosporulation-specific spo0A gene. Furthermore, a spo0A fragment was subjected to amplicon sequencing to define OTUs (operational taxonomic units) and the phylogenetic affiliation of the endospore formers. The results showed that despite the fact that the quantity of extracted DNA decreased with the age of the sediment, the abundance of endospore-forming bacteria remained constant. However, the diversity of this group of bacteria changed significantly, reflecting the eutrophication of the lake from 1960 to 1990. The shift in community composition was linked to the dominance of anaerobic clostridia-like endospore formers. This trend has reversed in the last 10 years of the record, suggesting a recovery after perturbation. This study shows that the abundance and diversity of endospore-forming bacteria can be used as proxies to reconstruct lake history. We hereby successfully introduce a new strategy for paleoecology that could also be applied to ocean sediments and long sediment cores.


Endospore-forming bacteria Paleoecological proxy Sediment record Lake Geneva Human impact Eutrophication 



This publication is part of the international, interdisciplinary research project ELEMO ( to investigate the deep waters of Lake Geneva using two Russian MIR submarines. Funding for this study was provided by the “Fondation pour l’Etude des Eaux du Léman” (FEEL; in particular by the Ferring Pharmaceuticals St Prex). Additional funding for the work described in this paper was provided by Swiss National Science Foundation grant No. 31003A-132358/1. We are grateful for the support. We thank the Russian MIR crew members ( for their excellent performance and the SAGRAVE team who provided and operated the platform from which the dives were carried out. We also thank Ulrich Lemmin and Jean-Denis Bourquin for project coordination and Samuel Arey for dive planning. The service of Mikhail Kranoperov (Russian Honorary Consulate) as liaison is greatly appreciated. The authors acknowledge Philippe Arpagaus and Angel Arantegui for their support during the fieldwork and laboratory analyses. The authors thank the two anonymous reviewers for their valuable comments for improving this manuscript.

Supplementary material

27_2013_329_MOESM1_ESM.doc (860 kb)
Supplementary material 1 (DOC 861 kb)


  1. Abecasis AB, Serrano M, Alves R, Quintais L, Pereira-Leal JB, Henriques AO (2013) A genomic signature and the identification of new sporulation genes. J Bacteriol 195(9):2101–2115PubMedCentralPubMedCrossRefGoogle Scholar
  2. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1):143–169PubMedCentralPubMedGoogle Scholar
  3. Anneville O, Pelletier JP (2000) Recovery of Lake Geneva from eutrophication: quantitative response of phytoplankton. Archiv für hydrobiologie 148(4):607–624Google Scholar
  4. Bartholomew JW, Paik G (1966) Isolation and identification of obligate thermophilic sporeforming bacilli from ocean basin cores. J Bacteriol 92(3):635–638PubMedCentralPubMedGoogle Scholar
  5. Bueche M, Wunderlin T, Roussel-Delif L, Junier T, Sauvain L, Jeanneret N et al (2013) Quantification of endospore-forming Firmicutes by quantitative PCR with the functional dene spo0A. Appl Environ Microbiol 79(17):5302–5312PubMedCentralPubMedCrossRefGoogle Scholar
  6. Corella JP, Amrani A, Sigro J, Morellon M, Rico E, Valero-Garces B (2011) Recent evolution of Lake Arreo, northern Spain: influences of land use change and climate. J Paleolimnol 46(3):469–485CrossRefGoogle Scholar
  7. Corella JP, Brauer A, Mangili C, Rull V, Vegas-Villarrubia T, Morellon M et al (2012) The 1.5-ka varved record of Lake Montcortès (southern Pyrenees, NE Spain). Quatern Res 78(2):323–332CrossRefGoogle Scholar
  8. de Rezende JR, Kjeldsen KU, Hubert CR, Finster K, Loy A, Jorgensen BB (2013) Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years. ISME J 7(1):72–84PubMedCentralPubMedCrossRefGoogle Scholar
  9. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461PubMedCrossRefGoogle Scholar
  10. Fichtel J, Koster J, Rullkotter J, Sass H (2007) Spore dipicolinic acid contents used for estimating the number of endospores in sediments. FEMS Microbiol Ecol 61(3):522–532PubMedCrossRefGoogle Scholar
  11. Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI, Rigden DJ (2012) Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes. Environ Microbiol 14(11):2870–2890PubMedCentralPubMedCrossRefGoogle Scholar
  12. Gerdeaux D, Perga ME (2006) Changes in whitefish scales 13C during eutrophication and reoligotrophication of subalpine lakes. Limnolo Oceanogr 51:772–780CrossRefGoogle Scholar
  13. Gorham E, Brush GS, Graumlich LJ, Rosenzweig ML, Johnson AH (2001) The value of paleoecology as an aid to monitoring ecosystems and landscapes, chiefly with reference to North America. Environ Rev 9(2):99–126CrossRefGoogle Scholar
  14. Hageman JH, Shankweiler GW, Wall PR, Franich K, McCowan GW, Cauble SM et al (1984) Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production. J Bacteriol 160(1):438–441PubMedCentralPubMedGoogle Scholar
  15. Hobbs WO, Lalonde SV, Vinebrooke RD, Konhauser KO, Weidman RP, Graham MD et al (2010) Algal-silica cycling and pigment diagenesis in recent alpine lake sediments: mechanisms and paleoecological implications. J Paleolimnol 44(2):613–628CrossRefGoogle Scholar
  16. Hubert C, Arnosti C, Bruchert V, Loy A, Vandieken V, Jorgensen BB (2010) Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature. Environ Microbiol 12(4):1089–1104PubMedCrossRefGoogle Scholar
  17. Kanz C, Aldebert P, Althorpe N, Baker W, Baldwin A, Bates K et al (2005) The EMBL nucleotide sequence database. Nucleic Acids Res 33:D29–D33PubMedCentralPubMedCrossRefGoogle Scholar
  18. Kindt R, Coe R (2005) Tree diversity analysis: a manual and software for common statistical methods for ecological and biodiversity studies. World Agroforestry Centre Eastern and Central Africa ProgramGoogle Scholar
  19. Klappenbach JA, Dunbar JM, Schmidt TM (2000) rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66(4):1328–1333PubMedCentralPubMedCrossRefGoogle Scholar
  20. Koinig A, Shotyk W, Lotter A, Ohlendorf C, Sturm M (2003) 9000 years of geochemical evolution of lithogenic major and trace elements 75 in the sediment of an alpine lake: the role of climate, vegetation, and landuse. J Paleolimnol 30(3):307–320CrossRefGoogle Scholar
  21. Kroos L (2007) The Bacillus and Myxococcus developmental networks and their transcriptional regulators. Annu Rev Genet 41:13–39PubMedCrossRefGoogle Scholar
  22. Lazzarotto J, Klein A (2012) Physical–chemical changes in the waters of Lake Geneva (major elements). Rapport de la Commission Internationale pour la Protection des Eaux du Léman contre la Pollution; Campagne 2011. Lausanne, SwitzerlandGoogle Scholar
  23. Loizeau J-L (1991) La sédimentation récente dans le delta du Rhône, Léman: processus et évolution. Université de Genève, GenevaGoogle Scholar
  24. Loizeau J-L, Dominik J, Luzzi T, Vernet J-P (1997) Sediment core correlation and mapping of sediment accumulation rates in Lake Geneva (Switzerland, France) using volume magnetic susceptibility. J Great Lakes Res 23(4):391–402CrossRefGoogle Scholar
  25. Lomstein BA, Langerhuus AT, D’Hondt S, Jorgensen BB, Spivack AJ (2012) Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment. Nature 484(7392):101–104PubMedCrossRefGoogle Scholar
  26. Meyers PA (2003) Applications of organic geochemistry to paleolimnological reconstructions: a summary of examples from the Laurentian Great Lakes. Org Geochem 34(2):261–289CrossRefGoogle Scholar
  27. Molinero JC, Anneville O, Souissi S, Balvay G, Gerdeaux D (2006) Anthropogenic and climate forcing on the long-term changes of planktonic rotifers in Lake Geneva, Europe. J Plant Res 28(3):287–296Google Scholar
  28. Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64(3):548–572PubMedCentralPubMedCrossRefGoogle Scholar
  29. Oksanen J, Kindt R, Legendre P, O’Hara B, Stevens MHH, Oksanen MJ, Suggests M (2007) The vegan package. Community Ecol Package 10(01):2008Google Scholar
  30. Onyenwoke RU, Brill JA, Farahi K, Wiegel J (2004) Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes). Arch Microbiol 182(2–3):182–192PubMedGoogle Scholar
  31. Ovreås L, Forney L, Daae FL, Torsvik V (1997) Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol 63(9):3367–3373PubMedCentralPubMedGoogle Scholar
  32. Pacton M, Ariztegui D, Wacey D, Kilburn MR, Rollion-Bard C, Farah R et al (2012) Going nano: a new step toward understanding the processes governing freshwater ooid formation. Geology 40(6):547–550CrossRefGoogle Scholar
  33. Plee K, Ariztegui D, Martini R, Davaud E (2008) Unravelling the microbial role in ooid formation–results of an in situ experiment in modern freshwater Lake Geneva in Switzerland. Geobiology 6(4):341–350CrossRefGoogle Scholar
  34. R TC (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  35. Rellstab C, Keller B, Girardclos S, Anselmetti FS, Spaak P (2011) Anthropogenic eutrophication shapes the past and present taxonomic composition of hybridizing Daphnia in unproductive lakes. Limnol Oceanogr 56(1):292–302CrossRefGoogle Scholar
  36. Renberg I, Nilsson M (1992) Dormant bacteria in lake sediments as paleoecological indicators. J Paleolimnol 7(2):125–135CrossRefGoogle Scholar
  37. Robles S, Rodriguez JM, Granados I, Guerrero MC (2000) Sulfite-reducing clostridia in the sediment of a high mountain lake (Laguna Grande, Gredos, Spain) as indicators of fecal pollution. Int Microbiol 3(3):187–191PubMedGoogle Scholar
  38. Rothfuss F, Bender M, Conrad R (1997) Survival and activity of bacteria in a deep, aged lake sediment (Lake Constance). Microb Ecol 33(1):69–77PubMedCrossRefGoogle Scholar
  39. Ryves DB, Battarbee RW, Juggins S, Fritz SC, Anderson NJ (2006) Physical and chemical predictors of diatom dissolution in freshwater and saline lake sediments in North America and West Greenland. Limnol Oceanogr 51:1355–1368CrossRefGoogle Scholar
  40. Sastre V, Loizeau J-L, Greinert J, Naudts L, Arpagus P, Anselmett F et al (2010) Morphology and recent history of the Rhone River Delta in Lake Geneva (Switzerland). Swiss J Geosci 103(1):33–42CrossRefGoogle Scholar
  41. Schaller T, Wehrli B (1996) Geochemical-focusing of manganese in lake sediments: an indicator of deep-water oxygen conditions. Aquat Geochem 2(4):359–378Google Scholar
  42. Schleifer KH (2009) Phylum XIII. Firmicutes Gibbons and Murray 1978, 5 (Firmacutes [sic] Gibbons and Murray 1978, 5). In: Vos P, Garrity G, Jones D et al (eds) Bergey’s Manual® of systematic bacteriology. Springer, New York, pp 19–1317Google Scholar
  43. Seaward MR, Cross T, Unsworth BA (1976) Viable bacterial spores recovered from an archaeological excavation. Nature 261(5559):407–408PubMedCrossRefGoogle Scholar
  44. Shida O, Takagi H, Kadowaki K, Komagata K (1996) Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int J Syst Bacteriol 46(4):939–946PubMedCrossRefGoogle Scholar
  45. Shida O, Takagi H, Kadowaki K, Nakamura LK, Komagata K (1997) Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47(2):289–298PubMedCrossRefGoogle Scholar
  46. Sneath PH (1962) Longevity of micro-organisms. Nature 195:643–646PubMedCrossRefGoogle Scholar
  47. Stackebrandt E, Sproer C, Rainey FA, Burghardt J, Pauker O, Hippe H (1997) Phylogenetic analysis of the genus Desulfotomaculum: evidence for the misclassification of Desulfotomaculum guttoideum and description of Desulfotomaculum orientis as Desulfosporosinus orientis gen. nov., comb. nov. Int J Syst Bacteriol 47(4):1134–1139PubMedCrossRefGoogle Scholar
  48. Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346PubMedCrossRefGoogle Scholar
  49. Thevenon F, Graham ND, Herbez A, Wildi W, Pote J (2011) Spatio-temporal distribution of organic and inorganic pollutants from Lake Geneva (Switzerland) reveals strong interacting effects of sewage treatment plant and eutrophication on microbial abundance. Chemosphere 84(5):609–617PubMedCrossRefGoogle Scholar
  50. Thevenon F, Adatte T, Wildi W, Pote J (2012) Antibiotic resistant bacteria/genes dissemination in lacustrine sediments highly increased following cultural eutrophication of Lake Geneva (Switzerland). Chemosphere 86(5):468–476PubMedCrossRefGoogle Scholar
  51. Traag BA, Pugliese A, Eisen JA, Losick R (2013) Gene conservation among endospore-forming bacteria reveals additional sporulation genes in Bacillus subtilis. J Bacteriol 195(2):253–260PubMedCentralPubMedCrossRefGoogle Scholar
  52. Utkin I, Woese C, Wiegel J (1994) Isolation and characterization of Desulfitobacterium dehalogenans gen. nov., sp. nov., an anaerobic bacterium which reductively dechlorinates chlorophenolic compounds. Int J Syst Bacteriol 44(4):612–619PubMedCrossRefGoogle Scholar
  53. Vonlanthen P, Bittner D, Hudson AG, Young KA, Muller R, Lundsgaard-Hansen B et al (2012) Eutrophication causes speciation reversal in whitefish adaptive radiations. Nature 482(7385):357–362PubMedCrossRefGoogle Scholar
  54. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16):5261–5267PubMedCentralPubMedCrossRefGoogle Scholar
  55. Willard DA, Cronin TM (2007) Paleoecology and ecosystem restoration: case studies from Chesapeake Bay and the Florida Everglades. Front Ecol Environ 5(9):491–498CrossRefGoogle Scholar
  56. Wunderlin T, Junier T, Roussel-Delif L, Jeanneret N, Junier P (2013) Stage 0 sporulation gene A (spo0A) as a molecular marker to study diversity of endospore-forming Firmicutes. Environ Microbiol Rep 5(6):911–924Google Scholar

Copyright information

© Springer Basel 2013

Authors and Affiliations

  • Tina Wunderlin
    • 1
  • Juan Pablo Corella
    • 2
    • 3
  • Thomas Junier
    • 1
  • Matthieu Bueche
    • 1
  • Jean-Luc Loizeau
    • 4
  • Stéphanie Girardclos
    • 2
  • Pilar Junier
    • 1
    Email author
  1. 1.Laboratory of MicrobiologyUniversity of NeuchâtelNeuchâtelSwitzerland
  2. 2.Department of Geology and Paleontology, Institute for Environmental Sciences (ISE)University of GenevaGeneveSwitzerland
  3. 3.Museo Nacional de Ciencias Naturales (MNCN/CSIC)MadridSpain
  4. 4.Institute F. A. Forel, Earth and Environmental Sciences Section, Faculty of SciencesUniversity of GenevaGenevaSwitzerland

Personalised recommendations