Microbial Ecology

, Volume 42, Issue 1, pp 56–68 | Cite as

Assessment of changes in biodiversity when a community of ultramicrobacteria isolated from groundwater is stimulated to form a biofilm

  • N. Ross
  • R. Villemur
  • É. Marcandella
  • L. DeschênesEmail author


The stimulation of groundwater bacteria to form biofilms, for the remediation of polluted aquifers, is subjected to environmental regulations that included measurement of effects on microbial biodiversity. Groundwater microorganisms contain a proportion of unidentified and uncharacterized ultramicrobacteria (UMB) that might play a major role in the bioclogging of geological materials. This study aimed to assess the changes in genetic and metabolic biodiversity when a community of UMB, isolated from groundwater, is stimulated to form biofilms on a ceramic surface. UMB were stimulated with aerobic conditions and injection of molasses, in reactors reproducing groundwater composition and temperature. Concentration of planktonic viable UMB, secretion of extracellular polymeric substances (EPS), and biofilm thickness were monitored. The assessment of changes in biodiversity was achieved by comparing the initial UMB community to the biofilm community, using the single strand conformational polymorphism (SSCP) method, the cloning and sequencing of 16S rRNA gene (16S rDNA) sequences, and the Biolog microplate system. The hypothesis stating that indigenous UMB would play a significant role of in the biofilm development was corroborated. Within 13 days of stimulation, the UMB produced 700 mg L−1 of planktonic EPS and formed a biofilm up to a thickness of 1100 μm. This stimulation led to a decrease in genetic diversity and an increase in metabolic diversity. The decrease in genetic diversity was shown by a reduced number of single strand DNA fragments in the SSCP profiles. As such, 16S rDNA sequences from the biofilm revealed the predominance of four bacterial groups: Zoogloea, Bacillus/Paenibacillus, Enterobacteriaceae, and Pseudomonads. A significant increase in metabolic diversity was shown by a highest substrate richness profile and a lower substrate evenness profile of the biofilm bacterial population (p=0.0 and p=0.09, respectively). This higher metabolic diversity might be a consequence of the stimulation that seemed to favor the growth of bacteria having a high nutritional versatility. Stimulation of UMB, isolated from groundwater, was effective to form a biofilm having a high metabolic biodiversity. This combination of molecular-based and metabolic-based methods expanded the insight into monitoring the changes in bacterial biodiversity.


Extracellular Polymeric Substance Metabolic Diversity Cell Surface Hydrophobicity Single Strand Conformational Polymorphism Synthetic Groundwater 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Gavaskar AR, Gupta N, Sass BM, Janosy RJ, O'Sullivan D (1998) Permeable Barriers for Groundwater Remediation—Design, Construction, and Monitoring. Battelle Press, Columbus, OH, pp 1–176Google Scholar
  2. 2.
    Bellamy KL, de Lint N, Cullimore DR, Abiola A (1993) In-Situ Intercedent Biological Barriers for the Containment and Remediation of Contaminated Groundwater. Droycon Bioconcepts Inc, Regina, SK, Canada, ppGoogle Scholar
  3. 3.
    Cunningham AB, Characklis WG, Abedeen F, Crawford D (1991) Influence of biofilm accumulation on porous media hydrodynamics. Environ Sci Technol 25:1305–1311CrossRefGoogle Scholar
  4. 4.
    Vandevivere P, Baveye P (1992) Relationship between transport of bacteria and their clogging efficiency in sand columns. Appl Environ Microbiol 58:2523–2530PubMedGoogle Scholar
  5. 5.
    Sharp RR, Gerlach R, Cunningham A (1999) Bacterial transport issues related to subsurface biobarriers. In: In Situ and On-Site Bioremediation, the Fifth International Symposium. Battelle Press, San DiegoGoogle Scholar
  6. 6.
    Morita RY (1982) Starvation-survival of heterotrophs in the marine environment. In: Marshall KC (ed) Advances in Microbial Ecology, vol 6. Plenum Press, New York, pp 171–193Google Scholar
  7. 7.
    Iizuka T, Yamanaka S, Nishiyama T, Hiraishi A (1998) Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil. J Gen Appl Microbiol 44:75–84PubMedCrossRefGoogle Scholar
  8. 8.
    Kemp PF, Sherr BF, Sherr EB, Cole JJ (1993) Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp 1–777Google Scholar
  9. 9.
    Janssen PH, Schuhmann A, Morschel E, Rainey FA (1997) Novel aerobic ultramicrobacteria belonging to the verrucomicrobiales lineage of bacterial descent isolated by dilution culture from anoxic rice paddy soil. Appl Environ Microbiol 63:1382–1388PubMedGoogle Scholar
  10. 10.
    Johnston CD, Trefry MG, Rayner JL, Ragusa SR, De Zoysa DS, Davis GB (1999) In situ bioclogging for the confinement and remediation of groundwater hydrocarbon plumes. In: Proceedings of the 1999 Contaminated Site Remediation Conference—Contaminated Site Remediation: Challenges Posed by Urban and Industrial Contaminants, March 21–25, Fremantle, Western AustraliaGoogle Scholar
  11. 11.
    Environment Canada (1997) New Substances Notification Regulations. In: Canadian Environmental Protection Act (CEPA) (available at: Scholar
  12. 12.
    United States Environmental Protection Agency (1997) Point to consider in the preparation of TSCA biotechnology submissions for microorganisms. In: Toxic Substances Control Act (TSCA) Biotechnology Program, Office of Pollution Prevention and Toxics, Washington, DC, p 71 (available at: Scholar
  13. 13.
    Ketcheson DR, Zwiers WG (1997) Evaluation of in-situ restoration technologies for a DNAPL impacted fractured carbonate rock site at Smithville, Ontario. In: Air and Waste Management Association's 90th Annual Meeting and Exhibition, Toronto, Ontario, CanadaGoogle Scholar
  14. 14.
    Schut F, Gottschal JC, Prins RA (1997) Isolation and characterization of the marine ultramicrobacterium Sphingomonas sp. Strain RB2256. FEMS Microbiol Rev 20:363–369CrossRefGoogle Scholar
  15. 15.
    Novitsky JA, Morita RY (1976) Morphological characterization of small cells resulting from nutrient starvation of a psychrophilic marine Vibrio. Appl Environ Microbiol 32:617–622PubMedGoogle Scholar
  16. 16.
    Power M, van der Meer JR, Tchelet R, Egli T, Eggen R (1998) Molecular-based methods can contribute to assessment of toxicological risks and bioremediation strategies. J Microbiol Methods 32:107–119CrossRefGoogle Scholar
  17. 17.
    White DC, Flemming CA, Leung KT, Macnaughton SJ (1998) In situ microbial ecology for quantitative appraisal, monitoring and risk assessment of pollution remediation in soils, the subsurface, the rhizosphere and in biofilms. J Microbiol Methods 32:93–105CrossRefGoogle Scholar
  18. 18.
    Lehman RM, Colwell FS, Ringelberg DB, White DC (1995) Combined microbial community-level analyses for quality assurance of terrestrial subsurface cores. J Microbiol Methods 22:263–281CrossRefGoogle Scholar
  19. 19.
    El Fantroussi S, Verschuere L, Verstraete W, Top EM (1999) Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles. Appl Environ Microbiol 65:982–988PubMedGoogle Scholar
  20. 20.
    Head IM, Saunders JR, Pickup RW (1998) Microbial evoluation, diversity, and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol 35:1–21PubMedCrossRefGoogle Scholar
  21. 21.
    Marilley L, Vogt G, Blanc M, Aragno M (1998) Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis. Plant Soil 198:219–224CrossRefGoogle Scholar
  22. 22.
    Garland JL, Mills AL (1991) Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl Environ Microbiol 57:2351–2359PubMedGoogle Scholar
  23. 23.
    Konopka A, Oliver L, Turco RFJ (1998) The use of carbon substrate utilization patterns in environmental and ecological microbiology. Microb Ecol 35:103–115PubMedCrossRefGoogle Scholar
  24. 24.
    Ross N, Deschênes L, Bureau J, Clément B, Comeau Y, Samson R (1998) Ecotoxicological assessment and effects of physicochemical factors on biofilm development in groundwater conditions. Environ Sci Technol 32:1105–1111CrossRefGoogle Scholar
  25. 25.
    Boulos L, Prévost M, Barbeau B, Coallier J, Desjardins R (1999) Live/dead Baclight: Application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. Microbiol Methods 37:77–86CrossRefGoogle Scholar
  26. 26.
    Hacking AJ, Taylor IWF, Jarman TR, Govan JRW (1983) Alginate biosynthesis by Pseudomonas mendocina. J Gen Microbiol 129:3473–3480Google Scholar
  27. 27.
    Characklis WG, Marshall KC (1990) Biofilms. John Wiley & Sons, Inc, New York, pp 1–796Google Scholar
  28. 28.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Biology a Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, ppGoogle Scholar
  29. 29.
    Lee DH, Zo YG, Kim SJ (1996) Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation-polymorphism. Appl Environ Microbiol 62:3112–3120PubMedGoogle Scholar
  30. 30.
    Bassam BJ, Caetano-Anollés G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196:80–83PubMedCrossRefGoogle Scholar
  31. 31.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 69:1408–1412Google Scholar
  32. 32.
    Felsenstein J (1989) Phylip: Phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  33. 33.
    Kimura M (1980) A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evolution 16:111–120CrossRefGoogle Scholar
  34. 34.
    Mathieu L, Paquin JL, Block JC, Randon G, Maillard J, Reasoner D (1992) Paramètres gouvernant la prolifération bacterienne dans les réseaux de distribution. Rev sci eau 5:91–112Google Scholar
  35. 35.
    Zak JC, Willig MR, Moorhead DL, Wildman HG (1994) Functional diversity of microbial communities: A quantitative approach. Soil Biol Biochem 26:1101–1108CrossRefGoogle Scholar
  36. 36.
    Janssen PH, Schuhmann A, Bak F, Liesak W (1996) Disproportionation of inorganic sulfur compounds by the sulfate-reducing bacterium Desulfocapsa thiozymogenes ge. Nov. Sp. Nov Arch Microbiol 166:184–192CrossRefGoogle Scholar
  37. 37.
    Rosado AS, Duarte GF, Seldin L, Van Elsas JD (1998) Genetic diversity of nifh gene sequences in Paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments. Appl Environ Microbiol 64:2770–2779PubMedGoogle Scholar
  38. 38.
    Brenner DJ, Muller HE, Steigerwalt AG, Whitney AM, O'Hara CM, Kampfer P (1998) Two new Rahnella genemospecies that cannot be phenotypically differentiated from Rahnella aquatilis. Int J Syst Bacteriol 48:141–149PubMedCrossRefGoogle Scholar
  39. 39.
    Bos R, van der Mei HC, Busscher HJ (1999) Physicochemistry of initial microbial adhesive interactions—its mechanisms and methods for study. FEMS Microbiol Rev 23:179–230PubMedCrossRefGoogle Scholar
  40. 40.
    Mueller RF, Characklis WG, Jones WL, Sears JT (1992) Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis. Biotechnol Bioeng 39:1161–1170CrossRefPubMedGoogle Scholar
  41. 41.
    Bergey DH (1984) Bergey's Manual of Systematic Bacteriology, J.G. Holt, pp 1–1599Google Scholar
  42. 42.
    Haack SK, Garchow H, Klug MJ, Forney LJ (1995) Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns. Appl Environ Microbiol 61:1458–1468PubMedGoogle Scholar
  43. 43.
    Smalla K, Wachtendorf U, Heuer H, Liu W-T, Forney L (1998) Analysis of Biolog GN substrate utilization patterns by microbial communities. Appl Environ Microbiol 64:1220–1225PubMedGoogle Scholar
  44. 44.
    Verschuere L, Fievez V, Van Vooren L, Verstraete W (1997) The contribution of individual populations to the Biolog pattern of model microbial communities. FEMS Microbiol Ecol 24:353–362CrossRefGoogle Scholar
  45. 45.
    Garland JL (1996) Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biol Biochem 28:213–221CrossRefGoogle Scholar
  46. 46.
    Howard PJA (1997) Analysis of data from Biolog plates: Comments on the method of Garland and Mills. Soil Biol Biochem 29:1755–1757CrossRefGoogle Scholar
  47. 47.
    Fuller ME, Scow KM, Lau S, Ferris H (1997) Trichloroethylene (TCE) and toluene effects on the structure and function of the soil community. Soil Biol Biochem 29:75–89CrossRefGoogle Scholar
  48. 48.
    Talpsep E, Heinaru E, Truu J, Laht T, Heinaru A, Wand H, Stottmeister U (1997) Functional dynamics of microbial populations in waters contaminated with phenolic leachate. Oil Shale 14:435–453Google Scholar
  49. 49.
    Bääth E, Diaz-Ravina M, Frostegard A, Campbell CD (1998) Effect of metal-rich sludge amendments on the soil microbial community. Appl Environ Microbiol 64:238–245PubMedGoogle Scholar
  50. 50.
    Marshall KC (1984) Advances in Microbial Ecology, Vol 7. Plenum Press, New York, pp 1–223Google Scholar
  51. 51.
    Horowitz A, Krichevsky MI, Atlas RM (1983) Characteristics and diversity of subarctic marine oligotrophic, stenoheterotrophic, and euryheterotrophic bacterial populations. Can J Microbiol 29:527–535CrossRefGoogle Scholar
  52. 52.
    Stocker JG (1968) The ecology of a diatom community in a thermal stream. Br Phycol Bul 3:501–514Google Scholar
  53. 53.
    Derry AM, Staddon WJ, Trevors JT (1998) Functional diversity and community structure of micro-organisms of uncontaminated and creosote-contaminated soils as determined by sole-carbon-source-utilization. World J Microbiol Biotechnol 14:571–578CrossRefGoogle Scholar
  54. 54.
    Cusack F, Singh S, McCarthy C, Grieco J, De Rocco M, Ngyen D, Lappin-Scott H, Costerton JW (1992) Enhanced oil recovery—three-dimensional sandpack simulation of ultramicrobacteria resuscitation in reservoir formation. J Gen Microbiol 138:647–655Google Scholar
  55. 55.
    Egli T (1995) The ecological and physiological significance of the growth of heterotrophic micro-organisms with mixtures of substrates. FEMS Microbiol Ecol 20:363–369Google Scholar
  56. 56.
    VanDemark PJ, Batzing BL (1987) The Microbes—an Introduction to Their Nature and Importance. Benjamin/Cummings Science, Inc, San Francisco, pp 1–991Google Scholar
  57. 57.
    Harder J, Probian C (1997) Anaerobic mineralisation of cholesterol by a novel type of denitrifying bacterium. Arch Microbiol 167:269–274PubMedCrossRefGoogle Scholar
  58. 58.
    Rossello-Mora RA, Wagner M, Amann R, Schleifer KH (1995) The abundance of Zoogloea ramigera in sewage treatment plants. Appl Environ Microbiol 61:702–707PubMedGoogle Scholar
  59. 59.
    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 47:289–298PubMedCrossRefGoogle Scholar
  60. 60.
    Marshall BJ, Ohye DF (1966) Bacillus macquariensis n.Sp., a psychrotrophic bacterium from sub-antarctic soil. J Gen Microbiol 44:41–46PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc 2001

Authors and Affiliations

  • N. Ross
    • 1
  • R. Villemur
    • 2
  • É. Marcandella
    • 3
  • L. Deschênes
    • 3
    Email author
  1. 1.National Water Research InstituteEnvironment CanadaBurlingtonCanada
  2. 2.Microbiologie et BiotechnologieINRS-Institut Armand-FrappierLavalCanada
  3. 3.NSERC Industrial Chair in Site Remediation and Management, Department of Chemical EngineeringÉcole Polytechnique de MontréalMontrealCanada

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