Cultivation of Environmental Bacterial Communities as Multispecies Biofilms

  • Marc A. Demeter
  • Joe Lemire
  • Susanne Golby
  • Monika Schwering
  • Howard Ceri
  • Raymond J. Turner
Part of the Springer Protocols Handbooks book series (SPH)


Microbes play an important role in the biogeochemistry of hydrocarbons and are frequently studied for potential applications in hydrocarbon biotechnologies. The empirical study of microbes often requires their cultivation, a problematic proposition as approximately only 1% of bacteria may be cultured by traditional methods. One promising strategy to grow the “unculturables” is to grow environmental microbes directly as mixed species biofilms – surface-bound, slime-encapsulated microbial communities. Mixed species biofilms can mimic natural environmental conditions and support microbial growth through beneficial social interactions between microbes, along with the inclusion of cell-cell signaling. Here, we describe a simple, flexible method for growing environmental mixed species biofilms in vitro using the Calgary Biofilm Device (CBD). Additionally, we describe a battery of assays for biofilm characterization. Using our approach, we have successfully grown mixed-species biofilms from a variety of hydrocarbon-contaminated environments, while demonstrating high retention of the original microbial diversity and the metabolic potential for biotechnology applications.


Biofilms Calgary Biofilm Device Unculturables 


  1. 1.
    Magot M, Ollivier B, Patel BKC (2000) Microbiology of petroleum reservoirs. Antonie Van Leeuwenhoek 77:103–116CrossRefPubMedGoogle Scholar
  2. 2.
    Head IM, Jones DM, Larter SR (2003) Biological activity in the deep subsurface and the origin of heavy oil. Nature 426:344–352CrossRefPubMedGoogle Scholar
  3. 3.
    Atlas RM, Hazen TC (2011) Oil biodegradation and bioremediation: a tale of the two worst spills in U.S. history. Environ Sci Technol 45:6709–6715CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Brar SK, Verma M, Surampalli RY et al (2006) Bioremediation of hazardous wastes—a review, practice periodical of hazardous. Toxic Radioactive Waste Manag 10:59–72CrossRefGoogle Scholar
  5. 5.
    Allard A-S, Neilson AH (1997) Bioremediation of organic waste sites: a critical review of microbiological aspects. Int Biodeterior Biodegradation 39:253–285CrossRefGoogle Scholar
  6. 6.
    Edwards SJ, Kjellerup BV (2013) Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals. Appl Microbiol Biotechnol 97:9909–9921CrossRefPubMedGoogle Scholar
  7. 7.
    Janssen PH (2006) Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719–1728CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Keller M, Zengler K (2004) Tapping into microbial diversity. Nat Rev Microbiol 2:141–150CrossRefPubMedGoogle Scholar
  9. 9.
    Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of “unculturable” bacteria. FEMS Microbiol Lett 309:1–7Google Scholar
  10. 10.
    Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–4160CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Costerton JW, Lewandowski Z, Caldwell DE et al (1995) Microbial biofilms. Annu Rev Microbiol 49:711–745CrossRefPubMedGoogle Scholar
  12. 12.
    Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108CrossRefPubMedGoogle Scholar
  13. 13.
    Beveridge TJ, Makin SA, Kadurugamuwa JL et al (1997) Interactions between biofilms and the environment. FEMS Microbiol Rev 20:291–303CrossRefPubMedGoogle Scholar
  14. 14.
    Burmølle M, Ren D, Bjarnsholt T et al (2014) Interactions in multispecies biofilms: do they actually matter? Trends Microbiol 22:84–91CrossRefPubMedGoogle Scholar
  15. 15.
    Nadell CD, Bucci V, Drescher K et al (2013) Cutting through the complexity of cell collectives. Proc R Soc B Biol Sci 280:1–11CrossRefGoogle Scholar
  16. 16.
    Nadell CD, Xavier JB, Foster KR (2009) The sociobiology of biofilms. FEMS Microbiol Rev 33:206–224CrossRefPubMedGoogle Scholar
  17. 17.
    Mitri S, Xavier JB, Foster KR (2011) Social evolution in multispecies biofilms. Proc Natl Acad Sci 108:10839–10846CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Parsek MR, Greenberg EP (2005) Sociomicrobiology: the connections between quorum sensing and biofilms. Trends Microbiol 13:27–33CrossRefPubMedGoogle Scholar
  19. 19.
    Joint I, Mühling M, Querellou J (2010) Culturing marine bacteria – an essential prerequisite for biodiscovery. Microb Biotechnol 3:564–575CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ceri H, Olson ME, Stremick CA et al (1999) The Calgary biofilm device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37:1771–1776PubMedPubMedCentralGoogle Scholar
  21. 21.
    Golby S, Ceri H, Gieg LM et al (2012) Evaluation of microbial biofilm communities from an Alberta oil sands tailings pond. FEMS Microbiol Ecol 79:240–250CrossRefPubMedGoogle Scholar
  22. 22.
    Demeter MA, Lemire J, George I et al (2014) Harnessing oil sands microbial communities for use in ex situ naphthenic acid bioremediation. Chemosphere 97:78–85CrossRefPubMedGoogle Scholar
  23. 23.
    Teitzel GM, Parsek MR (2003) Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl Environ Microbiol 69:2313–2320CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Harrison JJ, Turner RJ, Ceri H (2005) High-throughput metal susceptibility testing of microbial biofilms. BMC Microbiol 5:53CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Harrison JJ, Ceri H, Yerly J et al (2006) The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary biofilm device. Biol Proced Online 8:194–215CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Harrison JJ, Stremick CA, Turner RJ et al (2010) Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening. Nat Protoc 5:1236–1254CrossRefPubMedGoogle Scholar
  27. 27.
    Sule P, Wadhawan T, Carr NJ et al (2009) A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants. Lett Appl Microbiol 49:299–304CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Armitano J, Méjean V, Jourlin-Castelli C (2013) Aerotaxis governs floating biofilm formation in Shewanella oneidensis. Environ Microbiol 15:3108–3118PubMedGoogle Scholar
  29. 29.
    Lembke C, Podbielski A, Hidalgo-Grass C et al (2006) Characterization of biofilm formation by clinically relevant serotypes of group A Streptococci. Appl Environ Microbiol 72:2864–2875CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Xue Z, Lee WH, Coburn KM et al (2014) Selective reactivity of monochloramine with extracellular matrix components affects the disinfection of biofilm and detached clusters. Environ Sci Technol 48:3832–3839CrossRefPubMedGoogle Scholar
  31. 31.
    Lee KWK, Periasamy S, Mukherjee M et al (2013) Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J 2013:1–14Google Scholar
  32. 32.
    Horemans B, Breugelmans P, Hofkens J et al (2013) Environmental dissolved organic matter governs biofilm formation and subsequent linuron degradation activity of a linuron-degrading bacterial consortium. Appl Environ Microbiol 79:4534–4542CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Burmolle M, Webb JS, Rao D et al (2006) Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms. Appl Environ Microbiol 72:3916–3923CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Paulsen IT, Press CM, Ravel J et al (2005) Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat Biotechnol 23:873–878CrossRefPubMedGoogle Scholar
  35. 35.
    Ritalahti KM, Amos BK, Sung Y et al (2006) Quantitative PCR targeting 16S rRNA and reductive Dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Appl Environ Microbiol 72:2765–2774CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Golby S, Ceri H, Marques LLR et al (2013) Mixed-species biofilms cultured from an oil sand tailings pond can biomineralize metals. Microb Ecol 68:70–80CrossRefPubMedGoogle Scholar
  37. 37.
    Lehtinen J, Virta M, Lilius E-M (2003) Fluoro-luminometric real-time measurement of bacterial viability and killing. J Microbiol Methods 55:173–186CrossRefPubMedGoogle Scholar
  38. 38.
    Arretxe M, Heap JM, Christofi N (1997) The effect of toxic discharges on ATP content in activated sludge. Environ Toxicol 12:23–29Google Scholar
  39. 39.
    Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73:127–141CrossRefPubMedGoogle Scholar
  40. 40.
    Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedPubMedCentralGoogle Scholar
  41. 41.
    Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317–322CrossRefPubMedGoogle Scholar
  42. 42.
    Deng W, Xi D, Mao H et al (2007) The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review. Mol Biol Rep 35:265–274CrossRefPubMedGoogle Scholar
  43. 43.
    Marzorati M, Wittebolle L, Boon N et al (2008) How to get more out of molecular fingerprints: practical tools for microbial ecology. Environ Microbiol 10:1571–1581CrossRefPubMedGoogle Scholar
  44. 44.
    Galan M, Guivier E, Caraux G et al (2010) A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies. BMC Genomics 11:296CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Rothberg JM, Leamon JH (2008) The development and impact of 454 sequencing. Nat Biotechnol 26:1117–1124CrossRefPubMedGoogle Scholar
  46. 46.
    Ramos-Padrón E, Bordenave S, Lin S et al (2011) Carbon and sulfur cycling by microbial communities in a gypsum-treated oil sands tailings pond. Environ Sci Technol 45:439–446CrossRefPubMedGoogle Scholar
  47. 47.
    Liu R, Yu Z, Guo H et al (2012) Pyrosequencing analysis of eukaryotic and bacterial communities in faucet biofilms. Sci Total Environ 435–436:124–131CrossRefPubMedGoogle Scholar
  48. 48.
    Hong PY, Hwang C, Ling F et al (2010) Pyrosequencing analysis of bacterial biofilm communities in water meters of a drinking water distribution system. Appl Environ Microbiol 76:5631–5635CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Harrison JJ, Rabiei M, Turner RJ et al (2006) Metal resistance in Candida biofilms. FEMS Microbiol Ecol 55:479–491CrossRefPubMedGoogle Scholar
  50. 50.
    Schwering M, Song J, Louie M et al (2013) Multi-species biofilms defined from drinking water microorganisms provide increased protection against chlorine disinfection. Biofouling 29:917–928CrossRefPubMedGoogle Scholar
  51. 51.
    Lee ZMP, Bussema C, Schmidt TM (2009) rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea. Nucleic Acids Res 37:D489–D493CrossRefPubMedGoogle Scholar
  52. 52.
    Klappenbach JA, Dunbar JM, Schmidt TM (2000) rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66:1328–1333CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Tremaroli V, Vacchi Suzzi C, Fedi S et al (2010) Tolerance of Pseudomonas pseudoalcaligenes KF707 to metals, polychlorobiphenyls and chlorobenzoates: effects on chemotaxis-, biofilm- and planktonic-grown cells. FEMS Microbiol Ecol 74:291–301CrossRefPubMedGoogle Scholar
  54. 54.
    Sharp CE, Brady AL, Sharp GH et al (2014) Humboldt’s spa: microbial diversity is controlled by temperature in geothermal environments. ISME J 8:1166–1174CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Soh J, Dong X, Caffrey SM et al (2013) Phoenix 2: a locally installable large-scale 16S rRNA gene sequence analysis pipeline with Web interface. J Biotechnol 167:393–403CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Marc A. Demeter
    • 1
  • Joe Lemire
    • 1
  • Susanne Golby
    • 1
  • Monika Schwering
    • 1
  • Howard Ceri
    • 1
  • Raymond J. Turner
    • 1
  1. 1.Biofilm Research Group, Department of Biological SciencesUniversity of CalgaryCalgaryCanada

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