Applied Microbiology and Biotechnology

, Volume 99, Issue 14, pp 6035–6047 | Cite as

A three-step method for analysing bacterial biofilm formation under continuous medium flow

  • Karolin Schmutzler
  • Andreas Schmid
  • Katja BuehlerEmail author
Methods and protocols


For the investigation and comparison of microbial biofilms, a variety of analytical methods have been established, all focusing on different growth stages and application areas of biofilms. In this study, a novel quantitative assay for analysing biofilm maturation under the influence of continuous flow conditions was developed using the interesting biocatalyst Pseudomonas taiwanensis VLB120. In contrast to other tubular-based assay systems, this novel assay format delivers three readouts using a single setup in a total assay time of 40 h. It combines morphotype analysis of biofilm colonies with the direct quantification of biofilm biomass and pellicle formation on an air/liquid interphase. Applying the Tube-Assay, the impact of the second messenger cyclic diguanylate on biofilm formation of P. taiwanensis VLB120 was investigated. To this end, 41 deletions of genes encoding for protein homologues to diguanylate cyclase and phosphodiesterase were generated in the genome of P. taiwanensis VLB120. Subsequently, the biofilm formation of the resulting mutants was analysed using the Tube-Assay. In more than 60 % of the mutants, a significantly altered biofilm formation as compared to the parent strain was detected. Furthermore, the potential of the proposed Tube-Assay was validated by investigating the biofilms of several other bacterial species.


Pseudomonas taiwanensis VLB120 Tubular biofilm Biofilm dry weight Pellicle Single species biofilm Cyclic diguanylate 



We are grateful to Dr. Nick Wierckx of RWTH Aachen University, Institute of Applied Microbiology (iAMB), Aachen and Prof. Victor de Lorenzo of CNB, CSIC, Madrid for providing required plasmids. We thank Prof. Katja Ickstadt of TU Dortmund University, Faculty of Statistics, for her support during statistical data analysis.

Compliance with ethical standards


This study was funded by the Ministry of Innovation, Science and Research of North Rhine-Westphalia in the frame of CLIB-Graduate Cluster Industrial Biotechnology (contract no: 314–108 001 08).

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights and informed consent

This article does not contain any studies with human or animal subjects.

Supplementary material

253_2015_6628_MOESM1_ESM.pdf (579 kb)
ESM 1 (PDF 579 kb)


  1. An S, Wu J, Zhang LH (2010) Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-Di-GMP phosphodiesterase with a putative hypoxia-sensing domain. Appl Environ Microbiol 76:8160–8173PubMedCentralPubMedCrossRefGoogle Scholar
  2. Ausmees N, Mayer R, Weinhouse H, Volman G, Amikam D, Benziman M, Lindberg M (2001) Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity. FEMS Microbiol Lett 204:163–167PubMedCrossRefGoogle Scholar
  3. Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedCentralPubMedGoogle Scholar
  4. Bordeleau E, Fortier LC, Malouin F, Burrus V (2011) C-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases. PLoS Genet 7:e1002039PubMedCentralPubMedCrossRefGoogle Scholar
  5. Borucki MK, Peppin JD, White D, Loge F, Call DR (2003) Variation in biofilm formation among strains of Listeria monocytogenes. Appl Environ Microbiol 69:7336–7342PubMedCentralPubMedCrossRefGoogle Scholar
  6. Choi KH, Schweizer HP (2006) Mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 1:153–161PubMedCrossRefGoogle Scholar
  7. Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP (2005) A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443–448PubMedCrossRefGoogle Scholar
  8. Davey ME, O’Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867PubMedCentralPubMedCrossRefGoogle Scholar
  9. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193PubMedCentralPubMedCrossRefGoogle Scholar
  10. Duque E, de la Torre J, Bernal P, Molina-Henares MA, Alaminos M, Espinosa-Urgel M, Roca A, Fernandez M, de Bentzmann S, Ramos JL (2013) Identification of reciprocal adhesion genes in pathogenic and non-pathogenic Pseudomonas. Environ Microbiol 15:36–48PubMedCrossRefGoogle Scholar
  11. Eighmy TT, Maratea D, Bishop PL (1983) Electron microscopic examination of wastewater biofilm formation and structural components. Appl Environ Microbiol 45:1921–1931PubMedCentralPubMedGoogle Scholar
  12. Flemming HC, Neu TR, Wozniak DJ (2007) The EPS matrix: the “house of biofilm cells”. J Bacteriol 189:7945–7947PubMedCentralPubMedCrossRefGoogle Scholar
  13. Franklin FC, Bagdasarian M, Bagdasarian MM, Timmis KN (1981) Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci U S A 78:7458–7462PubMedCentralPubMedCrossRefGoogle Scholar
  14. Friedman L, Kolter R (2004) Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 51:675–690PubMedCrossRefGoogle Scholar
  15. Gjermansen M, Ragas P, Tolker-Nielsen T (2006) Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal. FEMS Microbiol Lett 265:215–224PubMedCrossRefGoogle Scholar
  16. Gross R, Hauer B, Otto K, Schmid A (2007) Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol Bioeng 98:1123–1134PubMedCrossRefGoogle Scholar
  17. Gross R, Lang K, Buehler K, Schmid A (2010) Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol Bioeng 105:705–717PubMedGoogle Scholar
  18. Halan B, Schmid A, Buehler K (2010) Maximizing the productivity of catalytic biofilms on solid supports in membrane reactors. Biotechnol Bioeng 106:516–527PubMedCrossRefGoogle Scholar
  19. Halan B, Schmid A, Buehler K (2011) Real-time solvent tolerance analysis of Pseudomonas sp. strain VLB120ΔC catalytic biofilms. Appl Environ Microbiol 77:1563–1571PubMedCentralPubMedCrossRefGoogle Scholar
  20. Halan B, Buehler K, Schmid A (2012) Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol 30:453–465PubMedCrossRefGoogle Scholar
  21. Hammar M, Bian Z, Normark S (1996) Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli. Proc Natl Acad Sci U S A 93:6562–6566PubMedCentralPubMedCrossRefGoogle Scholar
  22. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580PubMedCrossRefGoogle Scholar
  23. Hay ID, Remminghorst U, Rehm BHA (2009) MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol 75:1110–1120PubMedCentralPubMedCrossRefGoogle Scholar
  24. Hollander M, Wolfe DA, Chicken E (2014) Nonparametric statistical methods, 3rd edn. Wiley, HobokenGoogle Scholar
  25. Inglis TJ, Millar MR, Jones JG, Robinson DA (1989) Tracheal tube biofilm as a source of bacterial colonization of the lung. J Clin Microbiol 27:2014–2018PubMedCentralPubMedGoogle Scholar
  26. Karande R, Halan B, Schmid A, Buehler K (2014) Segmented flow is controlling growth of catalytic biofilms in continuous multiphase microreactors. Biotechnol Bioeng 111:1831–1840PubMedCrossRefGoogle Scholar
  27. Kim H, Ryu JH, Beuchat LR (2006) Attachment of and biofilm formation by Enterobacter sakazakii on stainless steel and enteral feeding tubes. Appl Environ Microbiol 72:5846–5856PubMedCentralPubMedCrossRefGoogle Scholar
  28. Kirisits MJ, Prost L, Starkey M, Parsek MR (2005) Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 71:4809–4821PubMedCentralPubMedCrossRefGoogle Scholar
  29. Koehler KA, Rueckert C, Schatschneider S, Vorhoelter FJ, Szczepanowski R, Blank LM, Niehaus K, Goesmann A, Puehler A, Kalinowski J, Schmid A (2013) Complete genome sequence of Pseudomonas sp. strain VLB120 a solvent tolerant, styrene degrading bacterium, isolated from forest soil. J Biotechnol 168:729–730CrossRefGoogle Scholar
  30. Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J, Miyata S, Lee DG, Neely AN, Hyodo M, Hayakawa Y, Ausubel FM, Lory S (2006) Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence. Proc Natl Acad Sci U S A 103:2839–2844PubMedCrossRefGoogle Scholar
  31. Lambert JM, Bongers RS, Kleerebezem M (2007) Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol 73:1126–1135PubMedCentralPubMedCrossRefGoogle Scholar
  32. Lappin-Scott HM, Bass C (2001) Biofilm formation: attachment, growth, and detachment of microbes from surfaces. Am J Infect Control 29:250–251PubMedCrossRefGoogle Scholar
  33. Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE (1991) Optical sectioning of microbial biofilms. J Bacteriol 173:6558–6567PubMedCentralPubMedGoogle Scholar
  34. Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S (2007) A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol Microbiol 65:1474–1484PubMedCentralPubMedCrossRefGoogle Scholar
  35. Li YH, Tian X (2012) Quorum sensing and bacterial social interactions in biofilms. Sensors (Basel) 12:2519–2538CrossRefGoogle Scholar
  36. Lynch AS, Robertson GT (2008) Bacterial and fungal biofilm infections. Annu Rev Med 59:415–428PubMedCrossRefGoogle Scholar
  37. Mann EE, Wozniak DJ (2012) Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol Rev 36:893–916PubMedCentralPubMedCrossRefGoogle Scholar
  38. Martinez-Garcia E, de Lorenzo V (2011) Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13:2702–2716PubMedCrossRefGoogle Scholar
  39. McDonald MJ, Gehrig SM, Meintjes PL, Zhang XX, Rainey PB (2009) Adaptive divergence in experimental populations of Pseudomonas fluorescens. IV. Genetic constraints guide evolutionary trajectories in a parallel adaptive radiation. Genetics 183:1041–1053PubMedCentralPubMedCrossRefGoogle Scholar
  40. Misiak K, Casey E, Murphy CD (2011) Factors influencing 4-fluorobenzoate degradation in biofilm cultures of Pseudomonas knackmussii B13. Water Res 45:3512–3520PubMedCrossRefGoogle Scholar
  41. Newell PD, Yoshioka S, Hvorecny KL, Monds RD, O’Toole GA (2011) Systematic analysis of diguanylate cyclases that promote biofilm formation by Pseudomonas fluorescens p f0–1. J Bacteriol 193:4685–4698PubMedCentralPubMedCrossRefGoogle Scholar
  42. Nicolella C, van Loosdrecht MC, Heijnen JJ (2000) Wastewater treatment with particulate biofilm reactors. J Biotechnol 80:1–33PubMedCrossRefGoogle Scholar
  43. O’Shea TM, Klein AH, Geszvain K, Wolfe AJ, Visick KL (2006) Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri. J Bacteriol 188:8196–8205PubMedCentralPubMedCrossRefGoogle Scholar
  44. O’Toole GA, Kolter R (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304PubMedCrossRefGoogle Scholar
  45. Panke S, Witholt B, Schmid A, Wubbolts MG (1998) Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl Environ Microbiol 64:2032–2043PubMedCentralPubMedGoogle Scholar
  46. Park JB, Buehler B, Panke S, Witholt B, Schmid A (2007) Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120ΔC. Biotechnol Bioeng 98:1219–1229PubMedCrossRefGoogle Scholar
  47. Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97:4885–4890PubMedCentralPubMedCrossRefGoogle Scholar
  48. Römling U, Gomelsky M, Galperin MY (2005) C-di-GMP: the dawning of a novel bacterial signalling system. Mol Microbiol 57:629–639PubMedCrossRefGoogle Scholar
  49. Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52PubMedCentralPubMedCrossRefGoogle Scholar
  50. Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinberger-Ohana P, Mayer R, Braun S, de Vroom E, van der Marel GA, van Boom JH, Benziman M (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281PubMedCrossRefGoogle Scholar
  51. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  52. Sanchez-Torres V, Hu H, Wood T (2011) GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli. Appl Microbiol Biotechnol 90:651–658PubMedCentralPubMedCrossRefGoogle Scholar
  53. Singh R, Paul D, Jain RK (2006) Biofilms: implications in bioremediation. Trends Microbiol 14:389–397PubMedCrossRefGoogle Scholar
  54. Spiers AJ, Kahn SG, Bohannon J, Travisano M, Rainey PB (2002) Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness. Genetics 161:33–46PubMedCentralPubMedGoogle Scholar
  55. Spiers AJ, Bohannon J, Gehrig SM, Rainey PB (2003) Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 50:15–27PubMedCrossRefGoogle Scholar
  56. Spurbeck RR, Tarrien RJ, Mobley HL (2012) Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of motility or sessility in Escherichia coli CFT073. MBio 3:e00307–e00312PubMedCentralPubMedCrossRefGoogle Scholar
  57. Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271PubMedCrossRefGoogle Scholar
  58. Starkey M, Hickman JH, Ma L, Zhang N, De Long S, Hinz A, Palacios S, Manoil C, Kirisits MJ, Starner TD, Wozniak DJ, Harwood CS, Parsek MR (2009) Pseudomonas aeruginosa rugose small-colony variants have adaptations that likely promote persistence in the cystic fibrosis lung. J Bacteriol 191:3492–3503PubMedCentralPubMedCrossRefGoogle Scholar
  59. Tal R, Wong HC, Calhoon R, Gelfand D, Fear AL, Volman G, Mayer R, Ross P, Amikam D, Weinhouse H, Cohen A, Sapir S, Ohana P, Benziman M (1998) Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J Bacteriol 180:4416–4425PubMedCentralPubMedGoogle Scholar
  60. Troeschel SC, Thies S, Link O, Real CI, Knops K, Wilhelm S, Rosenau F, Jaeger K-E (2012) Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida. J Biotechnol 161:71–79PubMedCrossRefGoogle Scholar
  61. Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ (2006) Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates. Environ Microbiol 8:1997–2011PubMedCrossRefGoogle Scholar
  62. Ueda A, Wood TK (2009) Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5:e1000483PubMedCentralPubMedCrossRefGoogle Scholar
  63. Weiner R, Seagren E, Arnosti C, Quintero E (1999) Bacterial survival in biofilms: probes for exopolysaccharide and its hydrolysis, and measurements of intra- and interphase mass fluxes. Methods Enzymol 310:403–426PubMedCrossRefGoogle Scholar
  64. Williams P, Winzer K, Chan WC, Camara M (2007) Look who’s talking: communication and quorum sensing in the bacterial world. Philos Trans R Soc Lond B Biol Sci 362:1119–1134PubMedCentralPubMedCrossRefGoogle Scholar
  65. Winn M, Casey E, Habimana O, Murphy CD (2014) Characteristics of Streptomyces griseus biofilms in continuous flow tubular reactors. FEMS Microbiol Lett 352:157–164PubMedCrossRefGoogle Scholar
  66. Wood TK, Gonzalez Barrios AF, Herzberg M, Lee J (2006) Motility influences biofilm architecture in Escherichia coli. Appl Microbiol Biotechnol 72:361–367PubMedCrossRefGoogle Scholar
  67. Wood TK, Hong SH, Ma Q (2011) Engineering biofilm formation and dispersal. Trends Biotechnol 29:87–94PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Karolin Schmutzler
    • 1
  • Andreas Schmid
    • 2
  • Katja Buehler
    • 1
    • 2
    Email author
  1. 1.Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical EngineeringTU Dortmund UniversityDortmundGermany
  2. 2.Department of Solar MaterialsHelmholtz-Centre for Environmental ResearchLeipzigGermany

Personalised recommendations