SpringerLink
Log in

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

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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 76:8160–8173

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 204:163–167

    Article  CAS  PubMed  Google Scholar 

  • Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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:e1002039

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Borucki MK, Peppin JD, White D, Loge F, Call DR (2003) Variation in biofilm formation among strains of Listeria monocytogenes. Appl Environ Microbiol 69:7336–7342

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Choi KH, Schweizer HP (2006) Mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 1:153–161

    Article  CAS  PubMed  Google Scholar 

  • 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 2:443–448

    Article  CAS  PubMed  Google Scholar 

  • Davey ME, O’Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 15:36–48

    Article  CAS  PubMed  Google Scholar 

  • Eighmy TT, Maratea D, Bishop PL (1983) Electron microscopic examination of wastewater biofilm formation and structural components. Appl Environ Microbiol 45:1921–1931

    CAS  PubMed Central  PubMed  Google Scholar 

  • Flemming HC, Neu TR, Wozniak DJ (2007) The EPS matrix: the “house of biofilm cells”. J Bacteriol 189:7945–7947

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 78:7458–7462

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Friedman L, Kolter R (2004) Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 51:675–690

    Article  CAS  PubMed  Google Scholar 

  • Gjermansen M, Ragas P, Tolker-Nielsen T (2006) Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal. FEMS Microbiol Lett 265:215–224

    Article  CAS  PubMed  Google Scholar 

  • Gross R, Hauer B, Otto K, Schmid A (2007) Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol Bioeng 98:1123–1134

    Article  CAS  PubMed  Google Scholar 

  • Gross R, Lang K, Buehler K, Schmid A (2010) Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol Bioeng 105:705–717

    CAS  PubMed  Google Scholar 

  • Halan B, Schmid A, Buehler K (2010) Maximizing the productivity of catalytic biofilms on solid supports in membrane reactors. Biotechnol Bioeng 106:516–527

    Article  CAS  PubMed  Google Scholar 

  • Halan B, Schmid A, Buehler K (2011) Real-time solvent tolerance analysis of Pseudomonas sp. strain VLB120ΔC catalytic biofilms. Appl Environ Microbiol 77:1563–1571

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Halan B, Buehler K, Schmid A (2012) Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol 30:453–465

    Article  CAS  PubMed  Google Scholar 

  • 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 93:6562–6566

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  CAS  PubMed  Google Scholar 

  • Hay ID, Remminghorst U, Rehm BHA (2009) MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa. Appl Environ Microbiol 75:1110–1120

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hollander M, Wolfe DA, Chicken E (2014) Nonparametric statistical methods, 3rd edn. Wiley, Hoboken

    Google Scholar 

  • Inglis TJ, Millar MR, Jones JG, Robinson DA (1989) Tracheal tube biofilm as a source of bacterial colonization of the lung. J Clin Microbiol 27:2014–2018

    CAS  PubMed Central  PubMed  Google Scholar 

  • Karande R, Halan B, Schmid A, Buehler K (2014) Segmented flow is controlling growth of catalytic biofilms in continuous multiphase microreactors. Biotechnol Bioeng 111:1831–1840

    Article  CAS  PubMed  Google Scholar 

  • 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 72:5846–5856

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kirisits MJ, Prost L, Starkey M, Parsek MR (2005) Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 71:4809–4821

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 168:729–730

    Article  CAS  Google Scholar 

  • 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 103:2839–2844

    Article  PubMed  Google Scholar 

  • 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 73:1126–1135

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lappin-Scott HM, Bass C (2001) Biofilm formation: attachment, growth, and detachment of microbes from surfaces. Am J Infect Control 29:250–251

    Article  CAS  PubMed  Google Scholar 

  • Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE (1991) Optical sectioning of microbial biofilms. J Bacteriol 173:6558–6567

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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 65:1474–1484

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li YH, Tian X (2012) Quorum sensing and bacterial social interactions in biofilms. Sensors (Basel) 12:2519–2538

    Article  CAS  Google Scholar 

  • Lynch AS, Robertson GT (2008) Bacterial and fungal biofilm infections. Annu Rev Med 59:415–428

    Article  CAS  PubMed  Google Scholar 

  • Mann EE, Wozniak DJ (2012) Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol Rev 36:893–916

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 13:2702–2716

    Article  CAS  PubMed  Google Scholar 

  • 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 183:1041–1053

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Misiak K, Casey E, Murphy CD (2011) Factors influencing 4-fluorobenzoate degradation in biofilm cultures of Pseudomonas knackmussii B13. Water Res 45:3512–3520

    Article  CAS  PubMed  Google Scholar 

  • 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 193:4685–4698

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nicolella C, van Loosdrecht MC, Heijnen JJ (2000) Wastewater treatment with particulate biofilm reactors. J Biotechnol 80:1–33

    Article  CAS  PubMed  Google Scholar 

  • O’Shea TM, Klein AH, Geszvain K, Wolfe AJ, Visick KL (2006) Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri. J Bacteriol 188:8196–8205

    Article  PubMed Central  PubMed  Google Scholar 

  • O’Toole GA, Kolter R (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304

    Article  PubMed  Google Scholar 

  • 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 64:2032–2043

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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 98:1219–1229

    Article  CAS  PubMed  Google Scholar 

  • 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 97:4885–4890

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Römling U, Gomelsky M, Galperin MY (2005) C-di-GMP: the dawning of a novel bacterial signalling system. Mol Microbiol 57:629–639

    Article  PubMed  Google Scholar 

  • Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52

    Article  PubMed Central  PubMed  Google Scholar 

  • 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 325:279–281

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • 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 90:651–658

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Singh R, Paul D, Jain RK (2006) Biofilms: implications in bioremediation. Trends Microbiol 14:389–397

    Article  CAS  PubMed  Google Scholar 

  • 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 161:33–46

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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 50:15–27

    Article  CAS  PubMed  Google Scholar 

  • 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–e00312

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271

    Article  CAS  PubMed  Google Scholar 

  • 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 191:3492–3503

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 180:4416–4425

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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 161:71–79

    Article  CAS  PubMed  Google Scholar 

  • Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ (2006) Biofilm formation and cellulose expression among diverse environmental Pseudomonas isolates. Environ Microbiol 8:1997–2011

    Article  CAS  PubMed  Google Scholar 

  • 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:e1000483

    Article  PubMed Central  PubMed  Google Scholar 

  • 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 310:403–426

    Article  CAS  PubMed  Google Scholar 

  • 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 362:1119–1134

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Winn M, Casey E, Habimana O, Murphy CD (2014) Characteristics of Streptomyces griseus biofilms in continuous flow tubular reactors. FEMS Microbiol Lett 352:157–164

    Article  CAS  PubMed  Google Scholar 

  • Wood TK, Gonzalez Barrios AF, Herzberg M, Lee J (2006) Motility influences biofilm architecture in Escherichia coli. Appl Microbiol Biotechnol 72:361–367

    Article  CAS  PubMed  Google Scholar 

  • Wood TK, Hong SH, Ma Q (2011) Engineering biofilm formation and dispersal. Trends Biotechnol 29:87–94

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

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

ᅟ

Funding

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.

Author information

Authors and Affiliations

  1. Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Strasse 66, 44227, Dortmund, Germany

    Karolin Schmutzler & Katja Buehler

  2. Department of Solar Materials, Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318, Leipzig, Germany

    Andreas Schmid & Katja Buehler

Authors
  1. Karolin Schmutzler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Schmid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katja Buehler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Katja Buehler.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 579 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schmutzler, K., Schmid, A. & Buehler, K. A three-step method for analysing bacterial biofilm formation under continuous medium flow. Appl Microbiol Biotechnol 99, 6035–6047 (2015). https://doi.org/10.1007/s00253-015-6628-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6628-8

Keywords

Search

Navigation