Applied Microbiology and Biotechnology

, Volume 86, Issue 6, pp 1941–1946 | Cite as

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria

  • Bryan A. Stubblefield
  • Kristen E. Howery
  • Bianca N. Islam
  • Ariel J. Santiago
  • Wendy E. Cardenas
  • Eric S. Gilbert
Applied Microbial and Cell Physiology


Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.


Biofilm Multispecies biofilm Biocatalysis Environmental microbiology Microbial ecology 


  1. Adriaens P, Focht D (1990) Continuous coculture degradation of selected polychlorinated biphenyl congeners by Acinetobacter spp. in an aerobic reactor system. Environ Sci Technol 24:1042–1049CrossRefGoogle Scholar
  2. Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium. Proc Natl Acad Sci U S A 104:17300–17304CrossRefGoogle Scholar
  3. Brenner K, You L, Arnold FH (2008) Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol 26:483–489CrossRefGoogle Scholar
  4. Cheng KC, Catchmark JM, Demirci A (2009) Enhanced production of bacterial cellulose by using a biofilm reactor and its material property analysis. J Biol Eng 3:12CrossRefGoogle Scholar
  5. Cowan SE, Gilbert E, Liepmann D, Keasling JD (2000) Commensal interactions in a dual-species biofilm exposed to mixed organic compounds. Appl Environ Microbiol 66:4481–4485CrossRefGoogle Scholar
  6. de Godos I, Gonzalez C, Becares E, Garcia-Encina PA, Munoz R (2009) Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor. Appl Microbiol Biotechnol 82:187–194CrossRefGoogle Scholar
  7. Diels L, Van Roy S, Taghavi S, Van Houdt R (2009) From industrial sites to environmental applications with Cupriavidus metallidurans. Antonie Van Leeuwenhoek 96:247–258CrossRefGoogle Scholar
  8. Dybas MJ, Hyndman DW, Heine R, Tiedje J, Linning K, Wiggert D, Voice T, Zhao X, Dybas L, Criddle CS (2002) Development, operation, and long-term performance of a full-scale biocurtain utilizing bioaugmentation. Environ Sci Technol 36:3635–3644CrossRefGoogle Scholar
  9. Eun YJ, Weibel DB (2009) Fabrication of microbial biofilm arrays by geometric control of cell adhesion. Langmuir 25:4643–4654CrossRefGoogle Scholar
  10. Flickinger MC, Schottel JL, Bond DR, Aksan A, Scriven LE (2007) Painting and printing living bacteria: engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity. Biotechnol Prog 23:2–17CrossRefGoogle Scholar
  11. Franco-Rivera A, Paniagua-Michel J, Zamora-Castro J (2007) Characterization and performance of constructed nitrifying biofilms during nitrogen bioremediation of a wastewater effluent. J Ind Microbiol Biotechnol 34:279–287CrossRefGoogle Scholar
  12. Gilbert E, Keasling J (2004) Bench scale flow cell for nondestructive imaging of biofilms. In: Spencer J, Ragout de Spencer A (eds) Environmental microbiology methods and protocols. Humana Press, TotowaGoogle Scholar
  13. Gilbert ES, Walker AW, Keasling JD (2003) A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion. Appl Microbiol Biotechnol 61:77–81Google Scholar
  14. Hansen SK, Rainey PB, Haagensen JA, Molin S (2007) Evolution of species interactions in a biofilm community. Nature 445:533–536CrossRefGoogle Scholar
  15. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll BK, Molin S (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146(Pt 10):2395–2407Google Scholar
  16. Ho KL, Chung YC, Tseng CP (2008) Continuous deodorization and bacterial community analysis of a biofilter treating nitrogen-containing gases from swine waste storage pits. Bioresour Technol 99:2757–2765CrossRefGoogle Scholar
  17. Kim G, Lee S, Kim Y (2006) Subsurface biobarrier formation by microorganism injection for contaminant plume control. J Biosci Bioeng 101:142–148CrossRefGoogle Scholar
  18. Kuboniwa M, Hendrickson EL, Xia Q, Wang T, Xie H, Hackett M, Lamont RJ (2009) Proteomics of Porphyromonas gingivalis within a model oral microbial community. BMC Microbiol 9:98CrossRefGoogle Scholar
  19. Lee J, Jayaraman A, Wood TK (2007) Indole is an inter-species biofilm signal mediated by SdiA. BMC Microbiol 7:42CrossRefGoogle Scholar
  20. Li XZ, Webb JS, Kjelleberg S, Rosche B (2006) Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production. Appl Environ Microbiol 72:1639–1644CrossRefGoogle Scholar
  21. Li XZ, Hauer B, Rosche B (2007) Single-species microbial biofilm screening for industrial applications. Appl Microbiol Biotechnol 76:1255–1262CrossRefGoogle Scholar
  22. Nielsen AT, Tolker-Nielsen T, Barken KB, Molin S (2000) Role of commensal relationships on the spatial structure of a surface-attached microbial consortium. Environ Microbiol 2:59–68CrossRefGoogle Scholar
  23. Niu C, Gilbert ES (2004) Colorimetric method for identifying plant essential oil components that affect biofilm formation and structure. Appl Environ Microbiol 70:6951–6956CrossRefGoogle Scholar
  24. O’Connell HA, Kottkamp GS, Eppelbaum JL, Stubblefield BA, Gilbert SE, Gilbert ES (2006) Influences of biofilm structure and antibiotic resistance mechanisms on indirect pathogenicity in a model polymicrobial biofilm. Appl Environ Microbiol 72:5013–5019CrossRefGoogle Scholar
  25. O’Connell HA, Niu C, Gilbert ES (2007) Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm. Biotechnol Bioeng 97:439–446CrossRefGoogle Scholar
  26. Pamp SJ, Sternberg C, Tolker-Nielsen T (2009) Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy. Cytometry A 75:90–103Google Scholar
  27. Parsek MR, Singh PK (2003) Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57:677–701CrossRefGoogle Scholar
  28. Periasamy S, Kolenbrander PE (2009) Mutualistic biofilm communities develop with Porphyromonas gingivalis and initial, early, and late colonizers of enamel. J Bacteriol 191:6804–6811CrossRefGoogle Scholar
  29. Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS (2003) Bacterial coaggregation: an integral process in the development of multi-species biofilms. Trends Microbiol 11:94–100CrossRefGoogle Scholar
  30. Rosche B, Li XZ, Hauer B, Schmid A, Buehler K (2009) Microbial biofilms: a concept for industrial catalysis? Trends Biotechnol 27:636–643CrossRefGoogle Scholar
  31. Shim HW, Lee JH, Kim BY, Son YA, Lee CS (2009) Facile preparation of biopatternable surface for selective immobilization from bacteria to mammalian cells. J Nanosci Nanotechnol 9:1204–1209CrossRefGoogle Scholar
  32. Suh KY, Khademhosseini A, Yoo PJ, Langer R (2004) Patterning and separating infected bacteria using host–parasite and virus–antibody interactions. Biomed Microdevices 6:223–229CrossRefGoogle Scholar
  33. Verduzco-Luque CE, Alp B, Stephens GM, Markx GH (2003) Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation. Biotechnol Bioeng 83:39–44CrossRefGoogle Scholar
  34. Xu T, Petridou S, Lee EH, Roth EA, Vyavahare NR, Hickman JJ, Boland T (2004) Construction of high-density bacterial colony arrays and patterns by the ink-jet method. Biotechnol Bioeng 85:29–33CrossRefGoogle Scholar
  35. Zhang S, Norrlow O, Wawrzynczyk J, Dey ES (2004) Poly(3-hydroxybutyrate) biosynthesis in the biofilm of Alcaligenes eutrophus, using glucose enzymatically released from pulp fiber sludge. Appl Environ Microbiol 70:6776–6782CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Bryan A. Stubblefield
    • 1
  • Kristen E. Howery
    • 1
  • Bianca N. Islam
    • 1
  • Ariel J. Santiago
    • 1
  • Wendy E. Cardenas
    • 1
  • Eric S. Gilbert
    • 1
  1. 1.Department of BiologyGeorgia State UniversityAtlantaUSA

Personalised recommendations