Skip to main content

Spatial Architecture of Nitrifying Bacteria Biofilm Immobilized on Polyurethane Foam in an Automatic Biodetector for Water Toxicity

  • Andrzej Woznica (a1), Jagna Karcz (a2), Agnieszka Nowak (a1), Aleksander Gmur (a3) and Tytus Bernas (a4) (a5)...

We describe the architecture of nitrifying bacteria biofilms immobilized on a three-dimensional (3D) polyurethane foam that permits efficient water flow through a bioreactor. The 3D spatial organization of immobilized bacterial colonies is characterized on three resolution levels with X-ray tomography, light confocal microscopy, and scanning electron microscopy (SEM). Using these techniques we demonstrate biofilm distribution in the foam and the existence of several modes of binding of bacteria to the foam. Computed X-ray tomography permits observation of the distribution of the biofilm in the whole open cellular polyurethane material volume and estimation of biofilm volume. SEM and confocal laser scanning microscopy techniques permit 3D visualization of biofilm structure. Three distinct immobilization patterns could be observed in the open cellular polyurethane material: (1) large irregular aggregates of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) spherical (pom-pom) aggregates of bacteria localized on the external surface of biofilm; and (3) biofilm threads adherent to the surface of polyurethane foam. Finally, we demonstrate that immobilized bacteria exhibit metabolic activity and growth.

Corresponding author
Corresponding author. E-mail:
Hide All
Al-Raoush R.I. & Willson C.S. (2005). Extraction of physically realistic pore network properties from three-dimensional synchrotron X-ray microtomography images of unconsolidated porous media systems. J Hydrol 300, 4464.
Barnes L.M., Lo M.F., Adams M.R. & Chamberlain A.H.L. (1999). Effect of milk proteins on adhesion of bacteria to stainless steel surfaces. Appl Environ Microb 61, 45434548.
Bothe H., Jost G., Schloter M., Ward B.B. & Witzel K-P. (2000). Molecular analysis of ammonia oxidation and denitrification in natural environments. FEMS Microbiol Rev 24, 673690.
Coskuner G., Ballinger S.J., Davenport R.J., Pickering R.L., Solera R., Head I.M. & Curtis T.P. (2005). Agreement between theory and measurement in quantification of ammonia-oxidizing bacteria. Appl Environ Microb 71, 63256334.
Daims H., Brühl A., Amann R., Schleifer K.-H. & Wagner M. (1999). The domain-specific probe EUB338 is insufficient for the detection of all bacteria: Development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22, 434444.
Daims H., Nielsen J.L., Nielsen P.H., Schleifer K-H. & Wagner M. (2001). In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants. Appl Environ Microb 67, 52735284.
Delatolla R., Tufenkji N., Comeau Y., Lamarre D., Gadbois A. & Berk D. (2009). In situ characterization of nitrifying biofilm: Minimizing biomass loss and preserving perspective. Water Res 43, 17751787.
Dogsa I., Kriechbaum M., Stopar D. & Laggner P. (2005). Structure of bacterial extracellular polymeric substances at different pH values as determined by SAXS. Biophys J 89, 27112720.
Eisenmann H., Letsiou I., Feuchtinger A., Beisker W.O., Mannweiler E., Hutzler P. & Arnz P. (2001). Interception of small particles by flocculent structures, sessile ciliates, and the basic layer of a wastewater biofilm. Appl Environ Microb 67, 42864292.
Flint S.H., Brooks J.D. & Bremer P.J. (2000). Properties of the stainless steel substrate, influencing the adhesion of thermo-resistant streptococci. J Food Eng 43, 235242.
Fratesi S.E., Lynch F.L., Kirkland B.L. & Brown L.R. (2004). Effects of SEM preparation on the appearance of bacteria and biofilms in the carter sandstone. J Sediment Res 74, 858867.
Garny K., Horn H. & Neu T.R. (2008). Interaction between biofilm development, structure and detachment in rotating annular reactors. Bioproc Biosyst Eng 31, 619629.
Gieseke A., Purkhold U., Wagner M., Amann R. & Schramm A. (2003). Structure and activity of multiple nitrifying bacteria population coexisting in a biofilm. Environ Microbiol 5, 355369.
Gieseke A., Tarre S., Green M. & de Beer D. (2006). Nitrification in a biofilm at low pH values: Role of in situ microenvironments and acid tolerance. Appl Environ Microb 72, 42834292.
Ivanov V., Stabnikova O., Sihanonth P. & Menasveta P. (2006). Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface World J Microb Biot 22, 807812.
Jahn A.P. & Nielsen H. (1995). Extraction of extracellular polymeric substances (EPS) from biofilm using a cation exchange resin. Water Sci Technol 32, 157164.
Jensen K., Sloth N.P., Risgaard-Petersen N., Rysgaard S. & Revsbech N.P. (1994). Estimation of nitrification and denitrification from microprofiles of oxygen and nitrate in model sediment systems. Appl Environ Microb 60, 20942100.
Jetten M.S.M. (2008). The microbial nitrogen cycle. Environ Microbiol 10, 29032909.
Jorand F., Boue-Bigne F., Block J.C. & Urbain V. (1998). Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances. Water Sci Technol 37, 307315.
Jorand F., Zartarian F., Thomas F., Block J.C., Bottero J.Y., Villemin G., Urbain V. & Manem J. (1995). Chemical and structural (2D) linkage between bacteria within activated sludge flocs. Water Res 29, 16391647.
Koenneke M., Bernhard A.E., de la Torre J.R., Walker C.B., Waterbury J.B. & Stahl D.A. (2005). Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437, 543546.
Lawrence J.R., Korber D.R., Hoyle B.D., Costerton J.W. & Caldwell D.E. (1991). Optical sectioning of microbial biofilms. J Bacteriol 173, 65586567.
Lee L.Y., Ong S.L. & Ng W.J. (2004). Biofilm morphology and nitrification activities: Recovery of nitrifying biofilm particles covered with heterotrophic outgrowth. Bioresource Technol 95, 209214.
Leis A.P., Schlicher S., Franke H. & Strathmann M. (2005). Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics. Appl Environ Microb 71, 48014808.
Lemaire R., Webb R.I. & Yuan Z. (2008). Micro-scale observation of the structure of aerobic microbial granules used for the treatment of nutrient-rich industrial wastewater. ISME J 2, 528541.
Ludwig W., Strunk O., Westram R., Richter L., Meier H., Yadhumakar , Buchner A., Lai T., Steppi S., Jobb G., Förster W., Brettske I., Gerber S., Ginhart A., Gross O., Grumann S., Hermann S., Jost R., König A., Liss T., Lümann R., May M., Nonhoff B., Reichel B., Strehlow R., Stamatakis A., Stuckmann N., Vilbig A., Lenke M., Ludwig T., Bode A. & Schleifer K.-H. (2004). ARB: A software environment for sequence data. Nucleic Acids Res 32, 13631371.
Martiny A.C., Jørgensen T.M., Albrechtsen H.J., Arvin E. & Molin S. (2003). Long-term succession of structure and diversity of a biofilm formed in a model drinking water distribution system. Appl Environ Microb 69, 68996907.
Massol-Deya A.A., Whallon J., Hickey R.F. & Tiedje J.M. (1995). Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater. Appl Environ Microb 61, 769777.
Matsumoto S., Terada A., Aoi Y., Tsuneda S., Alpkvist E., Picioreanu C. & van Loosdrecht M.C.M. (2007). Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm. Water Sci Technol 55, 283290.
Mshandete A.M., Björnsson L., Kivaisi K.A., Rubindamayugi M.S.T. & Mattiasson B. (2008). Performance of biofilm carriers in anaerobic digestion of sisal leaf waste leachate. Electron J Biotechnol 11, 18.
Nielsen P.H., Jahn A. & Palmgren R. (1997). Conceptual model for production and composition of exopolymers in biofilm. Water Sci Technol 36, 1119.
Okabe S., Satoh H. & Watanabe Y. (1999). In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes. Appl Environ Microb 65, 31823191.
Park H.-D. & Noguera D.R. (2007). Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations. J Appl Microbiol 102, 14011417.
Picioreanu C., Kreft J-U., Klausen M., Haagensen J.A.J., Tolker-Nielsen T. & Molin S. (2007). Microbial motility involvement in biofilm structure formation—A 3D modelling study. Water Sci Technol 55, 337343.
Picioreanu C., Kreft J-U. & van Loosdrecht M.C.M. (2004). Particle-based multidimensional multispecies biofilm model. Appl Environ Microb 70, 30243040.
Priester J.H., Horst A.M., van de Werfhorst L.C., Saleta J.L., Mertes L.A.K. & Holden P.A. (2007). Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. J Microbiol Meth 68, 577587.
Prosser J.I. & Nicol G.W. (2008). Relative contributions of archaea and bacteria to aerobic ammonia oxidation in the environment. Environ Microbiol 10, 29312941.
Pruesse E., Quast C., Knittel K., Fuchs B., Ludwig W., Peplies J. & Glöckner F.O. (2007). SILVA: A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nuceic Acids Res 21, 71887196.
Pynaert K., Smets B.F., Wyffels S., Beheydt D., Siciliano S.D. & Verstraete W. (2003). Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor. Appl Environ Microb 69, 36263635.
Ribeiro R., Varesche M.B.A., Foresti E. & Zaiat M. (2003). Influence of extracellular polymeric substances on anaerobic biofilms supported by polyurethane foam matrices. Environ Eng Sci 20, 249255.
Romaškevič T., Budriene S., Pielichowski K. & Pielichowski J. (2006). Application of polyurethanebased materials for immobilization of enzymes and cells: A review. Chemija 17, 7489.
Stehr G., Zoerner B., Boettcher B. & Koops H.P. (1994). Exopolymers: An ecological characteristic of a floc attached ammonia oxidizing bacterium. Microb Ecol 30, 115126.
Stoodley P., de Beer D. & Lappin-Scott H.M. (1997). Influence of electric fields and pH on biofilm structure as related to the bioelectric. Antimicrob Agents Ch 41, 18761879.
Stoodley P., de Beer D. & Lewandowski Z. (1994). Liquid flow in biofilm systems. Appl Environ Microb 60, 27112716.
Surman S.B., Walker J.T., Goddard D.T., Morton L.H.G., Keevil C.W., Weaver W., Skinner A., Hanson K., Caldwell D. & Kurtz J. (1996). Comparison of microscope techniques for the examination of biofilms. J Microbiol Meth 25, 5770.
Sutherland I.W. (2001). The biofilm matrix—An immobilized but dynamic microbial environment. Trends Microbiol 9, 222226.
Weber S.D., Ludwig W., Schleifer K-H. & Fried J. (2007). Microbial composition and structure of aerobic granular sewage biofilms. Appl Environ Microb 73, 62336240.
Woznica A., Nowak A., Beimfohr C., Karczewski J. & Bernas T. (2010). Monitoring structure and activity of nitrifying bacterial biofilm in an automatic biodetector of water toxicity. Chemosphere 78, 11211128.
Wuchter C., Abbas B., Coolen M.J.L., Herfort L., van Bleijswijk J. & Timmers P. (2006). Archaeal nitrification in the ocean. P Natl Acad Sci 103, 1231712322.
Xavier J., Dekreuk M., Picioreanu C. & Vanloosdrecht M.M. (2007). Multi-scale individual-based model of microbial and bioconversion dynamics in aerobic granular sludge. Environ Sci Technol 41, 64106417.
Xi C., Marks D., Schlachter S., Luo W. & Boppart S.A. (2006). High-resolution three-dimensional imaging of biofilm development using optical coherence tomography. J Biomed Opt 11, 16.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary Materials

Woznica Supplementary Material

 PDF (379 KB)
379 KB
Supplementary Materials

Woznica Supplementary Material

 Video (6.9 MB)
6.9 MB
Supplementary Materials

Woznica Supplementary Material

 Video (6.5 MB)
6.5 MB


Full text views

Total number of HTML views: 2
Total number of PDF views: 26 *
Loading metrics...

Abstract views

Total abstract views: 148 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 19th November 2017. This data will be updated every 24 hours.