Folia Microbiologica

, Volume 58, Issue 3, pp 235–243 | Cite as

The phylogenetic structure of microbial biofilms and free-living bacteria in a small stream

  • Lenka Brablcová
  • Iva Buriánková
  • Pavlína Badurová
  • Martin Rulík


The phylogenetic composition, bacterial biomass, and biovolume of both planktonic and biofilm communities were studied in a low-order Bystřice stream near Olomouc City, in the Czech Republic. The aim of the study was to compare the microbial communities colonizing different biofilm substrata (stream aggregates, stream sediment, underwater tree roots, stream stones, and aquatic macrophytes) to those of free-living bacteria. The phylogenetic composition was analyzed using fluorescence in situ hybridization for main phylogenetic groups. All phylogenetic groups studied were detected in all sample types. The stream stone was the substratum where nearly all phylogenetic groups were the most abundant, while the lowest proportion to the DAPI-stained cells was found for free-living bacteria. The probe specific for the domain Bacteria detected 20.6 to 45.8 % of DAPI-stained cells while the probe specific for the domain Archaea detected 4.3 to 17.9 %. The most abundant group of Proteobacteria was Alphaproteobacteria with a mean of 14.2 %, and the least abundant was Betaproteobacteria with a mean of 11.4 %. The average value of the CytophagaFlavobacteria group was 10.5 %. Total cell numbers and bacterial biomass were highest in sediment and root biofilm. The value of cell biovolume was highest in stone biofilm and lowest in sediment. Overall, this study revealed relevant differences in phylogenetic composition, bacterial biomass, and biovolume between different stream biofilms and free-living bacteria.


Bacterial Abundance Bacterial Biomass Domain Bacterium Sediment Organic Matter Content Cell Biovolume 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Free-living bacteria


Macroscopic stream aggregates


Stream sediment


Stream stone


Riparian underwater roots


Water buttercup leaves


4′,6-Diamidino-2-phenylindole, fluorescent dye


Indocarbocyanine fluorescent dye


Fluorescence in situ hybridization



The methods which required use of epifluorescence microscope were made available to us by the Department of Botany, Palacky University. We thank all the staff of this department for their cooperation. Mr. Simon Hooper and Mr. Alex Outlon are acknowledged for language correction, and the reviewers who amended the final version of the manuscript are thanked.


  1. Amalfitano S, Fazi S (2008) Recovery and quantification of bacterial cells associated with streambed sediments. J Microbiol Methods 75:237–243PubMedCrossRefGoogle Scholar
  2. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990a) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedGoogle Scholar
  3. Amann RI, Krumholz L, Stahl DA (1990b) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J Bacteriol 172:762–770PubMedGoogle Scholar
  4. Arino X, Saiz-Jimenez C (1996) Factors affecting the colonization and distribution of cyanobactera, algae and lichens in ancient mortars. In: Riederer J (ed) Proceedings of the eighth international congress on deterioration and conservation of stone. Rathgen Forschungslabor, Berlin, pp 725–731Google Scholar
  5. Augspurger C, Gleixner G, Kramer C, Kusel K (2008) Tracking carbon flow in a 2-week-old and 6-week-old stream biofilm food web. Limnol Oceanogr 53:642–650CrossRefGoogle Scholar
  6. Battin TJ, Wille A, Sattler B, Psenner R (2001) Phylogenetic and functional heterogenity of sediment biofilms along environmental gradients in a glacial stream. Appl Environ Microbiol 67:799–807PubMedCrossRefGoogle Scholar
  7. Bertoni R, Callieri C, Corno G, Rasconi S, Caravati E, Contesini M (2010) Long term trends of epilimnetic and hypolimnetic bacteria and organic carbon in a deep holo-oligomictic lake. Hydrobiologia 644:279–287CrossRefGoogle Scholar
  8. Bjerkan G, Witso E, Bergh K (2009) Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthopaedica 80:245–250PubMedCrossRefGoogle Scholar
  9. Böckelmann U, Manz W, Neu TR, Szewzyk U (2000) Characterization of the microbial community of lotic organic aggregates (“River snow”) in the Elbe River of Germany by cultivation and molecular methods. FEMS Microbiol Ecol 33:157–170CrossRefGoogle Scholar
  10. Boenigk J (2004) A disintegration method for direct counting of bacteria in clay-dominated sediments: dissolving silicates and subsequent fluorescent staining of bacteria. J Microbiol Methods 56:151–159PubMedCrossRefGoogle Scholar
  11. Boureau T, Jacques MA, Berruyer R, Dessaux Y, Dominguez H, Morris CE (2003) Comparison of the phenotypes and genotypes of biofilm and solitary epiphytic bacterial populations on broad-leaved endive. Microb Ecol 47:87–95Google Scholar
  12. Bouvier T, Giorgio PA (2003) Factors influencing the detection of bacterial cells using fluorescence in situ hybridization (FISH): a quantitative review of published reports. FEMS Microbiol Ecol 44:3–15PubMedCrossRefGoogle Scholar
  13. Bratbak G (1985) Bacterial biovolume and biomass estimations. Appl Environ Microbiol 49:1488–1493PubMedGoogle Scholar
  14. Brümmer IHM, Fehr W, Wagner-Dobler I (2000) Biofilm community structure in polluted rivers: abundance of dominant phylogenetic groups over a complete annual cycle. Appl Environ Microbiol 66:3078–3082PubMedCrossRefGoogle Scholar
  15. Buesing N, Gessner MO (2002) Comparison of detachment procedures for direct counts of bacteria associated with sediment particles, plant litter and epiphytic biofilms. Aquat Microb Ecol 27:29–36CrossRefGoogle Scholar
  16. Chrost RJ, Koton M, Siuda W (2000) Bacterial secondary production and bacterial biomass in four Mazurian lakes of differing trophic status. Polish J Environ Stud 9:255–266Google Scholar
  17. Cottrell MT, Kirchman DL (2000) Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization. Appl Environ Microbiol 66:5116–5122PubMedCrossRefGoogle Scholar
  18. Cottrell MT, Kirchman DL (2003) Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary. Limnol Oceanogr 48:168–178CrossRefGoogle Scholar
  19. Crump BC, Armbrust EV, Barros JA (1999) Phylogenetic analysis of particle attached bacteria and free-living bacterial communities in the Columbia river, its estuary and the adjacent coastal ocean. Appl Environ Microbiol 65:3192–3204PubMedGoogle Scholar
  20. Dakora DF, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245:35–47CrossRefGoogle Scholar
  21. Eggert SL, Wallace JB (2007) Wood biofilm as a food resource for stream detritivores. Limnol Oceanogr 52:1239–1245CrossRefGoogle Scholar
  22. Eisenmann H, Burgherr P, Meyer EI (1999) Spatial and temporal heterogenity of an epilithic streambed community in relation to the habitat templet. Can J Fish Aquat Sci 56:1452–1460Google Scholar
  23. Fazi S, Amalfitano S, Pernthaler J, Puddu A (2005) Bacterial communities associated with benthic organic matter in headwater stream microhabitats. Environ Microbiol 10:1633–1640CrossRefGoogle Scholar
  24. Fischer H, Wanner SC, Pusch M (2002) Bacterial abundance and production in river sediments as related to the biochemical composition of particulate organic matter (POM). Biogeochemistry 61:37–55CrossRefGoogle Scholar
  25. Gillan DC, Danis B, Pernet P, Joly G, Dubois P (2005) Structure of sediment-associated microbial communities along a heavy-metal contamination gradient in the marine environment. Appl Environ Microbiol 71:679–690PubMedCrossRefGoogle Scholar
  26. Golladay SW, Sinsabaugh RL (1991) Biofilm development on leaf and wood surfaces in a boreal river. Freshw Biol 25:437–450CrossRefGoogle Scholar
  27. Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2003) Physiological and community responses of established grassland bacterial populations to water stress. Appl Environ Microbiol 69:6961–6968PubMedCrossRefGoogle Scholar
  28. Grossart HP, Ploug H (2000) Bacterial production and growth efficiencies: direct measurement on riverine aggregates. Limnol Oceanogr 45:436–445CrossRefGoogle Scholar
  29. Haglund AL, Lantz P, Tornblom E, Tranvik L (2003) Depth distribution of active bacteria and bacterial activity in lake sediment. FEMS Microbiol Ecol 46:31–38PubMedCrossRefGoogle Scholar
  30. Hempel M, Blume M, Blindow I, Gross EM (2008) Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater. Microbiology 8:1–10Google Scholar
  31. Hintze J (2007) NCSS 2007. NCSS, LLC. Kaysville, Utah, USAGoogle Scholar
  32. Jones PR, Cottrell M, Kirchman DL, Dexter SC (2006) Bactrial community structure of biofilms on artificial surfaces in an estuary. Microb Ecol 53:153–162PubMedCrossRefGoogle Scholar
  33. Kang JI, Goulder R (1996) Epiphytic bacteria downstream of sewage-works outfalls. Wat Res 30:501–510CrossRefGoogle Scholar
  34. Kirchman DL, Yu L, Cottrell MT (2003) Diversity and abundance of uncultured Cytophaga-like bacteria in the Delaware estuary. Appl Environ Microbiol 69:6587–6596PubMedCrossRefGoogle Scholar
  35. Kloep F, Manz W, Roske I (2006) Multivariate analysis of microbial communities in the River Elbe (Germany) on different phylogenetic and spatial levels of resolution. FEMS Microbiol Ecol 56:9–94CrossRefGoogle Scholar
  36. Koutný J, Rulík M (2007) Hyporheic biofilm particulate organic carbon in a small lowland stream (Sitka, Czech Republic): structure and distribution. Int Rev Hydrobiol 92:402–412CrossRefGoogle Scholar
  37. Kreuzer K, Adamczyk J, Iijima M, Wagner M, Scheu S, Bonkowski M (2006) Grazing of a common species of soil protozoa (Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice (Oryza sativa L.). Soil Biol Biochem 38:1665–1672CrossRefGoogle Scholar
  38. Lamberti GA, Gregory SV, Ashkenas LR, Wildman RC, Moore KMS (1991) Stream ecosystem recovery following a catastrophic debris flow. Can J Fish Aquat Sci 48:196–208CrossRefGoogle Scholar
  39. Lehman RM, Colwell FS, Bala GA (2001) Attached and unattached microbial communities in a simulated basalt aquifer under fracture- and porous-flow conditions. Appl Environ Microbiol 67:2799–2809PubMedCrossRefGoogle Scholar
  40. Lock MA (1994) Attached microbial communities in rivers. In: Ford TE (ed) Aquatic microbiology—an ecological approach. Blackwell, Oxford, pp 113–138Google Scholar
  41. Lucker S, Doris S, Kasper U, Kjeldsen B, MacGregor BJ, Wagner M, Loy A (2002) Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization. Appl Environ Microbiol 68:5064–5081CrossRefGoogle Scholar
  42. Manz W, Amann RI, Ludwig W, Wagner M, Schleifer KH (1992) Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Appl Microbiol 15:593–600CrossRefGoogle Scholar
  43. Manz W, Amann RI, Ludwig W, Vancanneyt M, Schleifer KH (1996) Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga flavobacter bacteroides in the natural environment. Microbiology 142:1097–1106PubMedCrossRefGoogle Scholar
  44. Manz W, Wendt-Poohoff K, Neu TR, Szewzyk U, Lawrence JR (1999) Phylogenetic composition, spatial structure and dynamics of lotic bacterial biofilms investigated by fluorescent in situ hybridization and confocal laser scanning microscopy. Microb Ecol 37:137–155CrossRefGoogle Scholar
  45. McNamara CJ, Leff LG (2004) Bacterial community composition in biofilms on leaves in a northeastern Ohio stream. J N Am Bentholl Soc 23:677–685CrossRefGoogle Scholar
  46. Meyer JL, Likens GE, Sloane J (1981) Phosphorus, nitrogen, and organic carbon flux in a headwater stream. Arch Hydrobiol 91:28–44Google Scholar
  47. Morris CE, Monier JM (2003) The ecological significance of biofilm formation by plant-associated bacteria. Annu Rev Phytopathol 41:429–453PubMedCrossRefGoogle Scholar
  48. Norland S (1993) The relationship between biomass and volume of bacteria. In: Kemp PF, Sherr BF, Sherr EB, Cole JJ (eds) Handbook of methods in aquatic microbial ecology. Lewis Publishers, New Jersey, pp 303–307Google Scholar
  49. Nunan N, Daniell TJ, Singh BK, Papert A, McNicol JV, Prosser JI (2005) Links between plant and rhizoplane bacterial communities in grassland soils, characterized using molecular techniques. Appl Environ Microbiol 71:6784–6792PubMedCrossRefGoogle Scholar
  50. Olapade OA, Depas MM, Jensen ET, McLellan SL (2006) Microbial communities and fecal indicator bacteria associated with Cladophora mats on beach sites along Lake Michigan shores. Appl Environ Microbiol 72:1932–1938PubMedCrossRefGoogle Scholar
  51. Pernthaler J, Glöckner FO, Schönhuber W, Amann RI (2001) Fluorescence in situ hybridization. In: Paul J (ed) Methods in microbiology—marine microbiology vol. 30. Academic Press Ltd, LondonGoogle Scholar
  52. Porter KG, Feig YS (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 25:943–948CrossRefGoogle Scholar
  53. Raskin L, Stromley JM, Rittman BE, Stahl DA (1994) Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60:1232–1240PubMedGoogle Scholar
  54. Romaní AM, Giorgi A, Acuna V, Sabater S (2004) The influence of substratum type and nutrient supply on biofilm organic matter utilization in streams. Limnol Oceanogr 49:1713–1721CrossRefGoogle Scholar
  55. Schweitzer B, Huber I, Amann RI, Ludwig W, Simon M (2001) α- and β-Proteobacteria control the consumption and release of amino acids on lake snow aggregates. Appl Environ Microbiol 67:632–645PubMedCrossRefGoogle Scholar
  56. Shiraishi F, Zippel B, Neu TR, Arp G (2008) In situ detection of bacteria in calcified biofilms using FISH and CARD–FISH. J Microbiol Methods 75:103–108PubMedCrossRefGoogle Scholar
  57. Simon M, Azam F (1989) Protein-content and protein-synthesis rates of planktonic marine bacteria. Mar Ecol Progr Ser 51:201–213CrossRefGoogle Scholar
  58. Simon M, Grossart HP, Schweitzer B, Ploug H (2002) Microbial ecology of organic aggregates in aquatic ecosystems. Aquat Microb Ecol 28:175–211CrossRefGoogle Scholar
  59. Vadeboncouer Y, Lodge DM (2000) Periphyton production on wood and sediment: substratum-specific response to laboratory and whole-lake nutrient manipulations. J N Am Bentholl Soc 19:68–81CrossRefGoogle Scholar
  60. Whiteley AS, Griffiths RI, Bailey MJ (2003) Analysis of the microbial functional diversity within water stressed soil communities by flow cytometric analysis and CTC+ cell sorting. J Microbiol Methods 54:257–267PubMedCrossRefGoogle Scholar
  61. Wilmes P, Remis J, Hwang M, Auer M, Thelen MP, Banfield JF (2009) Natural acidophilic biofilm communities reflect distinct organismal and functional organization. ISME J 3:266–270PubMedCrossRefGoogle Scholar
  62. Wobus A, Bleul C, Maassen S, Scheerer C, Schuppler M, Jacobs E, Roske I (2003) Microbial diversity and functional characterization of sediments from reservoirs of different trophic state. FEMS Microbiol Ecol 46:331–347PubMedCrossRefGoogle Scholar
  63. Zubkov MV, Sleigh MA (2000) Comparison of growth efficiencies of protozoa growing on bacteria on surfaces and in suspension. J Eukaryot Microbiol 47:62–69PubMedCrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012

Authors and Affiliations

  • Lenka Brablcová
    • 1
  • Iva Buriánková
    • 1
  • Pavlína Badurová
    • 1
  • Martin Rulík
    • 1
  1. 1.Laboratory of Aquatic Microbial Ecology, Department of Ecology and Environmental Sciences, Faculty of SciencePalacky UniversityOlomoucCzech Republic

Personalised recommendations