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Applied and Environmental Microbiology, May 2005, p. 2677-2686, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2677-2686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Architecture of a Nascent Sphingomonas sp. Biofilm under Varied Hydrodynamic Conditions{dagger}

V. P. Venugopalan,1,{ddagger} M. Kuehn,1 M. Hausner,1,§ D. Springael,2 P. A. Wilderer,1 and S. Wuertz3*

Institute of Water Quality and Waste Management, Technical University of Munich, Am Coulombwall, D-85748 Garching, Germany,1 Laboratory of Soil Fertility and Soil Biology, Catholic University of Leuven, Kasteelpark, Arenberg 20, B-3001 Heverlee, and Environmental Technology, Flemish Institute for Technological Research, Boeretang 200, B-2400 Mol, Belgium,2 Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, California 956163

Received 2 July 2004/ Accepted 1 December 2004

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 105 µm2) or microcolony size (1 x 106 µm2) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 µm above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616. Phone: (530) 754-6407. Fax: (530) 752-7872. E-mail: swuertz{at}ucdavis.edu.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Water and Steam Chemistry Laboratory, BARC Facilities, Kalpakkam, Tamil Nadu 603 102, India.

§ Present address: Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL 60208-3109.

Applied and Environmental Microbiology, May 2005, p. 2677-2686, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2677-2686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Merod, R. T., Warren, J. E., McCaslin, H., Wuertz, S. (2007). Toward Automated Analysis of Biofilm Architecture: Bias Caused by Extraneous Confocal Laser Scanning Microscopy Images. Appl. Environ. Microbiol. 73: 4922-4930 [Abstract] [Full Text]  


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