Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs
Introduction
Microbial detachment plays a key role in determining the structure and function of both natural and engineered biofilm systems Characklis, 1981, van Loosdrecht et al., 2002, and detachment is important in a wide range of technical systems including heat exchangers and water distribution pipes, reducing the effects of biofouling and bio-corrosion (Flemming et al., 1996). Cells detach from a biofilm when an external force, such as fluid shear or particle abrasion, exceeds the cohesive strength of the matrix joining a cell or cluster to the remainder of the biofilm. Recent models of biofilm growth and development relate detachment to biofilm cohesive strength directly (Picioreanu et al., 2001) and as a ratio of cohesive strength to shear stress Hermanowicz, 1999, Hermanowicz, 2001. Unfortunately, measurements of biofilm cohesive strength are limited and current measurement approaches are problematic for various reasons, as described below. A method is presented in this paper which allows the direct measurement of the cohesive strength of biofilm fragments and other microbial aggregates.
A variety of methods have been used to estimate the cohesive strength of microbial biofilms. Ohashi and Harada (1994) used a centrifuge to estimate the tensile force required to separate fragments from a biofilm. A later effort used a collision-based device for estimating the shear forces involved in separating fragments from a biofilm (Ohashi and Harada, 1996). Data from both of these methods are difficult to interpret because the applied forces depend on the mechanism and geometry of the separation events, which were not evaluated. Ohashi et al. (1999) measured biofilm tensile strength as a bulk parameter over a separation area of several square millimeters by applying an axially directed force to a tubular biofilm. This approach yields direct measurements of biofilm strength, but only as a bulk property over a large separation area.
Other material properties related to cohesive strength have also been investigated. The elastic modulus and shear modulus of several biofilms were estimated by Stoodley et al. based on the observed deformation of biofilm streamers due to variations in fluid flow rates Stoodley et al., 1999, Stoodley et al., 2001. The forces acting on the biofilm streamers were not measured, but were estimated from theoretical correlations for fluid shear at a fluid-smooth surface interface. A uniaxial compression device was developed by Körstgens et al. (2001) for testing the elastic modulus and yield strength of bacterial lawns grown in unsaturated conditions. The authors suggest that the measured stress at failure for compression of the sample, as an average property over approximately 310 mm2 of surface area, may be related to the tensile properties of the aggregate.
For cohesive strength measurements to be most relevant to detachment processes, the scale of observation should be comparable to the scale at which detachment occurs. In the effluent from a trickling filter wastewater treatment facility, two-thirds of the particles observed were between 25 and 200 μm (longest dimension), and 80% had equivalent diameters of less than 100 μm (Zahid and Ganczarczyk, 1990). In a study of Pseudomonas aeruginosa biofilms grown in an annular reactor, Stewart et al. (1993) observed that a majority of the detached biomass occurred in particles ranging from 0.1 to 100 μm3, correlating with diameters of 1.2–12 μm. In a similar reactor system, Murga et al. (1995) observed effluent particles ranging in size from single cells to clusters of more than 100 μm. This paper describes a method for measuring the cohesive strength of microbial aggregates at scales comparable to the observed sizes of detached biofilm particles.
Section snippets
Materials and methods
A micro-mechanical technique, based on a method described by Yeung and Pelton (1996) for measuring the cohesive strength of abiotic flocs, was used to measure the tensile strength of bacterial aggregates. The method is based on the observed deflection of a cantilevered glass micropipette as it separates a microbial aggregate held by suction between the cantilever and a straight micropipette.
Return activated sludge
Most attempts to separate RAS flocs were unsuccessful, ending with the loss of suction between the floc and either the cantilever or the micropipette. Several locations on each floc were tested and, if none were measurable, the floc was discarded and another selected. A total of 49 successful separations were recorded from 21 separate RAS flocs. An initial 34 separations were recorded between 3.1 and 7.2 h of sample collection while the remaining 15 were recorded from 27 to 29 h after sample
Limits of applied force and stress
Microbial aggregates are held to the pipettes by the difference between atmospheric pressure (approximately 1×105 N/m2) and the partial vacuum within the pipette. The applied force is equal to the force which would be exerted on a projection of the opening in a plane perpendicular to the direction of the applied stress. Irregular or angled openings on the cantilever may affect the seal between the cantilever and the aggregate, but do not otherwise affect the amount of force that can be applied.
Acknowledgements
The authors thank Richard Poppele for the loan of equipment and for assistance in constructing the microforge electronics. The authors also thank Ed Bouwer for the loan of the precision drill press. Support for E. Poppele was provided in part by a U.S. Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship and a University of Minnesota fellowship.
References (19)
- et al.
Antifouling strategies in technical systems—a short review
Water Sci. Technol.
(1996) Two-dimensional simulations of biofilm development: effects of external environmental conditions
Water Sci. Technol.
(1999)A simple 2D biofilm model yields a variety of morphological features
Math. Biosci.
(2001)- et al.
Uniaxial compression measurement device for investigation of the mechanical stability of biofilms
J. Microbiol. Methods
(2001) - et al.
A novel concept for evaluation of biofilm adhesion strength by applying tensile force and shear force
Water Sci. Technol.
(1996) - et al.
A novel method for evaluation of biofilm tensile strength resisting erosion
Water Sci. Technol.
(1999) - et al.
Micromechanics: a new approach to studying the strength and breakup of flocs
J. Colloid Interface Sci.
(1996) - et al.
Suspended solids in biological filter effluents
Water Res.
(1990) Fouling biofilm development: a process analysis
Biotechnol. Bioeng.
(1981)
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