Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands1
Author for correspondence: Henny C. van der Mei. Tel: +31 50 363 31 40. Fax: +31 50 363 31 59. e-mail: h.c.van.der.mei{at}med.rug.nl
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ABSTRACT |
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Keywords: biofilms, adhesion mechanisms, deposition efficiency, cell surface hydrophobicity, marine bacteria
Abbreviations: PP, parallel plate; SP, stagnation point
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INTRODUCTION |
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Numerous studies have been conducted to determine the influence of the physical (Tsibouklis et al., 1999 ) and toxic (Dempsey, 1981
) properties of coatings on adhesion of marine bacteria, but it has seldom been realized that bacterial adhesion to surfaces depends in part on the type of system used to study adhesion in situ (Elimelech, 1994
). Both under laboratory conditions (slight rinsing and dipping to remove the notoriously undefined loosely bound organisms) and in real life (waves), surfaces with adhering bacteria are exposed to liquidair interfaces and other hydrodynamic detachment forces. It has been shown theoretically (Leenaars & OBrien, 1989
) and experimentally (Gomez-Suarez et al., 2001
) that a passing liquidair interface can remove up to 100% of all bacteria adhering to a substratum surface, depending on the surface properties of the bacterial strain and the substratum. For this reason, several devices have been developed (Adamczyk & Van de Ven, 1984
; Adamczyk, 1989
), allowing a well-controlled flow and enabling in situ observation of adhering bacteria. However, an extensive comparison of bacterial deposition in different flow devices and whether bacterial adhesion proceeds according to the same mechanisms under different modes of mass transport has not been made.
Theoretical comparisons of different flow devices are possible by solving the so-called convective-diffusion equation (Adamczyk & Van de Ven, 1981 ; Peters, 1990
) describing bacterial mass transport to a surface under flow. Exact analytical solutions of the convective-diffusion equation are often too difficult to obtain, but several approximate solutions exist. In the Von SmoluchowskiLevich approximation attractive LiftshitzVan der Waals forces between a bacterium and a substratum surface are thought to be counterbalanced by the hydrodynamic drag which a bacterium experiences when approaching a substratum surface, while electrostatic interactions are neglected (Brenner, 1961
). Consequently, bacterial deposition rates obtained from the Von SmoluchowskiLevich approximation are considered as an upper limit for bacterial mass transport in a given device, although Sjollema and coworkers (Sjollema et al., 1990a
) have presented bacterial deposition rates in excess of this theoretical upper limit. Bacterial strains showing such excessively high deposition rates could be identified as fibrillated strains and the conclusion was drawn that fibrils assist bacterial adhesion in a way not accounted for in the Von SmoluchowskiLevich approximation.
Theoretical comparisons of bacterial mass transport in a parallel plate (PP) (Adamczyk, 1989 ; Sjollema et al., 1989
) and a stagnation point (SP) (Dabros & Van de Ven, 1987
) flow chamber have demonstrated that mass transport in the PP flow chamber is slow, because convective flow is parallel to the substratum surface, while slow diffusional mass transport brings flowing bacteria toward the substratum surface; in contrast, convective flow is toward the substratum surface in the SP flow chamber as illustrated in Fig. 1
. By expressing experimental deposition rates relative to the upper limit for mass transport in the Von SmoluchowskiLevich approximation, a so-called deposition efficiency can be obtained that should theoretically account for the differences between different devices. However, it can also be anticipated that different adhesion mechanisms are operative under the different mass transport conditions. It is becoming increasingly apparent that microbial cell surfaces must be considered as possessing a soft surface layer (Morisaki et al., 1999
; Ohshima, 1995
; Van der Mei et al., 2000
) that may be somewhat compressed during head-on collisions with a substratum surface as in a SP flow chamber, allowing inner regions to interact. In the PP flow chamber, collisions are much gentler and only the outside of a microbial soft surface layer is anticipated to interact, which may yield a different adhesion mechanism.
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METHODS |
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Cell surface characterization.
Bacterial cell surfaces were characterized by their cell surface hydrophobicities, as determined by water contact angles, and by their zeta potentials in artificial seawater as a measure of their net surface charge. For zeta potentials (James, 1991 ), bacteria were washed twice and resuspended in artificial seawater to 1x108 bacteria ml-1. The electrophoretic mobilities of aliquots of these suspensions were measured at 150 V in a Lazer Zee Meter 501 (PenKem), which uses the scattering of incident laser light to detect the bacteria at relatively low magnification. The mobility of the bacteria under the applied voltage was converted to apparent zeta potentials according to the HelmholtzVon Smoluchowski equation (Hiemenz, 1977
). Cell surface hydrophobicities were assessed from water contact angles on lawns of bacteria using the sessile drop technique (Busscher et al., 1984
; Van Oss & Gillman, 1972
). Bacterial lawns were deposited on cellulose acetate membrane filters (Millipore, pore diameter 0·45 µm) which were positioned on a fritted glass support under negative pressure. The wet filters were fixed on sample holder plates with double-sided sticky tape and left to air-dry in ambient air until so-called plateau contact angles could be measured, indicated by stable water contact angles over time. Bacterial lawns were prepared by washing the bacteria twice with seawater and extensive washing with demineralized water after deposition on a filter. All cell surface characterization measurements were done in triplicate with separately cultured strains.
Substratum.
Glass surfaces were cleaned first by sponging with 2% Extran MA02 (Merck), followed by sonication for 10 min in 0·5% RBS25 (Fluka) and thorough rinsing with methanol, tap water and finally Millipore-Q water to obtain a fully water-wettable, hydrophilic surface, i.e. a water contact angle of zero degrees.
Flow chamber experiments and image analysis.
SP (Dabros & Van de Ven, 1987 ) and PP (Adamczyk & Van de Ven, 1981
) flow chambers were used. A flow chamber was incorporated between two communicating vessels, placed at different heights and containing the bacterial suspension, to create a pulse-free flow by hydrostatic pressure. Fluid was recirculated with a roller pump between the vessels. Briefly, for the PP flow chamber, deposition was observed in the centre of the bottom plate with a charge-coupled-device camera (CCD-MXR/5010, High Technology) mounted on a phase-contrast microscope (Olympus BH-2), equipped with a x40 ultra-long working distance objective (Olympus ULWD-CD Plan 40 PL). For the SP flow chamber, deposition in the area 1·1 mm away from the stagnation point was observed with a CCD-LDH camera (Philips) mounted on a dark-field microscope (Leitz) equipped with a x50 objective (Leitz Wetzlar). Live images were acquired with a PC-Vision+ frame grabber (Imaging Technology) and sharp filtered. For both systems, deposited bacteria were discriminated from the background by single grey value thresholding. This yielded binary black and white images which were stored on disk for later analysis. Experiments were carried out at a flow rate of 0·050 ml s-1 for the PP and 0·0088 ml s-1 for the SP flow chamber at 20 °C, corresponding to a common wall shear rate of 22·5 s-1 and a common Péclet number of 1·05x10-3. The Reynolds numbers for the PP and SP flow chambers were 1·3 and 2·6 respectively, proving a laminar flow in the flow chamber. Before each experiment the flow chamber and the collector surfaces were washed with artificial seawater for 15 min to condition the surface. All experiments were performed at 20 °C in triplicate, with separately cultured strains.
The total number of adhering bacteria per unit area n(t) was recorded as a function of time by image sequence analysis for at least 5 h. After a stationary end-point had been reached, an air bubble was passed through the flow chamber in order to obtain an indication of the adhesion force of attached bacteria, i.e. their retention capacity.
Data analysis.
The number of adhering bacteria in the stationary end-point and a characteristic adhesion time were determined by fitting the bacterial adhesion kinetics to
![]() | (1) |
(Elimelech et al., 1995 ), in which n
is the number of adhering bacteria in the stationary end-point, t is the time in seconds and
is a characteristic adhesion time.
The initial deposition rate, j0, was calculated directly by linear regression analysis from the initial increase of the numbers of adhering bacteria as a function of time, after which the entire adhesion kinetics over the full duration of an experiment was used to obtain the characteristic adhesion time . The characteristic adhesion time is composed of two components due to desorption and blocking according to
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(Elimelech, 1994 ), in which ß is the desorption rate and A1 is the area blocked by an adhering particle. The blocked area A can be derived from the radial pair distribution g(r) as can be calculated from the spatial distribution of the adhering bacteria. Radial pair distribution functions describe the relative density of adhering bacteria around a given centre bacterium as a function of their separation distance (Kamiti & Van de Ven, 1995
)
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where (r,dr) is the density of adhering particles in a shell with width dr at a distance r from a centre particle. Each adhering particle is taken once as a centre particle. As an example, the spatial arrangement of adhering bacteria and the resulting distribution function g(r) are given in Fig. 2
. The blocked area was calculated from the region for which g(r)<1 (Sjollema et al., 1990b
). Small blocked areas with a high relative density of adhering bacteria have been associated with a positive cooperativity for oral streptococci (Doyle et al., 1982
), while large blocked areas are indicative of repulsion between adhering bacteria.
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![]() | (4) |
(Leenaars & OBrien, 1989 ), in which r denotes the bacterial radius and
l is the surface tension of the suspending fluid. For most bacteria adhering from an aqueous fluid, this results in a force of about 10-7 N, which is relatively large compared with reported adhesion forces of bacteria to substratum surfaces (Rutter & Vincent, 1980
).
Statistical analysis.
A Students t-test was used to evaluate the significance of the differences observed, accepting statistical significance at P<0·05.
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RESULTS |
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Deposition experiments
Fig. 3 presents the deposition kinetics for M. hydrocarbonoclasticus, Psychrobacter sp. and H. pacifica to a glass surface from artificial seawater in the PP flow and SP flow chamber. As can be seen, the deposition kinetics differ not only among the strains, but also between the two flow chambers. In the SP flow chamber a stationary end-point is reached faster than in the PP flow chamber, while the final surface coverage reached in the two flow chambers was similar, at least for H. pacifica.
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Finally, with regard to the comparison of the flow chambers, Table 2 also shows that the experimental reproducibility is better in the SP than in the PP flow chamber. Initial deposition rates in the SP chamber are nearly four times more reproducibly measured than in the PP chamber, while stationary end-point numbers are obtained with the same reproducibility.
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DISCUSSION |
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All three strains showed different adhesion kinetics. The hydrophilic bacterium H. pacifica had the greatest affinity for the hydrophilic glass surface, in line with the thermodynamically predicted preference of hydrophilic strains for hydrophilic substrata (Absolom et al., 1983 ). Similarly, the hydrophobic M. hydrocarbonoclasticus was detached most easily from hydrophilic glass by a passing air bubble, also in line with the thermodynamically predicted dislike of hydrophobic strains for hydrophilic substrata, expressing the fact that the free energy required to detach hydrophobic bacteria from a hydrophilic surface is smaller than the energy needed to detach hydrophilic bacteria.
The initial deposition rates observed are higher in the SP flow chamber than in the PP flow chamber. In the PP flow chamber mass transport by convection is parallel to the substratum surface and bacteria in the flowing suspension have to bridge a relatively great distance by slow diffusion in order to reach the surface. On the other hand, in the SP flow chamber suspended bacteria are transported almost perpendicularly toward the surface by convection, and diffusion plays only a minor role. In most microbial habitats, including the marine environment, both modes of mass transport occur and the different deposition efficiencies observed for a given strain in the two flow chambers clearly indicate that different adhesion mechanisms are at work.
The deposition efficiencies are generally smaller in the SP flow chamber, as compared with the PP flow chamber, indicating a relatively high number of arrivals of bacteria at the substratum that do not result in eventual adhesion. This may indicate that the outermost cell surfaces gently interacting with the substratum surfaces are more adhesive than the inner surfaces of the bacteria after compression. On the basis of the Von SmoluchowskiLevich approach, a factor of 4·2 between initial deposition rates in both flow chamber devices is expected, which is larger than the experimental ratio of 2·5. The reason that deposition in the SP flow chamber does not exceed the theoretical maximum and the ratio is smaller might be that the theoretical initial deposition rate was calculated for the stagnation point, while measurements were done adjacent to the stagnation point. The shear rate at the stagnation point is 0 and increases to 22·5 s-1 at the measured point. For Psychrobacter sp. and H. pacifica in the PP flow chamber, the initial deposition rates exceed the theoretical maximum, possibly due to structural cell surface components assisting adhesion (Sjollema et al., 1990a ).
Higher ionic strength solutions, like seawater, lead to smaller blocked areas (Yang et al., 1999 ), due to decreased electrostatic repulsion between adhering and flowing particles. The slightly larger blocked areas as found in the PP flow chamber probably result from a disturbance of the flow lines behind an adhering bacterium yielding a local depletion of the suspension and a shadow zone of reduced adhesion downstream of an adhering bacterium (Bos et al., 1999
).
Finally, it has been shown that standard deviations over triplicate experiments in the PP flow chamber are never better than 2030% (Wit et al., 1997 ), while in the SP flow chamber standard deviations of 10% have been observed. Theoretically, accumulated errors in temperature and diffusion coefficients, concentration of bacterial suspension, flow chamber dimensions and flow rates could account for 1012% of the variations observed regardless of the type of flow chamber involved (Yang et al., 1999
), leaving a discrepancy between theoretically accountable and experimentally observed reproducibility in the PP flow chamber. Hypothetically, we attribute the lower reproducibility in the PP flow chamber to the fact that mass transport in this system depends much more on chance processes like diffusion and collisions between flowing and adhering bacteria than in the SP flow chamber, in which mass transport is mostly convection controlled.
It is difficult to give a preference for either of the two flow chamber devices evaluated in this paper, particularly as the adhesion mechanisms seem to differ under different modes of mass transport while both modes of mass transport are ecologically relevant. Although the SP flow chamber might appear more attractive due to its faster kinetics and the higher reproducibility of the results, the PP flow chamber poses less specific requirements for transparency of the substratum, and yields better image quality. Moreover, flow is homogeneous in the PP flow chamber over large areas, which allows adhesion to be studied over different microscopic fields of view. Also from the present results, it can be seen that larger differences are observed in the SP flow chamber than in the PP flow chamber for different combinations of strains and substrata, indicating that adhesion in the SP flow chamber is strongly interaction controlled. Considering the impact of the methodology on adhesion mechanisms, however, it is concluded that the choice for a specific flow chamber should a priori be made on the basis of relevance for the problem under investigation.
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ACKNOWLEDGEMENTS |
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Received 25 July 2001;
revised 5 October 2001;
accepted 10 October 2001.
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