Bacterial Retention on Superhydrophobic Titanium Surfaces

Feb 2, 2011 - A few recent studies have reported that mammalian cells interact in different ways with surfaces that have only their special and mechan...
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Bacterial Retention on Superhydrophobic Titanium Surfaces Fabricated by Femtosecond Laser Ablation Elena Fadeeva,*,† Vi Khanh Truong,‡ Meike Stiesch,^ Boris N. Chichkov,† Russell J. Crawford,‡ James Wang,§ and Elena P. Ivanova*,‡ †

Laser Zentrum Hannover e.V., Hollerithallee 8, D-30419 Hannover, Germany Faculty Life and Social Sciences and §Faculty of Engineering and Industrial Sciences, IRIS, Swinburne University of Technology, PO Box 218, Hawthorn, Victoria 3122, Australia ^ Medical High Scholl Hannover, Carl-Neuberg-Str. 1, D-30625 Hanover, Germany ‡

bS Supporting Information ABSTRACT: Two-tier micro- and nanoscale quasi-periodic self-organized structures, mimicking the surface of a lotus Nelumbo nucifera leaf, were fabricated on titanium surfaces using femtosecond laser ablation. The first tier consisted of large grainlike convex features between 10 and 20 μm in size. The second tier existed on the surface of these grains, where 200 nm (or less) wide irregular undulations were present. The introduction of the biomimetic surface patterns significantly transformed the surface wettabilty of the titanium surface. The original surface possessed a water contact angle of θW 73 ( 3, whereas the laser-treated titanium surface became superhydrophobic, with a water contact angle of θW 166 ( 4. Investigations of the interaction of S. aureus and P. aeruginosa with these superhydrophobic surfaces at the surface-liquid interface revealed a highly selective retention pattern for two pathogenic bacteria. While S. aureus cells were able to successfully colonize the superhydrophobic titanium surfaces, no P. aeruginosa cells were able to attach to the surface (i.e., any attached bacterial cells were below the estimated lower detection limit).

’ INTRODUCTION Titanium and its alloys are employed extensively as implant materials. These materials are used in manufacturing of orthopedic and dental prostheses and cardiac valves. They are also a standard solution for the microsurgical restoration of middle ear function.1-3 The use of titanium in such a wide range of medical applications is linked to its specific properties: it is light weight, possesses mechanical strength and excellent sound transmission properties, is corrosion-resistant against a range of aggressive fluidic environments, and spontaneously forms a highly biocompatible dioxide passivation film in both air and blood. In addition, there is an absence of reported allergic reactions when used in surgical applications.4-7 However, the performance of medical implants in both the short and long term can be adversely affected by the presence of bacterial biofilms that can cause infections.8-11 Currently, the formation of biofilms by human pathogenic bacteria on the surface of medical implants is one of most common causes of their failure, often resulting in the necessity to surgically remove the implant.12 This, in some cases, can be associated with the formation of a systemic infection, the loss of organ or limb function, resulting in amputation or death.13 The ability to remove biofilms from indwelling devices is an issue of great concern.12,14 For example, Troidle et al. demonstrated that S. aureus, E. coli, or P. aeruginosa biofilms had formed on all catheters that had been removed from the patients participating in their study.15 Therefore, understanding the mechanisms r 2011 American Chemical Society

by which human pathogens are retained on inanimate surfaces such as biomedical implant devices is critical when devising methods for improving the functionality of these devices. Different approaches for the prevention of bacterial attachment to medical implant surfaces have been developed, e.g., the functionalization of the implants using antimicrobial agents16 or metallic nanoparticles.17 Unfortunately, since many harmful bacteria have developed resistance to many common antimicrobial agents, functionalizing the surfaces of these implants may have limited or only temporary success. The application of metallic nanoparticles has the drawback that these particles may be toxic to both the bacterial and eukaryotic cells of surrounding human tissue.17 For these reasons, the design and fabrication of antifouling surfaces using the technique of surface structuring is an attractive solution for the prevention of bacterial adhesion and therefore the subsequent formation of biofilms. Surface structuring is known to be effective for reducing the adhesion between a surface and a fluid and/or cells due to the fact that a modification of the surface topography may allow control of the extent of contact area between solid surface and liquid/ cell.18,19 It has been shown that on flat surfaces water contact angles larger than 120 cannot be achieved.20 The fabrication of superhydrophobic water repelling surfaces therefore requires the employment of particular surface topographies. The contamination Received: November 22, 2010 Revised: December 22, 2010 Published: February 02, 2011 3012

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Figure 1. SEM images of structured and polished titanium surface taken at 1000, 3000, and 20000 magnification (top); AFM two- and threedimensional images of structured and polished titanium surface showing surface topography on approximately 1 μm  1 μm scanning areas (middle); sequential images of an 10 μL water drop skated on the superhydrophobic titanium surface (bottom).

resistance of the surface of some plant leaves and insects has been the focus of intensive investigations due to their superhydrophobicity and their ultralow adhesion properties, reflected in their so-called “self-cleaning” properties.21-24 A number of fabrication techniques have been developed to mimic these superhydrophobic surfaces.21-24 These include vacuum-ultraviolet lithography, e-beam lithography, soft lithography, template lithography, templating from anodizied alumina, replica molding, and microwave plasma-enhanced chemical vapor deposition micropatterning.25-30 Unfortunately, microarrays and in particular micropillar arrays, fabricated from a variety of soft materials, despite their many attractive properties, lack stability aganst adhesive and capillary forces.30 In this study, we have employed a femtosecond laser-based micro- and nanostructuring approach to fabricate superhydrophobic titanium surfaces with a water contact angle of θW = 166 and

mimicking the surface of the lotus leaf Nelumbo nucifera. Our aim was to fabricate metallic surfaces that mimicked the surface architechture of these contamination resistant lotus leaf surfaces, on the assumption that these properties would also be “antifouling” in nature. A few recent studies have reported that mammalian cells interact in different ways with surfaces that have only their special and mechanical features modified.31-33 The results reported here are in agreement with these observations, providing further evidence that surface features can be manipulated in order to control the extent of bacterial attachment, including the processes of bacterial ordering and oriented attachment.34-41 Despite these significant advances, the mechanisms by which bacterial cells attach and retain on to surfaces remains poorly understood. The aim of this study was to investigate the attachment behavior of two bacterial pathogens (rod-shaped P. aeruginosa and spherical S. aureus) onto 3013

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Figure 2. Dynamic wettability of the lotus-mimicked superhydrophobic titanium surfaces immersed in water. Air replacement by water (top). Frames are represented by images taken using on-axis color CCD camera coupled with 10 objective (OPTEM, MPLAN APO) (see also Supporting Information: air replacement by water movie). The graph (bottom) showing the pecentage of air trapped in superhydrophobic titanium surfaces after 11/2 h immersing in water.

superhydrophobic titanium surfaces in an aqueous system in order to evaluate whether a titanium surface that mimicks the lotus leaf architecture possesses antifouling properties.

’ RESULTS AND DISCUSSION A femtosecond laser ablation technique was used to fabricate the superhydrophobic self-organized structures on titanium surfaces (Figure 1). The term “self-organization” refers to the fact that the surface features were formed spontaneously under femtosecond laser irradiation, with the size of these surface features being much smaller than the irradiated area. The formation of self-organized microstructures on titanium by irradiation with femtosecond laser pulses has been already reported by Nayak et al. and Vorobyev and Guo.42,43 The first paper reported the formation of sharp, nearly regular, conical pillars covered with ripple structures under the influence of different ambient gases at low laser fluences (1.5-2.5 J/cm2).42 The second paper reported the femtosecond laser processing of titanium, resulting in the formation of self-organized columnar microstructures, formed after irradiation with 200 laser pulses at low fluences of 0.16-0.48 J/cm2.43 The wettability characteristics of the resulting microstructures, however, did not appear to have been measured in either study. In this work, we have designed and optimized the laser processing parameters to allow the generation of surface structures on the titanium surface, resulting in superhydrophobic properties. One naturally occurring hierarchical structure with superhydrophobic features is found on the surface of the lotus leaf Nelumbo nucifera. It is a convex microstructure (outside arced) covered with nanosized features which was taken as a model superhydrophobic surface on which our modified

titanium surfaces could be based. The fabrication of a convex microstructure covered with nanosized features was found to be possible using significantly higher laser fluences of 20-100 J/cm2 compared to those used in the studies mentioned above. To avoid ripple formation and to manufacture anisotropic nanoroughness, circular polarized light was used. This technique allowed the generation of a particular two-tier hierarchical surface structures. The first tier was comprised of large grainlike convex features with a size ranging from 10 to 20 μm. The second tier existed on the surface of these grains, where 200 nm (or less) wide irregular undulations were present. No chemical postprocessing was applied. High-resolution SEM and AFM images of these modified titanium surfaces (henceforth called “structured titanium”) are shown in Figure 1. Images, demonstrating the similarity of the surface topography and wettability characteristics of the structured titanium surface to those of lotus leaves (Nelumbo nucifera), can be found in the Supporting Information (Figures S1 and S2). Single and multipulse experiments, without moving the positioning system, were carried out in order to clarify the formation of spikes. The main processes responsible for the spike formation on titanium surfaces appeared to be similar to that known for silicon surfaces.44 These included melting, resolidification, and surface roughening at early stage formation, followed by ablation during the late-stage of formation. Titanium surfaces that were irradiated with a different number of laser pulses exhibited two particular regions (see Supporting Information: SEM images of multipulse experiment) due to the Gaussian intensity distribution in the laser focus. In the middle area, the melt depth was larger than that of the outer region, which led to the formation of 3014

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Figure 3. Representative S. aureus (a) and P. aeruginosa (a) attachment patterns on the structured and polished titanium surfaces after 18 h incubation: SEM images (top) represent an overview of the bacterial attachment pattern taken at 1000, 3000, and 20 000 magnifications showing S. aureus successful colonization of the superhydrophobic surfaces and lack of P. aeruginosa cells on the same surfaces. CSLM images (bottom) visualizing viable cells (stained red with SYTO 17 Red) and the production of EPS (stained green with concanavalin A 488).

surface features at the micrometer scale due to the melt dynamics producing the first tier. The melt depth and the size of micrometer features could be controlled by varying the laser fluence. During the late-stage formation, the self-organized structure was deepened by ablation. The second tier—roughness on the nanometer scale—was formed at low laser fluences (in the outer region of laser focus), where the conditions for nanostructure or ripple formation were fulfilled. When the laser focus was moved, regions of high and low laser fluence became superimposed, leading to the superimposition of nano- and microstructures. It is noteworthy that at a fixed laser focus position no nanofeatures could be observed in the middle area. The XPS (X-ray photoelectron spectrometry) analysis (see Supporting Information: an annotated survey spectrum, together with high-resolution spectra of the Ti 2p, O 1s, and C 1s regions; the atomic fractions of the elements calculated from the survey spectrum and relative contributions and binding energies are presented in Tables XS1 and XS2) indicated that the most abundant elements on the surfaces of both the polished and structured titanium are titanium and oxygen. As titanium forms a stable oxide (TiO2) on the surface, most of the oxygen detected can be expected to be bound in the surface oxide. The binding energy measured for the 2p3/2 component of the Ti 2p spectrum was 459.0 eV, which is within the range of values reported for TiO2.45 Three peaks were fitted to the spectrum and assigned as follows: the main carbon peak at 285.0 eV was assigned to

hydrocarbon species (C-H/C-C); the other two fitted peaks were attributed to C-O (i.e., alcohol) species at 286.6 eV and CdO (i.e., carbonates) species at 288.5 eV. A higher amount of titanium dioxide was found on the surfaces of the structured titanium. This is most likely due to the increase in surface area as a result of the laser surface structuring. The wettability measurements performed in this study demonstrated that the polished titanium samples were intermediate in their wettability, displaying a water contact angle of 73 ( 3. After femtosecond laser processing, the structured areas became superhydrophobic, displaying a water contact angle of 166 ( 4, which tended to repel the falling water droplets coming into contact with the surface (as shown in Figure 1). A bouncing of water droplets on structured surfaces was also observed (see Supporting Information: bouncing droplet movie). The structured surfaces exhibited a contact angle hysteresis (the difference between the advancing and receding contact angle) of 10.0 ( 4.5. Since the XPS analysis did not reveal any significant difference in the surface chemistry of the polished and structured titanium samples, it was concluded that the superhydrophobic properties of structured titanium arose as a direct consequence of the particular two-tier surface topography. We further investigated the dynamic wettability of the structured titanium surfaces while immersed in water. It was observed that on initial contact with the water ∼50% of the surface area of the structured titanium surface was comprised of air trapped in 3015

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Figure 4. S. aureus and P. aeruginosa biovolume and average biofilm thickness on surfaces of polished and structured titanium quantified using COMSTAT (n = 6, if p < 0.05).

the micro- and nanostructures on the surface. It was found that over time most of the air was gradually replaced by water, reaching a saturation point within 1 h, where only 6% of the surface area was comprised of trapped air (Figure 2 and Supporting Information: air replacement by water movie). Notably, a similar phenomenon was not observed on the surface of the control, polished titanium; i.e., no air was trapped on the surface. The contact angle of the structured titanium could be described by the Cassie-Baxter model46-48 (which relates the composite contact angle on heterogeneous surfaces to the area fractions and contact angles of its component parts) together with the Wenzel model (which relates the contact angle measured on a rough surface to that measured on a smooth surface of the same material).46-48 The Cassie-Baxter model states that49 cos θ ¼ f1 cos θ1 þf2 cos θ2

ð1Þ

where θ is the composite contact angle of the heterogeneous surface, f1 and f2 are the area fractions of surface components 1 and 2, and θ1 and θ2 are their respective contact angles. For the structured titanium surface, the two surface components are titanium and air. Since the water contact angle on air can be taken as 180 (i.e., θ2 = 180), and f2 = 1 - f1, then eq 1 becomes cos θ ¼ f1 ðcos θ1 þ1Þ-1

ð2Þ

Here, θ1 is the contact angle measured on the structured titanium, which is considered a “rough” surface. The contact angle measured on such a rough surface is related to that measured on a smooth surface by the Wenzel equation:50 cos θrough ¼ r cos θsmooth

ð3Þ

Therefore, the composite contact angle measured on the structured titanium surface (containing trapped air) can be related to the contact angle measured on a smooth titanium surface using a combination of the Cassie-Baxter and Wenzel equations (4). This equation also describes the “transition state” in the surface wettability of the structured titanium. cos θ ¼ f1 ðr cos θsmooth þ1Þ-1

ð4Þ

An analysis of the SEM and CSLM images of S. aureus and P. aeruginosa attached onto the super hydrophobic structured titanium surfaces (Figures 3 and 4) revealed different attachment responses for the two bacterial strains. As can be seen in the SEM and CSLM images, after 18 h of incubation S. aureus cells were able to successfully colonize the structured titanium surfaces. A statistically significant increase in the number of attached S. aureus cells (10.5  104 cells per mm2) was detected (t = 0.00, p < 0.05) on the structured titanium surfaces compared to that on the polished (smooth) titanium surfaces. In contrast to the attachment response of S. aureus, the P. aeruginosa cells were not able to 3016

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Langmuir colonize the structured titanium surfaces, with attached cell numbers being found to be below the lower detection limit (estimated as 1.1  103 cells mm-2). It should be noted, however, that the P. aeruginosa biovolume on both the polished and structured titanium surfaces was very similar (t = 0.29, p > 0.05) and comparable to that observed for the S. aureus cells (Figure 4). It is also noteworthy that the P. aeruginosa cells produced a considerable amount of EPS, which was retained on the surface of the titanium without associated bacterial cells (Figure 4 refers to the biofilm distribution maps). In the past, the physicochemical characteristics of substrate surfaces (such as wettability, surface charge, and surface chemistry) have been intensively studied in an attempt to predict trends in bacterial attachment behavior.51-58 A number of studies have reported the behavior of surfaces with antifouling properties; for example, “Sharklet AF” is a synthetic surface with a surface architecture that has been inspired by the skin of sharks, which deters colonization by certain disease-causing microbes such as algal spores and the marine bacterium Cobetia marina.59 Magina et al.60 reported the role of the Reynolds number in a model that predicts the degree of attachment of zoospores of Ulva and cells of Cobetia marina. Notwithstanding these advances, the mechanisms controlling the bacterial-surface interactions remain poorly understood. The results generated in this study indicated that the only property of the titanium surfaces that was affected by the femtosecond laser processing was the surface topography. As a result of the surface structuring, one would expect that the superhydrophobic, antifouling properties of the structured titanium surfaces, induced by the presence of the superimposed nano- and microtopography, would act as an “antiadhesive” for any bacterial strains coming into contact with these surfaces. Indeed, the rod-shaped P. aeruginosa cells were not able to colonize the structured titanium surface. It should be noted that while the total surface area of the surface increased as a result of the laser processing, not all of this area is available for bacterial adhesion; in fact, it is likely that the reason for this resistance to bacterial colonization has arisen from a greatly diminished surface area that is available for cell adhesion. In contrast, the spherical S. aureus appeared to be a successful colonizer of the structured titanium surfaces. It is likely that since the spherical bacteria would require a much lower degree of surface contact to allow successful adhesion compared to rod-shaped bacteria. The results obtained in this study suggest that bacterial adhesion to substrates may require the presence of an appreciable amount of initial cellular contact with the surface, together with the ability of EPS to further adhere the cells to the surface over this contact area. The smaller, spherical S. aureus cells appeared to be able to achieve these two requirements; however, the larger, rod-shaped P. aeruginosa cells were not. This may be due to the EPS formed being insufficient, given the surface morphology, to adequately anchor the rodlike cells to the structured titanium surface.

’ CONCLUSIONS Fabrication of superhydrophobic titanium surfaces using femtosecond laser ablation in a single processing step has allowed a structured titanium surface to be made, which possesses a surface topography mimicking that of the surface of the lotus leaf, Nelumbo nucifera. This surface topography appeared to selectively control the extent of bacterial attachment, the extent being dependent on the cellular morphology. The mechanism by which

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these surfaces exhibit such a selective antifouling behavior is not clear at this stage; however, it is postulated that the simple process of mechanically anchoring the bacterial cells to the surface may play an important role.

’ EXPERIMENTAL SECTION Manufacturing of Structured Titanium Samples. Commercial, pure grade 2 titanium disks with a diameter of 10 mm and thickness of 2 mm were used in this study. The samples were mechanically polished and further cleaned with acetone followed by methanol before laser treatment. The laser structuring was performed using a commercially available amplified Ti:sapphire femtosecond laser system (Femtopower Compact Pro, Femtolasers Produktions GmbH, Austria). The system delivers sub-30 fs pulses at 800 nm central wavelength with a pulse energy of up to 1 mJ and a repetition rate of 1 kHz. An x-y motorized translation stage (Physik Instrumente GmbH, Germany) was used for sample positioning and translation. A computer-controlled LCD element was used for setting the laser pulse energy. To fabricate the hierarchical superimposed nano- and microstructures, the titanium surfaces were uniformly irradiated with 50 fs laser pulses at the laser fluence of 100 J/cm2. The samples were processed under ambient air conditions. Structured samples were then cleaned using acetone in an ultrasonic bath. In order to exclude any possible influence of initial surface roughness on the formation of the final self-organized structure, the titanium samples were mechanically polished prior to further treatment. The polished but otherwise unmodified, titanium samples (called “polished titanium”) were used as a control for the characterization of the material properties (surface chemistry, wettability) and for evaluation of the bacterial strain response to the unstructured titanium surfaces. Characterization of Titanium Surfaces. An atomic force microscope (Solver P7LS, NT-MDT Co.) was used to study the surface morphology of samples and to analyze the surface roughness at the nanometer scale in a quantitative way by using the standard instrument software (LS7-SPM v.8.58). Sample surfaces were scanned to evaluate the overall homogeneity of the surface. The topographical profiles were studied in detail at five different locations. X-ray photoelectron spectrometry (XPS) was performed using an Axis Ultra spectrometer (Kratos Analytical Ltd., UK), equipped with a monochromatic X-ray source (Al KR, hν = 1486.6 eV) operating at 150 W. The spectrometer energy scale was calibrated using the Au 4f7/2 photoelectron peak at binding energy EB = 83.98 eV. Samples were flooded with low-energy electrons during the analysis to counteract surface charging. The hydrocarbon component of the C 1s peak (binding energy 285.0 eV) was used as a reference for charge correction. Photoelectrons emitted at 90 to the surface plane, from an area of 700 μm  300 μm, were analyzed with 160 eV for survey spectra and then with 20 eV for region spectra. Survey spectra were recorded at 1 eV/step, while the region spectra were taken at 0.1 eV/ step. The relative atomic concentration of elements detected by XPS was quantified on the basis of the peak area in the survey spectra with sensitivity factors for the Kratos instrument used. Peaks in the highresolution regions of spectra were fitted with synthetic Gaussian-Lorentzian components after removal of a linear background (using the Kratos Vision II software). Contact Angle Measurements. Wettability tests (sessile drop method) were performed using a video-based optical contact angle measuring system (OCA 40 Micro, DataPhysics Instruments GmbH, Germany). A tilting plate method was used to measure the advancing (θA) and receding (θR) water contact angles on structured surfaces. A water droplet was placed on the horizontal structured surface, and then the sample was slowly tilted while a CCD camera (25 Hz) recorded changes in the droplet’s shape. θA and θR were measured using the last recorded image before the droplet base started to move. All contact 3017

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angles were measured automatically using the software of the contact angle measuring system. Measurements have been performed under normal atmospheric conditions using water droplets of 10 μL volume. All data are given as a mean value from at least 10 independent measurements ( standard deviation. Sequential images of water droplets skating over the surface were recorded on the polished and structured titanium samples using a CCD camera with an interval of 10 ms. The dynamic wettability of the titanium surfaces was observed using an-axis color CCD camera coupled with 10 objectives (OPTEM, MPLAN APO). Titanium disks were immersed in 10 mL of Milli-Q water, and the images of real time air replacement by water on the superhydrophobic surfaces were taken (50 frames/s). Movies were processed using Microsoft Video Maker. Every single frame of the movie was converted into binary format using MATLAB version R2009a, which was employed to calculate the degree of air coverage on the titanium surfaces.

Microorganisms, Culture Conditions, and Sample Preparation. Pathogenic bacteria Staphylococcus aureus CIP 65.8 and Pseudomonas aeruginosa ATCC 9027 were used in this study. The bacterial strains were obtained from the American Type Culture Collection (ATCC, USA) and Culture Collection of the Institut Pasteur (CIP, France). Bacterial stocks were prepared in 20% glycerol nutrient broth (Oxoid) and stored at -80 C. Prior to each experiment, bacterial cultures were refreshed from stocks on nutrient agar (Oxoid), and a fresh bacterial suspension was prepared for each of the strains grown overnight in 100 mL of nutrient broth (Oxoid) (in 0.5 L Erlenmeyer flasks) at 37 C with shaking (120 rpm). Bacterial cells were collected at the logarithmic stage of growth. To ensure that the samples possessed a similar number of cells despite the variations in cell densities among the different strains used, the cell density of each strain was adjusted to OD600 = 0.3. To quantify the cell numbers in the adjusted bacterial suspensions before attachment experiments, a hemocytometer was used, as suggested by Mather and Roberts.61 The attachment response of S. aureus CIP 65.8 and P. aeruginosa ATCC 9027 to the polished titanium samples has been studied elsewhere,39,40 reporting that both bacteria were able to colonise the polished titanium surfaces. To allow the observation of the bacterial cells by scanning electron microscopy (SEM), the titanium disks were sputter-coated with gold using a Dynavac CS300 device using the protocol developed earlier in our laboratory.39,40 High-resolution images of the titanium disks with the adsorbed bacterial cells were taken by FESEM (ZEISS SUPRA 40VP) at 3 kV with 1000, 5000, and 20000 magnifications. The lower detection limit was estimated as 1.1  103 cells mm-2 according to Morono et al. using the following formula:62 n¼

Tfov lnð1-pÞ Gfov

where n is the number of cells required giving a probability p (p = 0.95, 95% chance to find one bacterial cell) of detecting a cell, Tfov is total area of fields of view, Cfov is the number of fields of view, and total area is 314 mm2. In order to visualize the viable bacterial cells, images of the cells attached to titanium surfaces and the EPS produced by bacterial cells were recorded with a confocal scanning laser microscope (CSLM) Olympus Fluorview FV1000 spectroscopic confocal system. The system included an inverted microscope Olympus IX81 (with 20, 40 (oil), 100 (oil) UIS objective lenses) and was operated with multiple Ar, He, and Ne laser lines (458, 488, 515, 543, and 633 nm). The 488 nm laser was used to image the concanavalin A Alexa 488 dye, and the 543 nm laser was used to image the SYTO 17 Red. The imaging software Fluorview FV 7.0 was employed to process the CSLM images and construct 3D images. To quantify 3D biofilm image stacks, the computer software COMSTAT was used.61 Six typical areas on each type of titanium surface were

exported into a stack of gray scale 8-bit images by Fluorview FV 7.0. Two quantitative parameters of biomass density were used to describe the biofilm formed on the titanium surfaces:63 (i) the biovolume, which encompasses both cells and EPS, i.e., the overall volume of the biofilm per unit area of substrate, and (ii) the average biofilm thickness.

’ ASSOCIATED CONTENT

bS

Supporting Information. Figures S1-S3, Tables XS1 and XS2, and two movies. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E.F.: Tel þ49-5112788378; Fax þ49-5112788100; e-mail [email protected]. E.P.I.: Tel þ613-92145137; Fax þ61392145921; e-mail [email protected].

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