Journal of Colloid and Interface Science 364 (2011) 213218

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Journal of Colloid and Interface Science

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Bacteria attachment to surfaces AFM force spectroscopy and physicochemical analyses

Ardiyan Harimawan, Aruliah Rajasekar, Yen-Peng Ting
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore

a r t i c l e

i n f o

a b s t r a c t
Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterialsurface interactions. Biolm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterialmetal adhesion force was quantied at different surface delay time from 0 to 60 s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53 1.40 nN and 7.88 0.94 nN) when compared to Gram-positive bacteria (1.44 0.21 nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and surface charges than B. subtilis along with the capability of producing extracellular polymeric substances (EPS). The higher hydrophobicity, surface charges, and greater propensity to form EPS by M. timonae and P. aeruginosa led to high adhesive force on the metal surface. 2011 Elsevier Inc. All rights reserved.

Article history: Received 12 April 2011 Accepted 7 August 2011 Available online 16 August 2011 Keywords: Biolm Hydrophobicity Adhesion force P. aeruginosa B. subtilis M. timonae

1. Introduction Biolms are complex clusters of microbial cells that grow on the surface of a solid material. The growth of biolm occurs in both animate and inanimate surfaces as well as in natural systems, industrial systems and even on the tissues of multicellular hosts [1]. The adhesion of single-cell microorganisms, such as bacteria, to surfaces is mediated by complex relationships between specic and nonspecic interactions [2,3]. Nonspecic interactions, such as electrostatic, hydrophobic, and van der Waals forces, are associated with physicochemical properties of the cells, surface, and solutions conditions in the surrounding environment. Specic interactions, on the other hand, involve specic recognition, such as the regulation of protein molecules that bind at the interacting surfaces [4,5]. Surface structures such as mbriae, agella [6,7], and capsules [8] are believed to be involved in bacterial adhesion to different types of surfaces and interfaces [9]. The presence and properties of these cell surface constituents depend on the nature of bacterial species and the growth conditions [10]. Both Gram-negative and Gram-positive bacteria have been shown to possess surface layers and appendages which can range in length from short laments to rigid or exible structures up to several times the diameter of the cell [11,12]. Atomic force microscopy (AFM) allows the probing of interfacial phenomena at the nanoscale, including specic and nonspecic

Corresponding author. Fax: +65 6779 1936.

E-mail address: chetyp@nus.edu.sg (Y.-P. Ting). 0021-9797/$ - see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.08.021

interactions that are involved in the attachment and molecular recognition events between cellsolid and cellcell surfaces [1315]. Microorganism immobilized on AFM cantilever tip has also been used to study the interfacial phenomena of a variety of surfaces. Bowen et al. [16] bound metabolically active Saccharomyces cerevisiae to the tip to examine the interaction force between the cells and various mica surfaces. Benoit et al. [4] measured discrete intercellular interactions of Dictyostelium discoideum attached to the tip by using lectin. Recently Kang and Elimelech [17] developed a method for preparing live cell probes for AFM using a bioinspired polydopamine wet adhesive. Numerous bacterial species are responsible for problems in industries such as food spoilage and contamination, and the biocorrosion of equipment including cooling towers [1821]. In this work, we investigate the interactions between biolm-forming bacteria and stainless steel surface. The bacteria Pseudomonas aeruginosa, Bacillus subtilis and Massilia timonae were chosen for the afore-mentioned reasons. M. timonae was isolated from cooling tower water and was found to be the dominant corrosive bacteria amongst the culturable organisms in a cooling water environment. This bacteria causes severe pitting corrosion in copper metal (unpublished data), and its DNA sequence (FJ755911) has been deposited in NCBI center (http://www.ncbi.nlm.nih.gov/nuccore/225196302). In this study, we utilized cell probes prepared using a polydopamine adhesive method described earlier [17]. Force measurement data were acquired from the functionalized AFM tips between each of the three (Gram-positive and Gram-negative) bacterial species and SS316 surfaces. Surface properties of the bacterial species,

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including zeta potential, contact angle and hydrophobicity (derived from bacteria adhesion to hydrocarbon (BATH) test and emulsication index) were also investigated to correlate the effects of physicochemical properties of the bacterial surfaces on adhesion forces, in order to better understand the mechanism of bacterial adhesion on SS316.

2.6. Zeta potential Zeta potential of the bacterial cells was measured at pH 7.0 using a Mavern Zetasizer Nano System (Brookhaven Instruments Corporation, USA) [22]. All measurements were carried out in triplicates. 2.7. Contact angle measurement (CAM)

2. Materials and methods 2.1. Microbial strains and growth conditions Three bacterial strains M. timonae ARTYP3, P. aeruginosa NRRL-B 3509 and B. subtilis NRRL-NRS 762 were used to prepare the cell probes. M. timonae was isolated from a cooling tower water at National University of Singapore (NUS) and identied by 16S rDNA gene sequence; the nucleotide gene sequencing has been deposited in NCBI under the accession number EU274637. P. aeruginosa NNRL-B3509 and B. subtilis NNRL-NRS762 were provided by Agricultural Research Service (ARS) Culture Collection, USDA. Each of the three bacteria were rst cultivated in Petri dishes and subsequently grown in Tryptone Glucose Yeast extract (TGY) medium containing 5 g/L tryptone, 5 g/L yeast extract, 1 g/L glucose, 1 g/L K2HPO4 and pH 6.8 at 30 C to their mid exponential phase. Cells were then washed twice with 10 mM PBS buffer (pH 7.2) and diluted to 104 cells/mL. Contact angle measurement was made using a VCA Optima sessile drop method goniometer (AST Products Inc.) with a 1 lL water droplet. Approximately 30 lL of concentrated bacterial suspension was spread onto glass cover slip to obtain a thick cell lawn and kept overnight. The cell lawn then was collected and dried for at least 4 h. Static water contact angle of cell lawn was measured at 25 C and 60% relative humidity [23]. The contact angle measurements were repeated at least 10 times on three separate occasions. 2.8. Bacterial adhesion to hydrocarbon (BATH) Bacterial adhesion to hydrocarbon was evaluated by mixing 5 ml of microbial suspension with 500 lL hexadecane and vortexing for 5 min. After 15 min, the absorbance of the aqueous phase was measured and compared to that prior to mixing [24]. All measurements were carried out in triplicates. 2.9. Emulsication index (E) 2.2. Substrate preparation Stainless steel 316 (SS316) coupons of 10 mm diameter were polished using sand paper of various grit sizes (P400P2500), then with 0.3 lm alumina paste until a smooth mirror nish was attained. Coupons were subsequently washed with copious amounts of water before being sonicated with acetone and nally rinsed with 70% ethanol. The cleaned coupons were then stored in 70% ethanol prior to use. The emulsifying capacity was evaluated using an emulsication index (E). 2 mL of hexadecane was added to the same amount of bacterial culture, vortexed for 2 min and the sample was allowed to stand for 24 h. The E index is dened as the percentage of height of emulsied layer (mm) divided by the total height of the liquid column (mm). Measurements were also conducted with cell-free supernatants [25]. All measurements were carried out in triplicates. 2.10. Preparation of cell probes 2.3. Biolm development Overnight cultures of bacteria were grown in Duran bottles containing TGY medium for static biolm development. Polished coupons were hung inside the bottles and subsequently withdrawn after 24 h for SEM and FTIR analysis. A triangular-shaped silicon nitride cantilever tip (Veecoprobes Nanofabrication Center, CA) with a spring constant of 0.12 0.01 N/m (calibrated using thermal tune method) was irradiated with UV for 15 min in a biological safety cabinet. The cantilever was exposed to 4 mg/mL dopamine hydrochloride (99%, Sigma) in 10 mM TRIS buffer (pH 8.5) solution for 1 h to coat the inner surface with polydopamine [17,26] before it was washed with DI water and dried under vacuum. To immobilize bacterial cells on the tip, 10 ll of prepared cell suspension was spread on a polished stainless steel surface placed onto an AFM Piezoholder, and active cells were then transferred to the prepared AFM tip by slowly moving the tip holder downwards until it touched the cell suspensions. The tip was held in position for one minute to allow bacterial cell adhesion. The successful coating of the bacterial-cell tip was subsequently conrmed by SEM. Polydopamine functionalized bare AFM tip was used as the control in the AFM force measurement. 2.11. AFM force measurements 2.5. Fourier Transform Infra Red (FTIR) spectroscopy The chemical composition of biolm surfaces formed by the three different bacterial strains on the SS316 coupons was examined by an FTIR analysis conducted in the reectance mode with wavenumber over the range of 4004000 cm1 using FTIR spectrometer (Bio-Rad Model: FTS 135) equipped with a narrow band of liquid nitrogen-cooled HgCdTe (MCT) detector. Bare SS316 surface was used as a blank. All force curves were obtained using a Multimode Picoforce AFM coupled to an upright microscope using uid cells buffered with PBS solution (pH 7.2) at room temperature. Cantilevers were moved at a velocity of 500 nm/s with a piezo movement of 1000 nm. The duration of contact time between the cell probe and SS316 surface (termed surface delays) was varied between 0 and 60 s. Data were sampled more than 15 times at each of three locations on the metal surface. The total duration of the experiment was about an hour to

2.4. Scanning Electron Microscopy (SEM) Biolm samples were xed with 3% glutaraldehyde (in PBS) for more than 4 h, rinsed with PBS, and dehydrated with an ethanol gradient (25%, 50%, 70%, 90% and 99%) before nal storage in desiccators. Prior to analysis, the coupons were coated with platinum at a voltage of 30 mV for 100 s. A scanning electron microscope (JEOL JSM-5600) with 15 kV beam voltage was used to visualize the morphology of the biolm.

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Fig. 1. SEM images of a bacterial cell probe.

ensure the viability of the cells at the end of each cantilever. Raw data were converted from cantilever deection during the approach and retraction into force versus distance curves using the software V613r1 (Nanoscope). A positive force is associated with repulsion, and a negative force indicates attraction between the probe and the surface. 3. Results and discussion Adhesion forces between the bacteria and SS316 surfaces were investigated using AFM force spectroscopy. Fig. 1 shows SEM image of an AFM cell probe prepared using the polydopamine technique, with a bacterial cell successfully coated on the cantilever tip. Unlike the gluteraldehyde immobilization technique, where cross-linking of proteins and amino acids in the exocellular polymeric layer significantly inuences the interaction of gluteraldehyde-treated cells [27], this technique is noninvasive and can be performed for a wide range of application without affecting cell viability [17]. Results of the AFM force measurement are presented in Fig. 2 and Table 1. Fig. 2 shows examples of the retraction force curves of the functionalized bare AFM tip (as control) and tip coated with P. aeruginosa, M. timonae, and B. subtilis on the SS316 substrate, which relate to their adhesion forces onto surfaces. With surface delays of up to 60 s, the adhesion forces and separation distances are gradually increased. As seen in this gure, the control manifested the least adhesion forces within the smallest separation distances as compared with bacterial coated tip. Average adhesion forces of bacterial cell probes on SS316 surface in 10 mM PBS buffer (pH 7.2) with surface delays varying from 0 to 60 s are shown in Table 1. Compared with B. subtilis (a Gram positive bacterium), both P. aeuginosa and M. timonae (Gramnegative bacteria) show larger adhesion forces. These forces also gradually increased with surface delay for all the bacteria. Increasing surface delay will lead to bond maturation between bacterial surfaces and SS316 surfaces. Bond maturation is purely physicochemical in nature and has little to do with metabolic processes inherent in microorganisms. This phenomenon has been associated with the removal of interfacial water, unfolding of bacterial surface structures or rotation of an entire particle to have its most favorable site facing a substratum surface (i.e., a rearrangement of microorganisms on the surface) [28,29]. Increasing surface delay also increases the interaction of the protein structures on the bacterial surface through the electrostatic or hydrogen bonding forces between charged amino acid and metal surface charges which increases the overall adhesion forces [30]. Higher adhesion forces on Gram-negative bacteria (P. aeruginosa and M. timonae) as compared to Gram-positive bacteria (B. subtilis) can be related to their hydrophobicity. In this study the hydropho-

bicity of the three microorganisms was investigated using CAM, BATH, and an emulsication index test, and the results are presented in Table 2. CAM provides a denitive overall hydrophobicity value of the microbial cell surfaces and allows a comparison between the surfaces. Results show that SS316 was intrinsically the most hydrophobic surface with a contact angle of 86.8o. High CAM value of stainless steel has also been reported elsewhere [31,32]. This measurement also indicated that all the three bacteria are hydrophilic (h < 90), with B. subtilis being the most hydrophilic. The bacteria cell surface hydrophobicity was further assessed using the BATH assay where hydrophilic organisms would remain in the aqueous phase while hydrophobic organism would adhere to the hydrophobic hydrocarbon phase [33]. Grown in n-hexadecane containing medium, P. aeruginosa and M. timonae showed higher hydrophobicity (at 39.25% and 70.13% respectively) compared to B. subtilis (7.20%), which corroborates the results of the contact angle measurement. However, it should be noted that BATH tests were carried out at pH of the growth medium (i.e., pH 6.8) where, at this pH, the test may also measure the complicated interplay of long-range van der Waals and electrostatic forces and of various short range interactions [34,35]. The signicant differences in hydrophobicity between the Gram-positive and Gram-negative bacteria might also be due to the efciency of biosurfactant production by the latter [36,37]. Biosurfactant produced by bacteria increases cell hydrophobicity and its ability to degrade hydrocarbons [38]. The emulsifying capacity evaluated using the emulsication index (E) at different time periods (i.e., 24, 48 and 72 h) was higher in P. aeruginosa and M. timonae as compared to B. subtilis. This suggests that the biosurfactant production of the two Gram-negative bacteria is higher than the Gram-positive bacteria. Higher production of biosurfactant, which contains mainly polysaccharides and lipoprotein [39], leads to higher uptake of hexadecane as the carbon source [40]. Microbial surface hydrophobicity has been noted to be a dominant factor inuencing its adhesion on surfaces. In biological systems, hydrophobic interactions are usually the strongest of all long range non-covalent interactions and can be dened as the attraction between apolar or slightly polar molecules, particles or cells, when immersed in water [41]. Hydrophobic groups on the microbial cells play a major role in removing water lms from between the interacting surfaces, enabling adhesion to occur. Since it was found that P. aeruginosa and M. timonae showed higher hydrophobicity than B. subtilis, it explains why the adhesion forces of the two former strains are stronger than the latter. Stronger adhesion forces experienced by Gram-negative bacteria are also related to the electrostatic interactions between microbial surfaces and SS316 surfaces. Table 2 shows the surface charges of the three bacteria in terms of zeta potential. B. subtilis recorded the most negative charges (18.1 0.7 mV in PBS buffer) compared to M. timonae (9.7 0.8 mV) and P. aeruginosa (12.4 0.8 mV). Using a single-factor analysis of variance (ANOVA), the differences in zeta potentials among these three bacteria are found to be significant (p < 0.05). Most bacteria possess negative surface charges which originate from the lipopolysaccharides of the cell envelope and/or acidic functional groups on the proteins of the pili [30,42]. Electrostatic interactions between negatively charged microbial surface and SS316 surface which is also negatively charged are expected to be repulsive. From this point of view, it is expected that M. timonae and P. aeruginosa will experience less electrostatic repulsion than B. subtilis when approaching SS316 surfaces. Despite both surfaces being negatively charged, electrostatic attraction may still occur. When a charged particle (such as bacteria) approaches a charged-conducting material (such as SS316), a so-called image charge will develop in the conducting material. This image charge will be opposite in sign to the charge of the approaching particle, and will form or disappear by charge

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Fig. 2. Examples of AFM retraction curves for: (a) functionalized bare AFM tip (control); (b) P. aeruginosa NRRL-B 3509; (c) M. timonae ARTYP3; and (d) B. subtilis NRRL-NRS 762 with various surface delays.

Table 1 Average adhesion forces between bacterial cell probes and SS316 in 10 mM PBS buffer solution (pH 7.2). No. Type of cell probe Adhesion force (nN) Duration (s) 0 1 2 3 4 Control P. aeruginosa M. timonae B. subtilis 0.59 0.16 3.84 0.28 7.52 0.37 0.65 0.17 10 0.76 0.28 5.31 0.30 7.82 0.41 0.99 0.16 30 0.88 0.19 5.66 0.68 8.91 0.51 1.16 0.13 60 0.99 0.24 8.53 1.40 7.88 0.94 1.44 0.21

() Represents the standard deviation over 30 adhesion force measurements from each type of cell probe.

rearrangement in the conducting materials upon approach or retraction of the charged particle from the surface, respectively. Upon contact, the attractive force between the negatively-charged particle and its image charge is maximal, hence the interactions with the image charges causes an additional attraction between SS316 and the negatively charged bacteria [43]. In order to observe the ability of each bacterial strain to adhere on the metal surfaces, SS316 coupons were hung inside a biolm reactor containing 108 cell/ml of each bacterial culture. After 24 h of growth, coupons were subsequently withdrawn and the biolm formed on the SS316 surfaces were then analyzed. Fig. 3 shows

evidence that after 24 h, P. aeruginosa and M. timonae formed denser biolm than B. subtilis. The surface properties of the biolm formed after 24 h were also analyzed using Fourier Transform-Infra Red (FTIR) Spectroscopy which has been successfully applied for the characterization of chemical composition of metabolites in bacterial samples [44]. Fig. 4 shows the FTIR spectra of 24 h-biolm formed by each bacterial strain, along with the bare SS316 as the control. The spectra were measured over the range 4000500 cm1, which would include the major characteristic bands pertinent for microorganism [45]. In the presence of P. aeruginosa and M. timonae, broad peaks were noted in the range 9003650 cm1, which may be assigned to adsorbed water molecule OH/NH group [46]. This band can also be attributed to symmetric stretching of CAH from CH3, asymmetric CAH stretching from CH2, CAH vibration of CH3 and CH2 functional groups dominated by fatty acid chains (e.g., phospholipids), and OAH stretching of hydroxyl group and NAH stretching[47,48]. The FTIR spectra also indicated the presence of proteins (1700 1500 cm1) as well as polysaccharides and nucleic acids (1200 900 cm1). During cell growth, both in planktonic and biolm state, bacteria are known to produce varieties of polymeric materials such as proteins, lipopolysaccharides, oligosaccharides and possibly a variety of other polymers [32]. The protein amide I (C@O stretch strongly coupled with CAN stretch and NAH bending)

Table 2 Contact angle measurement (CAM), bacterial adhesion to hydrocarbons (BATH), zeta potential, and emulsication index of bacteria. No. System CAM BATH, % Zeta potential f (mV) Emulsication index (E), (h) 24 1 2 3 4 SS316 P. aeruginosa M. timonae B. subtilis 86.8 6.0 47.9 8.7 39.6 3.7 31.1 9.6 39.25 7.2 70.13 6.7 7.20 1.2 337 4 12.4 0.8 9.7 0.8 18.1 0.7 44.79 3.6 18.75 3.1 11.46 1.8 48 48.96 1.8 22.92 7.2 15.63 0.0 72 48.96 1.8 22.92 7.2 16.67 1.8

() Represents the standard deviation from each analysis.

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Fig. 3. Typical SEM images of the bacterial biolm formed after 24 h on SS316 coupon surfaces: (a) P. aeruginosa NRRL-B 3509; (b) M. timonae ARTYP3 and (c) B. subtilis NRRLNRS 762.

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and amide II (CAN stretch strongly coupled with NAH bending) regions are found at approximately 1650 and 1540 cm1, respectively. These amide bands can be used as universal probes for proteins because the peptidic acid bond is a basic and repetitive functional group in proteins [49]. The amide I band is generally more intense than amide II band. However, Fig. 4 also shows intense amide II bands due to some polysaccharides, such as alginate, which give rise to an intense band associated with the CAO stretching vibrational mode [50] located at 1610 cm1. The amide I bands near 1650 cm1 shows prominent peaks and corresponds to C@O stretching with different conformation, and CAN bending of protein and peptides amide. The intense small peaks near 1500 cm1 can be attributed to the symmetric deformation of NH 3 absorbed onto stainless steel surfaces [51,52]. The peaks near 1400 cm1 represent the symmetric stretches mode of C@O and CAO bending from carboxylate group, which manifest the interaction between COO group and the metal surface [53]. In this range, FTIR conrms the presence of extracellular polymeric substances (EPS) (COO, amide groups) secreted by the Gram-

negative bacteria on the metal surface [52,54,55]. In the case of B. subtilis, FTIR peaks showed less intensity of CAO, and OH group compared to P. aeruginosa and M. timonae. The bands in the polysaccharide region (1200900 cm1) were attributed to CAOH stretching mode and CAOAC (1150 cm1), CAO ring vibrations of carbohydrates (oligo and polysaccharides), CAOAP and PAOAP in polysaccharides of the cell wall, and also symmetric stretching of P@O from PO2 in nucleic acid. [47,48]. Results from FTIR spectra showed that proteins and polysaccharides as components of extracellular polymeric substances (EPS) were produced by all the three bacteria during biolm formation. The results, which suggest that the changes in chemical bonding with the surface atoms during biolm formation involve COO and NH 3 interaction, are in good agreement with the previous ndings [56] which suggested that proteins often function as adhesives in specic attachment mechanism. The FTIR spectra revealed that B. subtilis showed less intense peaks than P. aeruginosa and M. timonae. This was due to the less

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dense biolm formed by B. subtilis during the same 24 h duration (as depicted in Fig. 3). These results corroborate our previous ndings that Gram-negative bacteria manifested stronger adhesion forces as compared to Gram-positive bacteria, thus creating greater attachment of bacteria onto the SS316 surfaces. The three bacterial strains produce EPS, with denser biolm formed by the P. aeruginosa and M. timonae which secrete more EPS than the Gram-positive B. subtilis [5759]. 4. Conclusions AFM spectroscopy with bacteria on the cantilever tip was used to explore the nature of adhesion mechanism of P. aeruginosa, M. timoniae, and B. subtilis on stainless steel. Compared to Grampositive B. subtilis, Gram-negative bacteria P. aeruginosa and M. timonae showed very high tendency to form biolm on stainless steel surface; a higher adhesion force was observed in the bacterial cell probe of P. aeruginosa and M. timoniae. These ndings are supported by physicochemical analysis which showed that the hydrophobicity and electrostatic interaction between bacteria and the metal surfaces have signicant effect on the cellmetal adhesion. The surface charges signicantly inuenced the adhesion forces and enhanced the surface hydrophobicity. The higher hydrophobic behavior of the bacterial isolates induces the strengthening of bacterial adhesion on SS316, which is corroborated by the AFM force measurement. Emulsication index and FTIR analysis also highlighted that bacterial adhesion on the metal surface is inuenced by the presence of EPS produced during the growth and biolm formation. This study provides insight into understanding biolm formation by the three bacterial isolate on SS316 metal surface, and further demonstrates the viability of using AFM spectroscopy for the analysis of biolm formed on metal surfaces. Acknowledgments One of the authors (A. Harimawan) would like to express his sincere gratitude to AUN/Seed-NET JICA and the National University of Singapore for the Research Scholarship and funding for this project, and to Prof. C.T. Lim and Dr. Zhong Shaoping from Nano Biomechanic Lab, Division of Bioengineering NUS for the usage of the AFM facility. References
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