Article pubs.acs.org/Langmuir
Bacteria−Polymeric Membrane Interactions: Atomic Force Microscopy and XDLVO Predictions Justice M. Thwala,*,†,⊥ Minghua Li,‡ Mavis C. Y. Wong,§ Seoktae Kang,∥,# Eric M. V. Hoek,§ and Bhekie B. Mamba⊥ †
University of Swaziland, Private Bag 4, Kwaluseni M201, Swaziland, Southern Africa Nalco Company, Naperville, Illinois 60563, United States § Department of Civil and Environmental Engineering and California NanoSystems Institute (CNSI), University of California, Los Angeles, California 90095, United States ∥ Department of Civil and Environmental Engineering, University of Alberta, 3-096 Markin/CNRL Natural Resources Engineering Facility, Edmonton, Alberta T6G 2W2, Canada ⊥ Department of Applied Chemistry, Faculty of Science, University of Johannesburg Doornfontein Campus, Johannesburg, South Africa ‡
S Supporting Information *
ABSTRACT: Atomic force microscopy (AFM) in conjunction with a bioprobe developed using a polydopamine wet adhesive was used to directly measure the adhesive force between bacteria and different polymeric membrane surfaces. Bacterial cells of Pseudomonas putida and Bacillus subtilis were immobilized onto the tip of a standard AFM cantilever, and force measurements made using the modified cantilever on various membranes. Interaction forces measured with the bacterial probe were compared, qualitatively, to predictions by the extended Derjaguin−Landau−Verwey− Overbeek (XDLVO) theory with steric interactions included. The XDLVO theory predicted attractive interactions between low energy hydrophobic membranes with high energy hydrophilic bacterium (P. putida). It also predicted a shallow primary maximum with the most hydrophilic bacterium, B. subtilis. Discrepancies between predictions using the XDLVO theory and theory require involvement of factors such as bridging effects. Differences in interaction between P. putida and B. subtilis are attributed to acid−base interactions and steric interactions. P. putida is Gram negative with lipopolysaccharides present in the outer cell membrane. A variation in forces of adhesion for bacteria on polymeric membranes studied was interpreted in terms of hydrophilicity and interfacial surface potential calculated from physicochemical properties.
1. INTRODUCTION
Bacterial adhesion has, in previous studies, been attributed to properties of the bacterial cell surface characteristics such as hydrophobicity, zeta potential, extracellular polymeric substances such as lipopolysacharides (LPS), flagella, fimbriae, proteins, and biosurfactants.2,5 The properties of the bacterial surface constituents depend on the species of bacteria and the growth media involved. Gram-negative bacteria are known to possess LPS molecules on their outer membrane surface, and Gram-positive bacteria have been found to have teichoic and lipoteichoic molecules instead of LPS molecules. The substratum surface structure has also been reported to influence bacterial adhesion.6 Substratum properties that affect bacterial adhesion are hydrophobicity, zeta potential, and surface roughness.7
Knowledge of bacterial attachment to surfaces is important for the control and application of bacterial adhesion and biofilms in engineering, environmental, and biomedical industries. Bacteria adhere to different kinds of surfaces including the human body, plants, soil, metals, and plastics to form a fluid microbial structure called a biofilm.1 In biomedical fields failure of devices such as orthopedic implants (joint prosthetics), dentures, heart valves, contact lenses, and vascular catheters is due to bacterial adhesion, which may prevent host immune mechanisms and limit treatment by antibiotics. Frequently, the infected device implants are removed surgically.2 Bacteria also adhere to on reverse osmosis (RO) and nanofiltration (NF) membrane surfaces in water treatment plants, increasing operating costs needed to produce potable water and to clean the membranes.3 Bacterial adhesion to soil has been used for in situ bioremediation.4 © 2013 American Chemical Society
Received: July 19, 2013 Revised: September 23, 2013 Published: September 24, 2013 13773
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
2. MATERIALS AND METHODS
Atomic force microscopy (AFM) has been used in various ways to study the interactions between bacteria and different surfaces. This includes chemical modification of AFM tips with specific molecules to study nanoscale interaction of bacterial cells, colloidal particles and organic molecules.1−19,26 Razatos et al.35 developed methods of directly measuring the forces of interaction between AFM cantilevers with standard silicon nitride (Si3N4) tips and bacterial lawn immobilized on flat glass substrates.4 However, this technique was restricted to the study of micrometer-sized beads of low size disparity. To address this problem, Razatos et al. then developed a procedure for coating the (Si3N4) tips with a confluent of bacteria.25 In the current study, a single cell AFM tip, earlier developed by Kang and Elimelech,10 was utilized to study bacterialmembrane interactions. The use of a single cell probe gives a much more accurate assessment of the forces of interaction with substrates since the probe has a more defined geometry compared to a confluent of bacteria. Force measurements were taken using polydopamine functionalized tips for Gram-positive bacteria, Bacillus subtilis, and Gram-positive bacteria, Pseudomonas putida, on various membrane surfaces that span from low to high hydrophilicities. Surface properties of bacteria species, such as zeta potential, contact angle, and hydrophobicity (calculated from interfacial free energy of cohesion or hydrophilicity), were also determined to investigate whether a correlation exists between physicochemical properties of bacterial surfaces and bacterial adhesion on membrane surfaces. Despite numerous studies that have been undertaken to understand the processes of bacterial adhesion, the mechanism has not yet been fully resolved. Theories on adhesion of colloidal particles such as the Derjaguin−Landau−Verwey− Overbeek (DLVO)7 and extended DLVO model8,9 have been used to explain microbial adhesion. The classical DLVO theory accounts for two main interactions, the attractive Liftshitz−van der Waals (LW) and the repulsive electrostatic double layer (EL) interactions. The XDLVO theory by van Oss8,9 added an acid−base interaction term to account for Lewis acid−base interaction involved where bacterial cells interacts with hydrophilic surfaces through hydrogen bonding. In both DLVO and XDLVO theories, the adhesive forces may be determined using the classical nonretarded van der Waals approach that uses the Hamaker constant or a thermodynamic approach obtained from contact angles measurements to quantify hydrophobic interactions. Both approaches have been used in this study and compared with AFM measurements. Furthermore, most studies exclude steric interactions that originate from bacterial surface features such as LPS molecules. Steric interactions may be repulsive in instances where the substrate is hydrophilic and may be attractive (bridging interaction) where the substrate is hydrophobic.15 In this paper, a comparison of the force of adhesion between a single bacterial cell probe (prepared on AFM cantilever tips) and a variety of RO and NF membrane surfaces including mica is made. Experimental results using the AFM to measure the forces of interaction between bacteria and membrane surfaces are compared to theoretical predictions based on the extended DLVO model. In this model, in addition to hydrophobic forces, steric interactions have been added to van der Waals and electrostatic components for Gram-negative bacteria, P. putida. For Gram-positive bacteria, B. subtilis, only hydrophobic, van der Waals and electrostatic components were considered.
2.1. Bacterial Strains and Growth Conditions. Two bacterial strains, P. putida (strain KT2440) and B. subtilis (strain JH642) were used as model microbes. B. subtilis cells were grown in Luria−Bertani (LBat 20 g/L Fisher Scientific, Hampton, NH) medium at 37 °C to their midexponential growth phase. Cells were then washed in Tris buffer (pH8.0) and diluted to 104/mL prior to attachment on the AFM tips. P. putida cells were grown in Trypticase Soy Broth (BD, Franklin Lakes, NJ), which is a mixture of 5 g/L bacteriological peptone and 1 g/L yeast extract, and then grown for 24 h at 30 °C, with vigorous shaking (180 rpm). 2.2. Preparation of Single Cell Probes. Polydopamine was prepared by dissolving 0.4 g of decarboxylated L-3,4-dihydroxyphenylalanine (DOPA (OH)2C6H3CH2CH2NH2Br), Sigma-Aldrich, St. Louis, MO) in 50 mL of deionized (DI) water to give a 8 mg/mL solution. This was stored in a refrigerator at 4 °C until use. Then 200 mL of a 20 mM Tris buffer solution was prepared by dissolving 0.5 g of (hydroxymethyl)aminomethane ((HOCH2)3CNH2, Sigma-Aldrich) in 200 mL of deionized water to give a solution of pH 9.75. Equal volumes of DOPA and Tris buffer were mixed to prepare the polydopamine solution (4 mg/mL), and the pH was adjusted to 8.5− 8.8 using 2 N HCl and NaOH. A triangularly shaped cantilever was cleaned in strong oxidizing “piranha solution” (H2SO4/H2O2at a 4:1 ratio) to remove organic residue for 30 min and rinsed in DI water before it was dried in a vacuum. The tip was then cleaned in a UV/ozone reactor (PSD-UV series, Novascan, Ames, IA) for 30−40 min to oxidize all remaining organics. The furthest end of the tip was dipped in the polydopamine solution for 1 h using a micromanipulator to ensure that the part on which the AFM laser focuses is not covered. Finally, the tip was rinsed in DI water and dried in vacuum. A second layer may be formed with the same method to ensure complete coverage of the tip and successful bacterial adhesion. The bacterium was then incubated for 1 h, after which the AFM experiments were run. To attach the single bacterial cell on the cantilever, a 10 μL bacterial suspension was spread on a clean glass slide and observed through a microscope (Olympus, BX51WI fixed-stage with an upright microscope). A single active cell was isolated and attached to a DOPA modified cantilever tip by touching the tip to the cell with the aid of a micromanipulator. The bioprobe was prepared for scanning electron microscopy (SEM), after it has been used for adhesion experiments, by dipping in 20, 50, 75, 95, and 99% ethanol/water mixtures for 5 min each to dehydrate the bacteria as the microscope operates in high vacuum. It was then dried in a Petri dish in a desiccator followed by sputter coating using gold at 20 mA for 20 s. The SEM images were taken at voltages in the range of 7.5−10 kV. 2.3. Bacterial Viability Experiments. An SEM image of the AFM tip was taken after adhesion force measurements were taken to check the presence of the cells. Fluorescence microscopy was also run after force data were collected and prior to SEM imaging to check viability of the bacteria using a SYTO 9/propidium iodide stain (SYTO 9/PI, Molecular Probes, Inc., Eugene, OR) procedure on control glass slides and on the cantilever tip after each AFM bioprobe experiment. Silica glass surfaces were washed in methanol followed by a wash in piranha solution (H2SO4/H2O2) and put in the UV/ozone reactor for 40 min after drying in vacuum. The surfaces are then coated in polydopamine. Cells in Tris buffer are then suspended in the surfaces for 1 h. After incubation, the surfaces are gently washed to remove excess loosely bound cells and fluorescence viability is run after staining cells for 15 min in SYTO/PI using 10 μL drop of stain. 2.4. Membranes. The commercial membranes, selected to give a range of hydrophilicity values for this study, were SW3+ (Hydranautics, Oceanside, CA), NF90 (Dow-FilmTec Corp., Edina, MN), and SWHR (Dow-FilmTec). NF90PVA was prepared by modifying NF90 through dip coating in polyvinyl alcohol using standard procedures developed by Peng et al.22 Mica (AFM-grade, Novascan) was also included to represent highly hydrophilic substrate. Membrane roughness was obtained from AFM measurements, and the 13774
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
hydrophilicity of each membrane was evaluated using contact angle measurements. 2.5. AFM Interaction Force Measurements. Membrane roughness and force curves were obtained via Novascan 3D SPM microscope. Force data were collected in a liquid cell buffered with 10 mM Tris HCl buffer at 22 °C. Force measurements were taken with the piezoelectric scanner moving at 500 nm/s over 100 nm at a resolution of 2000 data points. For each membrane, data was collected at 50 locations taking a minimum of at least 20 measurements at each location. The total experimental time was kept to within 1 h to ensure viability of the bacterium at the end of the tip. Data was recorded as force versus cantilever deflection during approach and retraction. A negative force indicated attraction between the cell and the membrane and a positive force is associated with repulsion. The overall quality of a measured force curve was assessed by the scatter in the data following contact between the bacteria and the membrane surface.24 The cantilever deflection data was converted to a force measurement using the detector response (deflection in nA), the cantilever optical sensitivity (nA/nm), along with the calibrated spring constant (0.06 N/m), which was converted from raw data (photodiode voltage versus z-position of the piezoelectric scanner) to force-separation curves by the AFM software. 2.6. Contact Angle and Zeta Potential Measurements. Contact angles (θ) were measured by the sessile drop technique using a contact angle goniometer (DSA10, KRUSS GmbH, Hamburg, Germany). The equilibrium value was the steady state average of right and left angles of a deionized water droplet on the membranes placed in an environmental chamber of the goniometer. Contact angles for bacterial cells were determined by preparing cell lawns on a SCW3+ membrane to complete coverage through dead end filtration. These were then dried in a vacuum chamber before the measurements were made. Zeta potential was determined for the bacterial cells in 10 mM Tris buffer. Cells were harvested in the midexponential phase (108cells/mL), washed, and resuspended in 10 mM buffer (pH 8.85). Zeta potential was measured using a ZetaPALS potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). The number concentration of cells in each suspension was quantified using a hemacytometer. 2.7. Interaction Energy Calculations. Two models were employed in order to interpret AFM force data: the extended DLVO theory and a model for steric repulsion between bacterial lipopolysacharide molecules and membrane surfaces. 2.7.1. XDLVO Interaction. The interaction energy between the bacterium and membrane surfaces can be described using classical DLVO theory as a sum of van der Waals and electrostatic double layer interactions: DLVO Van EL Vmlb = Vmlb + Vmlb
the surface free energies can be separated into an apolar or dispersion component (due to Lifshitz−van der Waals force) and a polar or Lewis acid−base component comprising an electron-donor and electronacceptor parameter. A three probe liquid contact angle method is used. Two of the probe liquids should be polar and one of the probe liquids must be apolar. The extended Young−Dupré equation:11 ⎛ 1 + cos θ ⎞ TOT ⎜ ⎟γ = ⎝ ⎠l 2
XDLVO
γm−γl+
(3)
,γ , γ and γ are total liquid surface tension, Lifshitz−van where γ der Waals (LW), electron-donor, and electron-acceptor components of the solid surface tension, respectively. An apolar liquid, such as diiodomethane, is used to calculate the nonpolar component of surface tension (γLW S ) The extended Young−Dupré equation for the apolar liquid becomes: TOT
LW
+
⎛ 1 + cos θ ⎞ TOT ⎜ ⎟γ = ⎝ ⎠l 2
γsLWγlLW
where γTOT is the total surface tension of a pure substance obtained as L a sum of LW and AB components, yielding:
γ TOT = γ LW + γ AB
(5)
And the acid−base component, AB, is given by γ AB = 2 γ −γ +
(6)
The Lifshitz−van der Waals interfacial free energy component, ΔGLW, of the XDLVO equation may then be written as LW ΔGmlb = 2( γlLW −
γmLW )( γbLW −
γlLW )
(7)
AB
while the acid−base component ,ΔG , follows from AB ΔGmlb = 2 γl+ ( γm− +
+ 2 γl− ( γm+ +
γb− − γb+ −
γl− ) γl+ ) − 2( γb−γm+ γm−γb+ )
(8) If, instead of the bacteria (b), the membrane (m) is inserted in eqs 7 and 8 (e.g., b = m), ΔGTOT mlb gives the interfacial free energy of adhesion. If the free energy is positive, the membrane surface is considered hydrophilic, whereas if the energy is negative, the membrane structure is considered hydrophobic.12 2.7.3. Interaction Energy between Bacterium and Membrane Surfaces. In order to map the Liftshitz and acid−base interaction energy profiles for bacteria and membrane surfaces with varying separating distance between them, van Oss proposed the equation:
(1)
LW ΔG(plate −plate) = −
whereVDLVO is the total interaction energy between membrane and bacteria immersed in water,VVan is the van der Waals interaction term, and VEL is the electrostatic interaction term. Subscripts m, l, and b stand for the membrane surface, liquid, and the bacterial surface, respectively. According to the XDLVO model, the free energy of interaction is a sum of Liftshitz−van der Waals, electrostatic double layer, and Lewis acid−base interactions. The total interfacial free energy of interaction becomes:7 XDLVO LW EL AB Vmlb = Vmlb + Vmlb + Vmlb
‑
γm+γl− +
γmLWγlLW +
A 12πy 2
(9)
where A is the Hamaker constant and y is the separation distance between two planar surfaces. The expression for Hamaker constant is LW A = − 12πy0 2 ΔGmlb
(10)
where y0 is the equilibrium cut off distance (distance where bacterium contact the membrane surface). The interaction free energy per unit area between two flat surfaces is scaled to the corresponding interaction energy between bacteria and membrane surfaces using Derjaguins’ technique7 to yield the equation:
(2)
TOT
(=V ) is the total interaction energy between where V membrane and bacteria immersed in water, VLW is the Liftshitz−van der Waals interaction term, and VAB is the Lewis acid−base interaction term. The acid−base interaction term accounts for hydrophobic attraction, hydrophilic repulsion, and structural forces. 2.7.2. Thermodynamic Approach: Liftshiftz−van der Waals Acid−Base Model. The XDLVO model requires that surface energy parameters of the membrane and bacteria be obtained from experiment. Thermodynamically, interfacial free energies can be derived from contact angle (θ) measurements between a liquid and a membrane surface or bacterial lawn. According to van Oss et al.,8,9
LW V(sphere −plate) = −
AR 6h
(11) 27,29
and h is the separation where R is the equivalent bacterial radius between bacteria and the membrane surface. In order to compare AFM force measurements data with predictions from the XDLVO theory, eq 11is differentiated via F=− 13775
dV dh
(12) dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
Figure 1. SEM images of (a) B. subtilis bacteria cell probe and (b) P. putida bacterial cell probe.
Figure 2. (a) AFM and (b) SEM images for NF90 nanofiltration membrane. the electrostatic force of interaction is obtained:
Upon differentiation, the force interaction term for bacteria− membrane LW interaction term becomes LW F(sphere −plate) = −
EL Fmlb = πεrε0R(ψ12 + ψ2 2)
AR 6h2
(13)
y0 2 R h2
(14)
The acid−base force of interaction is obtained by applying Derjaguin’s technique on the free energy of interaction between flat surfaces:13 AB AB ΔG(plate −plate) = ΔGmlb
⎡ y − y⎤ exp⎢ 0 ⎣ λ ⎥⎦
1 = κ
(15)
(16)
The AB force of interaction, obtained through the derivative of eq 16, becomes
⎡ y − y⎤ AB AB Fmlb exp⎢ 0 = 2πR ΔGmlb ⎥ ⎣ λAB ⎦ From the derivative of the interaction energy equation
(17)
2niυ2e 2
(20)
3/4 ⎤ ⎡ ⎛ 2L ⎞9/4 ⎛ h ⎞ ⎥ steric Fmlb = kBT Γ 3/2LR ⎢⎜ 0 ⎟ − ⎜ ⎟ ⎢⎝ h ⎠ ⎝ 2L0 ⎠ ⎥⎦ ⎣
7
⎛ ⎛ 1 + exp(− κh) ⎞ EL V(sphere ⎟ ⎜2ψ1ψ2 ln⎜ −plate) = πεrε0R ⎜ ⎝ 1 − exp(− κh) ⎠ ⎝ ⎞ + (ψ12 + ψ2 2) ln[1 − exp( −2κh)]⎟⎟ ⎠
εrε0kBT
where e is the electron charge, ni is the number of ions per m3, υ is the valence of ions, kB is the Boltzmann constant, and T is absolute temperature. 2.7.4. Electrosteric Model. A steric model developed for polymers grafted on surfaces at high coverages was used to model steric interaction between bacteria on AFM tip and membrane surfaces. For high surface coverage, de Gennes23 calculated the force between two equal surfaces with one surface having a polymer coating (bacterial surface with LPS), to obtain:
where λ is the decay length. This yields
⎡ y0 − y ⎤ AB AB V(sphere −plate) = 2πRλΔGmlb exp⎢ ⎣ λ ⎥⎦
(19)
where ε0 is the permittivity of vacuum, εr is the relative dielectric permittivity of water, κ is the inverse Debye screening length, ψ1 is the surface potential of the bacterium, and ψ2 is the surface potential of the membrane surface. The surface potentials were estimated from measured zeta potentials of the surfaces involved. The Debye screening length is determined using the equation:
Combining eqs 10 and 11 gives the LW interaction force between bacteria and a membrane surface in aqueous media: LW LW Fmlb = 2π ΔGmlb
2κ exp(2κh) − 1
(21)
with kB being the Boltzmann constant and T the absolute temperature; Γ is adsorption density in m−2, L0 is the equilibrium thickness of the polymer brush, R is the bacterium radius, and h is the separation distance between bacteria and the membrane surface.
(18) 13776
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
Table 1. Adhesion Forces for B. subtilis and P. putida on NF90 Nanofiltration Membrane NF90 BS PP a
contact angle
mean force (nN)
SD
n
ζ potential/mV
θwater
θEGa
θDIb
−2.44 −3.01
2.42 1.55
143 184
−39.15 ± 0.05 −24.86 ± 0.28
16.6 ± 0.29 29.3 ± 0.6
26.3 ± 0.58 34.0 ± 1.6
40.0 ± 3.5 52.7 ± 2.8
Ethylene glycol. bDiiodomethane.
Table 2. Surface Tension and Hydrophilicity of Various Substrates and Bacterial Lawns Expressed as mJ/m2
a
surface
γLW
γ+
γ−
γAB
γTOT
ΔGLW
ΔGAB
ΔGmlm
SWC3+ NF90 SHWR NF90PVA mica P. putida B. subtilis
39.4 42.5 49.2 43.1 35.8 42.7 34.0
2.7 1.8 5.7 5.6 2.9 5.2 0.0
1.9 6.7 72.7 70.0 111.9 89.7 79.8
4.6 6.9 40.7 39.6 35.8 43.3 3.7
44.1 49.5 89.9 82.6 71.6 86.0 37.7
−5.2 −6.8 −11.0 −7.2 −3.4 −6.9 −2.7
−49.5 −36.4 36.9 35.6 74.3 48.9 75.2
−54.7 −43.2 25.9 28.4 70.8 42.0a 72.5a
ΔGmlm is represented by ΔGblb for bacteria
3.3. Measured Physicochemical Properties of Bacterial Cells. Table 1 summarizes the physicochemical properties of bacterial strains used in the study. Briefly, the average hydrodynamic radii of P. putida and B. subtilis were about 1.02 ± 0.15 and 0.75 ± 0.05 μm, respectively. P. putida and B. subtilis have contact angles (θwater) of 29.3 ± 0.6 o and 16.6 ± 0.29°, respectively. These contact angles indicate that both bacteria are quite hydrophilic with P. putida showing less hydrophilicity than B. subtilis. B. subtilis and P. putida also have negatively charged zeta potentials of −39.15 and −24.86 mV in Tris buffer, respectively. Bacterial cells under physiological conditions of this study are negatively charged due to the presence of anionic surface groups such as carboxyl and phosphate.14,15 3.4. Interfacial Interaction Parameters for Bacteria. Thermodynamic interfacial interaction parameters for membranes and bacteria are provided in Table 2. The Liftshitz−van der Waals component of surface energy (γLW) for P. putida and B. subtilis is 42.7 and 34.0 mJ/m2, respectively. Electron-donor components of surface energy (γ−) are 2 orders of magnitude larger than electron-acceptor (γ+) values for B. subtilis and 10 times larger for P. putida. The electron-donor surface energy values are consistent with previous studies. It has been reported that microorganisms living in the Earth’s lower atmosphere, where levels of oxygen are high, tend to have hydrated cell surfaces and have a predominance of electron-donor functionality.16−18 The interfacial free energy of cohesion, ΔGblb, represents the free energy when two surfaces of the same material, in this case a bacterial lawn, are immersed in water. The bacteria studied showed positive interfacial free energy of cohesion values, ΔGblb, of 42.0 mJ/m2 for P. putida and 72.5 mJ/m2 for B. subtilis. These positive values indicate that both bacteria are hydrophilic in the order P. putida < B. subtilis. P. putida is Gram-negative and has lipids and proteins, flagellum, and lipopolysacharides (LPS) in the outer membrane structure.15,19 These molecules render the bacteria less hydrophilic than the Gram-positive B. subtilis that are without LPS molecules. B. subtilis has a higher Lewis acid−base activity, ΔGAB, than P. putida, rendering it more hydrophilic. 3.5. Interfacial Interaction Parameters for Membrane Surfaces. Table 2 shows the interfacial free energy of interaction, ΔGmlm, for all the membranes and bacterial lawns tested. The hydrophilicity of the membranes, using the
3. RESULTS AND DISCUSSION 3.1. Bacterial Cell Probe. Figure 1 shows SEM images of single bacterial cell probes prepared using polydopamine for both B. subtilis and P. putida. An accidentally crushed B. subtilis bacterial cell can be seen on the edge of the AFM tip in Figure 1b. This shows the difficulty that is at times encountered in selecting single bacterium from the slides on which they are placed during the cell probe preparation. Confluent bacterial probes were initially prepared to verify the viability of using polydopamine as an adhesive. These were then observed under an optical microscope. The optical images are shown in the Supporting Information (Figure 1S). Figure 1Sd also shows a schematic representation of the bacterium attached to the AFM cantilever tip. Previous studies reported immobilization of bacteria using glutaraldehyde and observed that the attached cells were inactivated and thus altered the force of adhesion between the studied bacteria and substrates of interest.25 The use of polydopamine in the technique reported in this study does not affect cell viability and can be applied in a wide range of cells and applications.26 3.2. Measured Physicochemical Properties of Membranes. The membranes investigated were reverse osmosis membranes, namely, SW3+, SW4+, NF90, SWHR, NF90-PVA, and mica listed in order of increasing hydrophilicity. Figure 2 shows typical AFM tapping mode and SEM images of the NF90 nanofiltration membrane. AFM images of the other membranes are shown in Supporting Information Figure 2S. Measured physicochemical properties of the five test substrates are listed in Table 1S in the Supporting Information. Briefly, the water contact angles are 76° for SWC3+, 62° for NF90, 41° for SHWR, 48°for NF90PVA, and 0° for mica; increasing in the order mica < SHWR < NF90PVA < NF90 < SWC3+. The surface to area ratio (SAD (%)) values for SWC3+, NF90, SHWR, and NF90PVA are 29%, 37%, 16%, and 16%, respectively. Surface roughness increases in the order mica < NF90PVA = SHWR < SW3+ < NF90 as reflected by SAD (%) values. Previous studies have shown that more hydrophobic and rough surfaces have higher fouling propensity,3 which would be expected to have higher adhesion forces with bacterial probes. 13777
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
reported ΔGmlm values, increases in the order of SWC3+ < NF90 < SHWR < NF90PVA < mica. Electron donor functionality, (γ−), predominates on SHWR, NF90PVA, and mica with values of 72.7, 70.0, and 111.9 mJ/m2, respectively. These values are larger than the acceptor functionality of the membranes resulting in large acid−base functionality. The high acid base functionality is due to active chemical functionality of these membranes. SHWR is a reverse osmosis membrane with a polyamide thin film made of m-phenylenediamine (MPD) and trimesoyl chloride (TMC) monomers backbone with amide (−NH), hydroxyl (−OH), and carboxylic acid groups (−COOH). SHWR also contains vinyl alcohol (−CCOH) functionalities.20 The nanofiltration membrane, NF90, also consists of a fully aromatic polyamide thin film made of MPD and TMC monomers, less cross-linked than SHWR with a thickness of 50−250 nm. High cross-linking results in high hydrophobicity in membranes as the number of carboxyl functionalities is reduced.21 Polyvinyl alcohol (PVA) modified nanofiltration membrane, NF90PVA, has predominately hydroxyl groups (−OH) and cross-linked C−O−C backbone, increasing the membrane hydrophilicity.22 Table 3 shows interfacial free energy between membrane surfaces and bacteria. The true interaction free energy between
Figure 3. Force−distance profile for B. subtilis on a nanofiltration membrane (NF90) in 10 mm Tris buffer.
interactions that involve bond formation and cell deformation allowing contact between bacteria and the membranes. Ting et al. reported similar trends in the force of adhesion with increase in delay times, and attributed these to bond maturation, removal of interfacial water, unfolding of bacterial surface structures and rearrangement of microorganisms on substrates.26 Adhesion values obtained by Ting et al. for B. subtilis on a metal surface SS316 in 10 mM PBS buffer were in the range −8 ± 2 nN. Increasing surface delay was also reported to increase interaction of protein structures on the bacterial surface through electrostatic or hydrogen bonding forces between amino acid and membrane surface charges.27 Figure 4 shows force per unit separation distance, F/R, profiles of P. putida on various membranes. The measured adhesion force is related to the adhesion energy per unit area Wadh by Fadh = 1.5πRWadh for soft deformable surfaces.31 A plot of F/R therefore gives a direct assessment of the interaction energy between surfaces. To accurately analyze AFM force data the contact point between the bacterial probe and the membrane surfaces needs to be established.28 The contact point was defined as the point where the cantilever deflection becomes a linear function of piezo scanner position (compliance).28 As expected, Figure 4 shows that at large distances from the surface (zero piezo position on the figure) no interaction between the bacteria and the membrane was detected (adhesion forces close to zero). As the bioprobe approaches the membrane surface, there is a repulsive force, namely, electrostatic repulsion. Once contacted, some of the surface proteins, teichoic and lipoteichoic acids, normally found in Gram-negative bacteria, attach the bacteria to the membrane surface. This gives rise to the observed force minimum in the retraction curves, followed in some instances by multiple unbinding events, which indicate formation of specific interactions after initial contact and existence of exocellular polymeric layers on the cell probe even at distances of 500 nm.10 Figure 4 presents the force of adhesion per unit separation distance, F/R, of P. putida for SW3+, NF90PVA, and SHWR. Figure 5S in the Supporting Information also shows both approach and retraction force curves versus piezo position for P. putida on the various membranes including histograms for the force-curve profiles. Figure 3S shows the variation of adhesion forces against contact angle of selected membranes and Table 4 the mean values and standard deviations. All the membrane surfaces showed attractive interactions with P. putida except mica. As shown in Table 4, P. putida gave average attractive forces of −4.88, −3.01, −1.49, and −1.2 nN with
Table 3. Interfacial Free Energies between Membrane Surfaces and Bacteria B. subtilis
P. putida
surface
ΔGLW
ΔGAB
ΔGBS mlb
ΔGLW
ΔGAB
ΔGPP mlb
SW3+ NF90 SHWR NF90-PVA Mica
−3.7 −4.3 −5.5 −4.4 −3.0
−8.9 5.0 54.3 52.9 79.6
−12.7 0.7 48.9 48.6 76.6
−6.0 −6.9 −8.8 −7.1 −4.8
9.9 19.3 42.8 42.1 60.2
3.9 12.3 34.0 35.0 55.4
membranes and bacterial cells is described by the free energy of adhesion, ΔGmlb (mJ/m2). Even though the bacteria-membrane interfacial free energy of adhesion is not predictive, it gives insight into the likelihood of bacteria being attracted or repelled by the membranes.8,24−26 For B. subtilis, the bacteria-membrane interfacial free energy of adhesion, ΔGBS mlb, shows an increase in adhesion in the order: mica < SHWR < NF90PVA < NF90 < SWC3+. P. putida-membrane interfacial free energy of adhesion, ΔGPP mlb, shows an increase in adhesion in the order: mica < NF90PVA < SHWR < NF90 < SWC3+. For both bacteria, the increase in the bacteria-membrane interfacial free energy of adhesion, ΔGmlb, follows the same order of increase in hydrophobicity and decrease in electron-donor functionality. Furthermore, the bacteria-membrane interfacial free energies of adhesion for P. putida are lower than those of B. subtilis for hydrophilic membranes (NF90PVA and SHWR) and mica, implying a higher likelihood for P. putida to adhere to those membranes than B. subtilis. 3.6. AFM Results. Figure 3 presents a typical AFM force− distance curve of a B. subtilis bioprobe approaching a nanofiltration polymeric membrane (NF90) at various probe dwell times. Figure 3 shows the variation of the force of adhesion as a function of separation distance at different probe contact times. An optimum dwell time of 5 ms was found to be sufficient for all force measurements carried out in this study. This data shows that biological adhesion to surfaces is a dynamic process and bacterial cells adhere to surfaces through specific 13778
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
Figure 4. F/R versus separation distance for (a) P. putida bacteria on SHWR, NF90PVA, NF90, and SWC3+ membrane and mica. (b) Polydopamine coated bare AFM cantilever tip (control) on a NF90 membrane surface showing representative approaching and retraction profile.
correspond to interaction energies of −2.03, −1.25, −0.62, −0.50, and +1.11 mJ/m2 for SWC3+, NF90, SHWR, NF90PVA, and mica, respectively. This suggests that the more hydrophobic the membrane surface is, the higher the adhesion force. The SWC3+ RO membrane is the most hydrophobic, and it showed the highest adhesive force of −4.88 nN; and the least hydrophobic surface (NF90PVA) showed the least adhesion force (−1.2 nN). Interaction forces of the two bacteria strains studied were also compared. The average interactive force obtained for P. putida was −3.01 ± 1.55 nN and −2.44 ± 2.42 nN for B. subtilis on NF90 in 10 mM Tris buffer (shown in Table 4). Higher adhesion forces of Gram-negative bacteria (P. putida) as compared to Gram-positive bacteria (B. subtilis) can be explained in terms of hydrophilicity. B. subtilis is more
Table 4. Summary of Adhesion Forces between P. putida Bacterial Cell Probe and RO Membranes in 10 mm Tris buffer at pH 8.85a membrane
mean force (nN)
SDb
Nc
SW3+ NF90 SHWR NF90-PVA mica
−4.48 −3.01 −1.49 −1.21 +2.66
3.40 1.55 1.61 1.26 2.63
168 184 204 186 150
a
Representative adhesion force curves and force distributions are shown in Figure 2. bStandard deviation of measurements. cTotal number of measurements.
SWC3+, NF90, SHWR, and NF90PVA, respectively, whereas a repulsive force of +2.66 nN was observed for mica. These
Figure 5. Calculated interaction profiles for P. putida. (a) Force−distance curve with a primary maximum on NF90. (b) Force−distance curves with a primary minimum on NF90. (c) Force−distance curves compared to AFM approach force curves for NF90 (r = 0.72, SRR=4430). (d) Force− distance curve for interaction with mica in 10 mM buffer. 13779
dx.doi.org/10.1021/la402749y | Langmuir 2013, 29, 13773−13782
Langmuir
Article
Figure 6. Calculated force−distance curves for B. subtilis interacting with NF90 membrane surface in 10 mM buffer showing (a) XDLVO, DLVO, LW, EL, and AB interactions. (b) XDLVO interaction on an expanded scale.
where the fitting parameters are A, that represents the amplitude, and λ, the decay length. According to Oss et al., the equilibrium distance of closest approach between bacteria and membrane surfaces, y0, is normally set at 0.157 nm. A value of y0 + z (z = 0.5 nm)28 which yields 0.657 nm was used in this study due to the fact that the distance is measured from the shear plane, as the zeta potential, ζ, is used instead of surface charge (ψ0) while evaluating electrostatic attraction.11,12,19 The graft density, Γ, obtained is comparable to that of P. putida KT2442 strain of 2.4 × 1015 m−2 reported by Camesano et al. in 1 mM ionic strength. The polymer lengths reported by Camesano et al. using the steric repulsion model ranged from 230 to 1040 nm. Simoni et al. reported LPS lengths of 20 nm for Pseudomonas sp B13.36 In Figures 5 and 6, the DLVO and XDLVO predictions are different at low separations (
