Article pubs.acs.org/est

Electrokinetic Control of Bacterial Deposition and Transport Jinyi Qin,† Xiaohui Sun,‡ Yang Liu,‡ Tom Berthold,† Hauke Harms,† and Lukas Y. Wick*,†,‡ †

Department of Environmental Microbiology, UFZ - Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Saxony, Germany ‡ Department of Civil and Environmental Engineering, 3-133 Markin/CNRL Natural Resources Engineering Facility, University of Alberta, Edmonton, Alberta T6G 2W2, Canada S Supporting Information *

ABSTRACT: Microbial biofilms can cause severe problems in technical installations where they may give rise to microbially influenced corrosion and clogging of filters and membranes or even threaten human health, e.g. when they infest water treatment processes. There is, hence, high interest in methods to prevent microbial adhesion as the initial step of biofilm formation. In environmental technology it might be desired to enhance bacterial transport through porous matrices. This motivated us to test the hypothesis that the attractive interaction energy allowing cells to adhere can be counteracted and overcome by the shear force induced by electroosmotic flow (EOF, i.e. the water flow over surfaces exposed to a weak direct current (DC) electric field). Applying EOF of varying strengths we quantified the deposition of Pseudomonas f luorescens Lp6a in columns containing glass collectors and on a quartz crystal microbalance. We found that the presence of DC reduced the efficiency of initial adhesion and bacterial surface coverage by >85%. A model is presented which quantitatively explains the reduction of bacterial adhesion based on the extended Derjaguin, Landau, Verwey, and Overbeek (XDLVO) theory of colloid stability and the EOF-induced shear forces acting on a bacterium. We propose that DC fields may be used to electrokinetically regulate the interaction of bacteria with surfaces in order to delay initial adhesion and biofilm formation in technical installations or to enhance bacterial transport in environmental matrices.

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INTRODUCTION It is believed that the majority of all bacteria, particularly when living exposed to flowing water, exist in the sessile state. They inhabit biofilms, i.e. surface-associated, metabolically cooperating communities. Whereas natural biofilms provide important ecosystem services, biofilms cause offense when infesting technical systems, a phenomenon referred to as biofouling as it may give rise to microbially influenced corrosion of metals and clogging of filters and membranes or pose risks to human safety water treatment processes being colonized. On the other hand environmental biotechnology, for instance bioremediation, might rely on our ability to enhance the transport of bacteria to places where their catabolic activity is desired. There is, hence, strong interest in methods to prevent microbial adhesion. The initial step of biofilm formation is cell attachment to a surface by unspecific interactions that are influenced by physicochemical properties of the microbe, the collector surface, and the aqueous medium.1 Generally, the deposition of bacteria-sized particles on solid surfaces can be approximated by the extended Derjaguin, Landau, Verwey, and Overbeek (XDLVO) theory of colloid stability.2 This quantitative approach accounts for the three forces van der Waals attraction (GLW), electrostatic repulsion (GEDL), and the acid−base interactions (GAB) and has been successfully applied to explain the deposition of bacterial cells on collector surfaces.3,4 When bacteria move along a collector attachment © XXXX American Chemical Society

requires that their kinetic energy is lower than the interaction energy with the surface.1 In this study we thus tested the hypothesis that the attractive interaction energy leading to cell adhesion can be counteracted and overcome by the shear force induced by surface charge-induced, so-called electroosmotic water flow (EOF). When an electric field is applied to an electrolyte solution in a supporting matrix, electromigrating ions concentrated close to surfaces of opposite charge exert a viscous drag on water evoking EOF, usually from the anode to the cathode.5 Discovered by Reuss,6 EOF has been used to disperse poorly mobile, dissolved compounds, to dry or to decontaminate soil, and to transport solutes in microfluidic devices. Previous studies addressed the influence of weak electric fields and EOF on bacterial physiology7 or the mechanisms governing microbial retardation and biofilm detachment from matrices including the transport of biocides, electrolytically formed reactive oxygen species and gas bubbles.8−10 In 2001, Poortinga et al. postulated that EOF may influence the detachment of attached cells from electrode surfaces.11 In contrast to the parabolic profile of pressure-driven hydraulic flow, the velocity profile of EOF is quasi planar Received: December 22, 2014 Revised: March 26, 2015 Accepted: April 6, 2015

A

DOI: 10.1021/es506245y Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology beginning at the so-called electrical double layer located a few nanometers above the surface12 and, hence, in the range of XDLVO forces. In the present study we applied different DC conditions and electrolyte concentrations to investigate the influence of EOF on the deposition efficiency of Pseudomonas f luorescens Lp6a on glass collectors in percolated columns and on a quartz crystal microbalance with dissipation monitoring (QCM-D) technology. These data were quantitatively explained using a novel model based on the XDLVO theory of colloid stability and calculated EOF-induced shear forces.

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MATERIALS AND METHODS Experimental Procedures. Cultivation of Bacteria and Preparation of Inocula. Pseudomonas f luorescens LP6a is a rodshaped bacterium of 2 μm length and 1 μm width that is known to degrade polycyclic aromatic hydrocarbons (PAH).13 It was cultivated in 70 mL of minimal medium14 containing glucose (1 g L−1) in 300 mL Erlenmeyer flasks until the late exponential phase (≈14 h; 30 °C; rotary shaker at 170 rpm). The cultures were centrifuged at 3000 × g for 10 min, and the pellet was resuspended in either 10 mM, 50 mM, or 100 mM potassium phosphate buffer (i.e., K2HPO4, KH2PO4) at pH = 7 to obtain optical densities (OD578 nm; Cary 400Scan, Varian, USA) of 0.3 or 0.03. Characterization of Surface Properties. The zeta-potentials (ζ) of bacteria and glass beads in 10 mM and 100 mM buffer (pH = 7) were approximated from the electrophoretic mobility measured by Doppler electrophoretic light scattering analysis (Zetamaster, Malvern Instruments, Malvern, UK)15 using a Dip Cell Kit (Malvern Instruments, Malvern, UK). To estimate the ζ of clean glass beads and the effect of adhered bacteria on ζ of glass surfaces, the beads were immersed for given periods (t = 0−300 min) into an LP6a cell suspension of OD578 nm = 0.3. Thereafter, the beads were sieved, rinsed cautiously with 3 mL of 100 mM buffer, resuspended with 100 mM buffer, and used for ζ analysis. In order to calculate the surface free energies of glass and bacteria, contact angles of water θw, formamide θf, and methylene iodide θmi were measured using a DSA 100 dropshape analysis system (Krüss GmbH, Hamburg, Germany) as described earlier.16 Bacterial lawns were prepared by depositing bacteria from inoculated suspensions on cellulose acetate membrane filters (Millipore, 0.45 μm) and applying one droplet of each liquid per filter. Experiments were performed in triplicate. Column Deposition Experiments in Electrokinetically Percolated Columns. The effect of DC electric fields on bacterial deposition and transport was tested in percolated columns. The experiments were performed in triplicate at 25 °C in heat-sterilized vertical percolation columns (i.d.: 1.3 cm; l.: 10 cm) (Figure 1) made of borosilicate glass. The columns were wet packed with clean, sterile glass beads of 0.1−0.25 mm diameter (Retsch, Germany) resulting in a porosity of ≈0.42 (estimated gravimetrically) and a total pore volume (PV) of 3.97 mL. Beads had been sterilized by soaking them for 1 h in 10 mL of 2% aqueous Wofasteril (Kesla Pharma Wolfen GmbH) and subsequent washing three times with 45 mL of sterile distilled water. The columns were allowed to equilibrate by circulating clean buffer for 30 min. Then homogenized bacterial suspensions (OD578 nm = 0.3 (≈4.5 × 108 cells mL−1) in 10 mM, 50 mM, or 100 mM buffer) were circulated with a peristaltic pump at a hydraulic flow rate of 3.4 × 10−4 m s−1 (19.3 mL h−1) from the top to the bottom (i.e., in opposite

Figure 1. Schematic view of the percolation column setup used to assess the effect of DC electric fields on bacterial transport and adhesion in porous media. It consists of a reservoir (1) containing a bacterial suspension which was circulated by a peristaltic pump (2) from the top to the bottom of the column (3). The column was confined at the bottom by a glass frit (4). The column was packed with a bed (5) of small glass beads and contained two disk-shaped Ti/Li electrodes (6) at its top and bottom end. The electrodes were connected to a DC power pack (7) allowing for a constant DC electric field. In order to avoid an accumulation of gas released from the bottom electrode in the frit chamber (8), the oxygen bubbles were allowed to discharge via a bypass glass tube (9), which simultaneously acted as a sampling port (10) for column effluents. Hydrogen bubbles formed at the cathode were allowed to escape by an intended leakage at the top of the column (11).

direction of the EOF). By placing the anode at the outflow of the column potential impact of anodic reactive oxygen species on bacterial deposition could be avoided. The column was confined at the bottom by a glass frit (pore size: 160−250 μm) and contained two disk-shaped Ti/Ir electrodes (De Nora Deutschland GmbH) at the top and bottom ends. The electrodes were connected to a DC power pack (P333, Szczecin, Poland) applying a constant DC electric field at X = 0 (control), 1, 2, or 3 V cm−1 and a current of 4, 8, and 12 mA, respectively. The lid at the column top allowed the release of electrolytically formed gas bubbles to and hence avoided the passage of gas bubbles through the glass beads. Cell deposition was determined by comparing the OD578 nm of the influent and effluent. After pumping of 25 PV of cell suspensions, the columns were rinsed with 10 PV of the corresponding cell-free buffer solution. Deposition on a Quartz Crystal Microbalance with Dissipation (QCM-D) Monitoring. The frequency shift (Δf5, at overtone 5) and dissipation shift (ΔD5, at overtone 5) of a silica coated sensor were monitored simultaneously using the experimental setup described in Figure S6. Underpressure was applied to draw bacterial suspensions of LP6a (OD578 nm = 0.03 or 0.3) at a flow rate of 9 mL h−1 from a well-mixed reservoir through Teflon tubing (2) to the silica coated QCM-D sensor B

DOI: 10.1021/es506245y Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

Figure 2. Parts A and C: Breakthrough curves and calculated fractions of P. f luorescens LP6a transported through percolation columns filled with glass beads in the absence (open circle) and presence (filled symbols) of DC electric fields of X = 1 V cm−1 (diamonds), X = 2 V cm−1 (circles), and X = 3 V cm−1 (triangles) using 100 mM (Part A) phosphate buffer. Parts B and D: Breakthrough curves and calculated fractions of P. f luorescens LP6a transported through percolation columns filled with glass beads in the absence (open symbols) and presence (filled symbols) of DC electric fields of X = 2 V cm−1 using 10 mM (squares) and 50 mM (triangles) phosphate buffer. All data represent averages and standard deviations of triplicate experiments. Vertical dashed lines mark the times at which buffer only was flushed through the columns.

Theory. Calculation of Collision Efficiency, Fraction of Bacteria Retained, and Surface Coverage. Filtration of strain LP6a was quantified using various column conditions in the presence and absence of DC electric fields assuming spherical bacteria (of 2 μm diameter). For a detailed explanation of the calculation method, the reader is referred to the work by Martin et al.,19 the filtration equation of Rajagopalan and Tien,19 and the SI. Shortly, the collision efficiency (αt) of bacteria was defined as the ratio of the rate of attachment (ηt; cf. eq S2) to the rate of bacterial transport to the collector surfaces (ηtrans; cf. eq S3):20 For columns packed with quasi uniform glass spheres, the calculation of ηtrans took into account the contributions of convection, diffusion, van der Waals attraction, and sedimentation.20 The fraction of bacteria retained in the column was calculated based on the difference of inlet and outlet cell concentration.3 Prediction of the Effect of XDLVO, Hydraulic, and EOF Shear Forces on Bacterial Adhesion. We attempted to calculate the net effect of the XDLVO forces (FXDLVO) and the shear forces induced by hydraulic FHF and electroosmotic flow FEOF in the presence of a DC electric field. We therefore assumed that the net force FNet acting on a bacterium located at the distance of the actual XDLVO secondary energy minimum

(Q-Sense AB, Gothenburg, Sweden) and further to a recipient vessel. Two copper wires (i.d.: 0.2 mm) were inserted through the Teflon tubing at the inlet and outlet of the sensor chamber, serving as electrodes to apply a DC electric field of X = 2 V cm−1. The copper wires were connected via Ti/Li electrodes to a power pack. This assembly allowing to induce an EOF opposite17 to the hydraulic flow was applied to the sensor chamber (cf. Figure S2B in 100 mM X = 2 V cm−1). The silica sensor was cleaned by 3-fold rinsing with 50 mL of 2% aqueous SDS followed by 150 mL of deionized water, dried under a nitrogen stream, and UV-sterilized for 10 min in a laminar flow bench. Prior to the experiment, ca. 30 mL of sterile buffer solution was pumped through the sensor until the system approached the steady state. Then, cell suspension (OD578 = 0.3 or 0.03) was pumped in the direction of the anode (cf. Figure S7) through the silica coated sensor in the presence and absence of 2 V cm−1 and f and D automatically analyzed. At the end of each experiment, the QCM-D sensor was carefully rinsed using a sterile buffer solution, and the cell number of attachment to the sensor was quantified by epifluorescence microscopy (Axioskop II microscope, Zeiss, Jena, Germany) after staining with DAPI as described elsewhere.18 C

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(hs) is influenced by the XDLVO interaction force FXDLVO and the shear forces FHF and FEOF according to eq 1 FNet , hs = FEOF , hs + FHF , hs − FXDLVO , hs

GXDLVO h

(1)

(2)

According to the XDLVO theory,4 GXDLVO is composed of the electrostatic repulsion (GEDL) and the Lifshitz−van der Waals (GLW) and the acid−base (GAB) interaction energy (eq 3; for calculations of the dependency of the GXDLVO on the ionic strength I, θ, and ζ of the surfaces cf. the SI): GXDLVO = GEDL + GLW + GAB

(3) 4,21

The shear forces FHF and FEOF acting on a bacterium located at hs were calculated from the shear rates (σ),22 induced by the velocity of hydraulic (υHF) and the electroosmotic (υEOF) water flow using FHF , hs = σHF , hs*η*Ab /2 =

υHF , h hs

*η*Ab /2

(4)

and FEOF , hs = σEOF , hs*η*Ab /2 =

υEOF , h hs

*η*Ab /2

(5)

where η is the viscosity of the liquid (η = 3.19 kg m−1 h−1), and Ab/2 (Ab/2 = 6.3 × 10−12 m2) is the effective surface of the adhered bacterium, i.e. assuming that half of the cell surface area is subject to the shear flow. The EOF velocity (υEOF,h) at given distance h from the collector surface is calculated by eq 6, which is the combination of a simplified EOF expression23 of the Navier−Stokes equation with the potential distribution from the Gouy−Chapman model24,25 υEOF , h = −

2·J1(κh) ⎞ εr ·ε0 ·E ·ζ ⎛ ⎟⎟ ⎜⎜1 − η κrJ0 (κh) ⎠ ⎝

(6)

where J0 and J1 are the zero- and first-order modified Bessel functions, κ−1 is the thickness of the double layer, εr is the dielectric constant of water (78.5), ε0 (8.85 × 10−12 F m−1) is the vacuum permittivity, ζ is the actual zeta potential of the glass bead at the experimental conditions, and E is the electric field strength (1, 2, and 3 V cm−1) applied. Assuming laminar flow as calculated from the Reynold number (Re < 2000) at the given bulk flow velocity (υavg) and based on R ≫ κ‑1, the velocity profile of hydraulic flow υHF(h) in the pore cross section can be described by the Hagen− Poiseuille approach,26 using R-h instead of original h in order to unify the measurement of h ⎛ 2h h2 ⎞ υHF (h) = 2·υavg ⎜ − 2⎟ ⎝R R ⎠

RESULTS

Quantification of Cell Attachment in Percolation Columns. The effect of DC fields on bacterial deposition was studied in columns percolated with cell suspensions either in 100 mM buffer at various electric field strengths (X = 0, 1, 2, and 3 V cm−1) (Figures 2A and B) or in 10 and 50 mM buffer at a fixed electric field strength (X = 2 V cm−1) (Figures 2C and D). In the absence of DC, the initial adhesion efficiency (αo) depended clearly on the ionic strength I with αo of 0.26 (100 mM), 0.07 (50 mM), and 0.09 (10 mM) and the resulting total retention of bacteria in the column summing up to 62% (100 mM) and 15% (10 and 50 mM) after 25 PV (Figures 2B and D). Exposing the collector surfaces to DC fields decreased the αo values and the number of bacteria retained as compared to DC free controls. DC fields, by contrast, had no apparent effect on the detachment of bacteria when the influent was switched to the corresponding cell-free buffer solution after 25 PV. The electrokinetically induced reduction of bacterial adhesion depended on both the electric field and the ionic strength: At X = 1, 2, and 3 V cm−1 and 100 mM buffer, αo attained values of 0.19, 0.18, and 0.04, respectively (i.e., reduced by 25, 30, and 85% relative to the corresponding controls (Figure 3B)). DC field induced reduction of αt was observed at all DC fields and any time (Figure S5A) with highest reductions at X = 3 V cm−1 (>85% at PV = 0). At X = 2 V cm−1 a biphasic behavior of the collision efficiency was observed (Figure 3B) with αo ≈ 0.19 at 0 PV and αt = 0.01 at 6 PV. At this time, no further increase of the collector surface coverage (≈6%) was observed (Figure S5B). With 10 mM and 50 mM buffer insignificant DC effects (X = 2 V cm−1) on αo were calculated, i.e. αo ≈ 0.06 and 0.07 for 10 and 50 mM buffer, respectively as compared to the control (αo ≈ 0.09). In contrast to DC-free controls, no or only a minor accumulation of bacteria was observed after 1 and 6 PV at X = 3 and 2 V cm−1 (Figures 2B and D). A discontinuity was also observed in the calculated relative collector surface area covered by bacteria (Figure 3A) and the evolution of the collision efficiency (Figure 3B). Whereas the coverage continuously increased in the DC-free control and at X = 1 V cm−1, it constantly remained below 1% at X = 3 V cm−1 and 6% at X = 2 V cm−1. Interestingly, changes in αt and coverage coincided with an increase of ζglass from −9 mV to −15 mV indicating substantial coverage with bacteria (ζbacteria = −30 mV). Both DC fields tested had neither an effect on the apparent viability of cells in the outflow of the columns (as measured by colony forming units) nor on the charge and hydrophobicity of the cells (Figure S6). Quantification of Cell Attachment by QCM-D. Using QCM-D, we studied the influence of a DC field on the deposition dynamics of P. fluorescens LP6a suspensions of two different cell densities to the silica-covered sensor (Figures 4 A−D). Figures 4 A−D depict the QCM-D frequency shifts and dissipation energy changes during bacterial deposition to the sensor. In the absence of DC, the QCM frequency declined monotonically at cell-density dependent rates, indicating continuous bacterial adhesion to the sensor surface. In the presence of a 2 V cm−1 electric field, the frequency shifts stabilized after 60 min (OD578 = 0.3) and 250 min (OD578 = 0.03) and remained constant for the rest of the experiment indicating no further deposition. A similar trend was observed with the dissipation energy, which in the absence of DC increased to ΔD = 4 × 10−6 indicating that soft and viscoelastic biomass was loaded onto the silica surface. In the presence of

For a given distance (h) to the collector surface, FXDLVO can be calculated by the XDLVO energy distribution (GXDLVO) as given by eq 2:

FXDLVO(h) =

Article

(7)

where R is the radius of the pore equaling (√2−1) · aS27 with aS being an estimate for the minimum pore radius confined by spheres. D

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whether the attractive interaction energy of initial cell adhesion can be overcome by the shear force induced by EOF. If this was the case, EOF would hold potential as a means to suppress bacterial adhesion. In the electric double layer, unlike hydraulic flow, the plug shaped velocity profile EOF may cause substantial shear right above the electric double layer (e.g., on a silica surface for 100 mM buffer at ≈ 0.65 nm), i.e. at a distance where the forces governing bacterial adhesion act. Figure S2 depicts the extents and velocity profiles of the EOF and the hydraulic flow. To test this concept, we varied EOF strengths (varying electric field strengths (X) and electrolyte ionic strengths (I)) and quantified the attachment of P. f luorescens Lp6a in percolation column deposition experiments. We found that the deposition efficiency depended on the electrokinetic conditions applied with >85% reduced collision efficiency at the highest X tested. We also observed that the relative change of the DC-induced collision efficiency depended on the coverage of the collectors (Figure S5A). Interestingly, a change in αt coincided with a decrease of the ζ of glass collector surfaces from −9 mV to −15 mV (Figure 3A). This is likely to be the result of increasing deposition of the more negatively charged LP6a cells and may also go along with surface contamination by bacterial extracellular polymeric substances.28,29 According to the XDLVO theory higher surface charges will promote higher electrostatic repulsion leading to slightly reduced XDLVO attraction (GXDLVO; Table 1; Figure S3C). The GXDLVO accounts for the three forces: van der Waals attraction (GLW), electrostatic repulsion (GEDL), and the acid− base interactions (GAB). While GEDL and GLW are functions of the separation distance between a particle and a surface, GAB compares the energy status between attached and nonattached situations, and there is scientific debate whether to include GAB in the calculation of FXDLVO for given distances to the collector surface. In Table 1 we hence both give the empirical FXDLVO and the theoretically sound FDLVO (according to eq 2 FDLVO equals the ratio of GDLVO and the distance h; with GDLVO = GEDL +GLW). This comparison reveals no significant influence ( 2 V cm−1 > 1 V cm−1 > NO DC. Effect of DC Fields in QCM-D. Quartz crystal microbalance with dissipation (QCM-D) monitoring is a highly sensitive analysis method (resolution of ng per cm2) by which complex interfacial processes such as bacterial adhesion can be monitored.30,31 The frequency shift (Δf) thereby corresponds to the biomass loaded on the quartz sensor surface, whereas the

Figure 3. Measured zeta potential (dashed lines; Part A), calculated coverage of the glass bead (dotted lines; Part A), and collision efficiencies (Part B) of P. f luorescens LP6a in percolation experiments in the absence (open circles) and presence (filled symbols) of DC electric fields of X = 1 V cm−1 (filled squares), X = 2 V cm−1 (filled circles), and X = 3 V cm−1 (filled triangles) using 100 mM phosphate buffer. The initial collision efficiency (αo) was calculated by extrapolation of the effective collision efficiency (αt) to the clean collector surface. Data are presented for time points at which the ζ of the collector surface was measured.

DC, ΔD decreased to −2 × 10−6 after 60 min (OD578 = 0.3) and to 1.8 × 10−6 after 250 min (OD578 = 0.03). The number of bacterial cells adhered to the sensor surface was quantified by fluorescence microscopy at different time intervals (Figure 4E). The results are in agreement with QCMD results (Figure 4A). In the first 40 min, no significant difference could be observed on bacterial adhesion to a sensor surface in the presence or absence of the DC field (Figure 4E, first column). After 90 and 300 min, the attenuating effect of the DC field on frequency shifts and ΔD could also be approximated from the overall reduced numbers of bacteria adhered to the sensor surface: in the presence of the DC field, the bacterial cells counted on the sensor surface were reduced from 0.26 × 107 cells cm−2 (90 min) and 0.55 × 107 cells cm−2 (300 min) to 0.19 × 107 cells cm−2 (90 min) and 0.34 × 107 cells cm−2 (300 min), respectively.

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DISCUSSION Effect of DC Fields on Deposition and Transport in Percolation Columns. Deposition of bacteria is supposed to take place only when their kinetic energy is lower than their interaction energy with the surface.1,20 Hence, we tested E

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Figure 4. QCM-D signals and cell count of P. f luorescens LP6a attaching to a silica-coated QCM sensor in the absence (empty symbols) and presence (filled symbols) of a DC electric field (X = 2 V cm−1; 100 mM buffer) for cell suspensions of OD578 nm = 0.3 (Parts A and B) and OD578 nm = 0.03 Parts C and D). Parts A and C depict the frequency shifts as a measure of the biomass loaded on the silica sensor surface, whereas Parts B and D show the dissipation energy shift (ΔD) as an indicator for the viscoelastic properties of the material attaching to the sensor surface.32,33 Experiments with OD578 nm = 0.3 were either performed for 40 min (down triangle), 90 min (up triangle), or 300 min (circle). Part E depicts the number of cells accumulated on the silica sensor surface after these intervals (stars refer to statistically significant differences between presence and absence of DC electric field.

Table 1. Comparison of the Calculated Adhesion Efficiency (α) at Given Pore Volumes (PV) to the Calculated Electroosmotic (υEOF) and Hydraulic Flow (υHF) Velocities, the XDLVO Force (FXDLVO), the DLVO Force (FDLVO), and the Electroosmotic and Hydraulic Shear Forces (FHF, FEOF) Acting on a P. f luorescens LP6a Bacterium Positioned in 100 mM Phosphate Buffer at the Distance of the Secondary Minimum (hs) above a Surface of a Given Zeta Potential (ζ) ζ = −9 mV −7

−1

υEOF (× 10 ms ) υHF (× 10−7 ms−1) FEOF (pN) FHF (pN) FDLVO (pN) FXDLVO (pN) αt (−)

ζ = −15 mV

3 V cm‑1

2 V cm‑1

1 V cm‑1

NO DC

3 V cm‑1

2 V cm‑1

1 V cm‑1

NO DC

17.3 2.4 1.5 0.2 1.5 1.4 0 (green to red colors) significant reduction of the bacterial deposition efficiency is expected, whereas at FNET,hs < 0 (blue to dark blue colors), FXDLVO will dominate the effect of FNET,hs on bacterial deposition and transport. Figure 5 also visualizes how high X, high I, and highly negative ζ of the involved surfaces jointly increase FNET,hs while reducing FXDLVO (Table 1). Relevance for Environmental Technology. Our experimental findings and model calculations culminating in Figure 5 suggest that EOF may be used for the electrokinetic control of bacterial adhesion to collector surfaces and therefore be of relevance for the manipulation of biofouling and bacterial transport. Our data also suggest that priming of collector surfaces with highly charged materials in conjunction with a suited electrokinetic treatment may help to prevent further microbial deposition to collector surfaces and, hence, limit biofilm formation. However, the roles of bacterial growth stage

Figure 5. Dependency of the net force FNET,hs = FEOF,hs + FHF,hs − FDLVO,hs (cf. eq 1) acting on P. f luorescens LP6a bacteria positioned at the secondary minimum above a glass surface in response to variations of the ionic strength (I), electric field strength (X) strength, and the zeta potential (ζ) of the glass surface, respectively. Positive values, as symbolized by light colors signify that FNET > FDLVO, i.e. that a significant reduction of bacterial deposition on surfaces is predicted.

and the evolution of surface macromolecules on electrokinetically operated cell adhesion are still unclear and awaits further G

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Environmental Science & Technology research.38 Likewise, deepened knowledge on the joint action of the (likely insignificant) electrophoretic and the EOF-driven dispersal of bacteria on their adhesion is still poorly understood.8,9,39 In the past, several DC-based approaches have been proposed, which, however, aimed at disrupting biofilms forming on electrodes either by applying biocidal current40,41 or using electricity to promote the penetration of biocides into the biofilm.42 Our research adds a new, preventive way of controlling biofilm with electric current. It should be noted that in agreement with an earlier study,9 the weak DC fields applied in the present study neither affected the apparent bacterial viability nor changed their physicochemical surface properties relevant for adhesion and transport (Figure S5). The inoffensiveness of the DC fields also opens possibilities for the desired enhancement of bacterial transport in porous matrices in the presence of DC and the controlled deposition upon removal of DC.

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monas sp LB126 in a DC-electrical field typical for electrobioremediation measures. Microb. Biotechnol. 2008, 1 (1), 53−61 DOI: 10.1111/j.1751-7915.2007.00006.x. (8) Shi, L.; Muller, S.; Harms, H.; Wick, L. Y. Factors influencing the electrokinetic dispersion of PAH-degrading bacteria in a laboratory model aquifer. Appl. Microbiol. Biotechnol. 2008, 80 (3), 507−515 DOI: 10.1007/s00253-008-1577-0. (9) Shi, L.; Muller, S.; Harms, H.; Wick, L. Y. Effect of electrokinetic transport on the vulnerability of PAH-degrading bacteria in a model aquifer. Environ. Geochem. Health 2008, 30 (2), 177−182 DOI: 10.1007/s10653-008-9146-0. (10) Del Pozo, J. L.; Rouse, M. S.; Patel, R. Bioelectric effect and bacterial biofilms. A systematic review. Int. J. Artif. Organs 2008, 31 (9), 786−795. (11) Poortinga, A. T.; Smit, J.; van der Mei, H. C.; Busscher, H. J. Electric field induced desorption of bacteria from a conditioning film covered substratum. Biotechnol. Bioeng. 2001, 76 (4), 395−399 DOI: 10.1002/bit.10129. (12) Ross, D.; Johnson, T. J.; Locascio, L. E. Imaging of electroosmotic flow in plastic microchannels. Anal. Chem. 2001, 73 (11), 2509−2515 DOI: 10.1021/ac001509f. (13) Hazrin-Chong, N. H.; Marjo, C. E.; Das, T.; Rich, A. M.; Manefield, M. Surface analysis reveals biogenic oxidation of subbituminous coal by Pseudomonas f luorescens. Appl. Microbiol. Biotechnol. 2014, 98 (14), 6443−6452 DOI: 10.1007/s00253-014-5832-2. (14) Wick, L. Y.; Wattiau, P.; Harms, H. Influence of the growth substrate on the mycolic acid profiles of mycobacteria. Environ. Microbiol. 2002, 4 (10), 612−616 DOI: 10.1046/j.14622920.2002.00340.x. (15) Hiemenz, P. C. Principles of colloid and surface chemistry, 2nd ed.; M. Dekker: New York, 1986; p xv, 815p; DOI 10.1002/ pol.1984.130220910. (16) Sharma, P. K.; Rao, K. H. Adhesion of Paenibacillus polymyxa on chalcopyrite and pyrite: surface thermodynamics and extended DLVO theory. Colloids Surf., B 2003, 29 (1), 21−38 DOI: 10.1016/S09277765(02)00180-7. (17) Reddy, K. R.; Cameselle, C. Electrochemical remediation technologies for polluted soils, sediments and groundwater; John Wiley & Sons: 2009; DOI 10.1002/9780470523650. (18) Zhou, K.; Sun, G. Z.; Bernard, C. C.; Thouas, G. A.; Nisbet, D. R.; Forsythe, J. S. Optimizing interfacial features to regulate neural progenitor cells using polyelectrolyte multilayers and brain derived neurotrophic factor. Biointerphases 2011, 6 (4), 189−199 DOI: 10.1116/1.3656249. (19) Martin, R. E.; Bouwer, E. J.; Hanna, L. M. Application of CleanBed Filtration Theory to Bacterial Deposition in Porous-Media. Environ. Sci. Technol. 1992, 26 (5), 1053−1058 DOI: 10.1021/ es00029a028. (20) Velasco-Casal, P.; Wick, L. Y.; Ortega-Calvo, J. J. Chemoeffectors decrease the deposition of chemotactic bacteria during transport in porous media. Environ. Sci. Technol. 2008, 42 (4), 1131− 1137 DOI: 10.1021/es071707p. (21) Roosjen, A.; Boks, N. P.; van der Mei, H. C.; Busscher, H. J.; Norde, W. Influence of shear on microbial adhesion to PEO-brushes and glass by convective-diffusion and sedimentation in a parallel plate flow chamber. Colloids Surf., B 2005, 46 (1), 1−6 DOI: 10.1016/ j.colsurfb.2005.08.009. (22) Mampallil, D.; van den Ende, D. Electroosmotic shear flow in microchannels. J. Colloid Interface Sci. 2013, 390, 234−241. (23) Cummings, E. B.; Griffiths, S. K.; Nilson, R. H.; Paul, P. H. Conditions for similitude between the fluid velocity and electric field in electroosmotic flow. Anal. Chem. 2000, 72 (11), 2526−2532 DOI: 10.1021/ac991165x. (24) Rice, C. L.; Whitehea, R. Electrokinetic Flow in a Narrow Cylindrical Capillary. J. Phys. Chem. 1965, 69 (11), 4017−4024 DOI: 10.1021/j100895a062. (25) Ghosal, S. Fluid mechanics of electroosmotic flow and its effect on band broadening in capillary electrophoresis. Electrophoresis 2004, 25 (2), 214−228 DOI: 10.1002/elps.200305745.

ASSOCIATED CONTENT

S Supporting Information *

Text containing theory and calculations, Tables S1 and S2, Figures S1−S8, a description and graphic of Movie 1, and a video containing Movie 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 49 341 235 1316. Fax: 49 341 235 1351. E-mail: lukas. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been performed in the frame of the Helmholtz Alberta Initiative and contributes to research program topic CITE of the Helmholtz Association. The authors wish to thank Jana Reichenbach, Rita Remer, Birgit Würz, and Marta Vallejo Montes for wonderful and skilled technical help. Provision of the Pseudomonas f luorescens LP6a by Prof. Julia Foght, University of Alberta, Edmonton, Canada is greatly acknowledged.

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