Interaction forces between waterborne bacteria and activated carbon particles

Journal of Colloid and Interface Science 322 (2008) 351–357 www.elsevier.com/locate/jcis

Interaction forces between waterborne bacteria and activated carbon particles Henk J. Busscher a,b , Rene J.B. Dijkstra a , Don E. Langworthy c , Dimitris I. Collias d , David W. Bjorkquist c , Michael D. Mitchell d , Henny C. Van der Mei a,∗ a Department of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1,

9713 AV Groningen, The Netherlands b SASA BV, G.N. Schutterlaan 4, 9797 PC Thesinge, The Netherlands c The Procter & Gamble Company, 8700 Mason-Montgomery Road, Cincinnati, OH, USA d The Procter & Gamble Company, Corporate R&D, 8611 Beckett Rd., West Chester, OH 45069, USA

Received 22 November 2007; accepted 12 March 2008 Available online 19 March 2008

Abstract Activated carbons remove waterborne bacteria from potable water systems through attractive Lifshitz–van der Waals forces despite electrostatic repulsion between negatively charged cells and carbon surfaces. In this paper we quantify the interaction forces between bacteria with negatively and positively charged, mesoporous wood-based carbons, as well as with a microporous coconut carbon. To this end, we glued carbon particles to the cantilever of an atomic force microscope and measured the interaction forces upon approach and retraction of thus made tips. Waterborne Raoultella terrigena and Escherichia coli adhered weakly (1–2 nN) to different activated carbon particles, and the main difference between the activated carbons was the percentage of curves with attractive sites revealed upon traversing of a carbon particle through the bacterial EPS layer. The percentage of curves showing adhesion forces upon retraction varied between 21% and 69%, and was highest for R. terrigena with positively charged carbon (66%) and a coconut carbon (69%). Macroscopic bacterial removal by the mesoporous carbon particles increased with increasing percentages of attractive sites revealed upon traversing a carbon particle through the outer bacterial surface layer. © 2008 Elsevier Inc. All rights reserved. Keywords: AFM; Activated carbon; Bacterial adhesion; Water filtration; Interaction force; Raoutella terrigena; Escherichiae coli; EPS

1. Introduction Activated carbon particles have been used for a long time for water purification purposes in rural areas around the world because of their large surface area and high adsorption capacity [1,2]. Activated carbons can remove waterborne bacteria, like Aeromonas spp. and Pseudomonas aeruginosa, Raoultella terrigena and Escherichia coli from potable water supply systems [3,4]. Microorganisms attach to activated carbon particles through strong Lifshitz–van der Waals forces despite electrostatic repulsion between negatively charged cells and carbon surfaces [5]. In low ionic strength solutions, such as potable water systems, electrostatic interactions can be sizable and at the same time offer possibilities to enhance the efficacy of activated car* Corresponding author. Fax: +31 50363159.

E-mail address: [email protected] (H.C. Van der Mei). 0021-9797/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2008.03.018

bons to remove microorganisms from water by positive charge modification of the carbon surfaces. Once there is charge reversal, the electrostatic attraction between negatively charged microbial cell surfaces and positively modified carbon particles will be strong [6,7]. Moreover, positively charged activated carbon particles, as can be created by coating with a quaternary ammonium compound, have bacteriocidal properties upon bacterial contact [7] and yield less biofilm growth. In addition to bacterial cell surface charge, the hydrophobicity of the interacting surfaces is important in adhesion [6]. Initial microbial adhesion to surfaces generally involves weak interaction forces and by consequence is reversible, especially under conditions of electrostatic repulsion as on negatively charged surfaces. However, after initial adhesion, most adhering organisms manage to rapidly attach themselves in a less reversible fashion through the production of extracellular polymeric substances (EPS) [8], that assists the maintenance of structural integrity of a biofilm and protects the organisms in-

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side against antimicrobial agents. More than 90% of the EPS volume consists of water [9] which is located in pores, with a minor portion bound to the EPS. EPS has long been considered to consist only of polysaccharides, but recently considerable amounts of proteins, glycoproteins, lipopolysaccharides, lipids and other components [10] have been identified as EPS constituents. The atomic force microscope (AFM) has been developed not only as an imaging tool, but moreover as an instrument to measure interaction forces between different surfaces. The adhesive forces between immobilized microorganisms and the silicon nitride tip of AFM instruments, usually with a radius of curvature around 20 nm, have been measured upon retraction of the tip from microbial cell surfaces by a variety of groups [11–17] and found to amount up to approximately 5 nN in most cases, but can go up to 30 nN [15] in extreme cases, extending over several hundreds of nm. Alternatively, in most cases, electrosteric repulsion has been measured upon approach of the interaction surfaces that ranges over several tens of nm and amounting up to 10–20 nN upon contact. Vadillo-Rodriguez et al. [18] have determined relationships between these interaction forces as measured by microscopic AFM tips and macroscopic adhesion of bacteria to surfaces in a parallel plate flow chamber. Initial deposition rates to silicon nitride surfaces were found to be highest when the repulsion forces upon approach as probed by AFM were smallest, whereas adhesion forces measured upon retract were suggested to be determinant as to whether bacteria, once adhering, are able to remain adhering during shear and other environmental detachment challenges. Clearly, in real life and most certainly in activated carbon filtration, deposition, adhesion and detachment are occurring simultaneously. As a further step in establishing a relationship between macroscopic bacterial deposition, adhesion and interaction forces determined using AFM, the microscopic tip should be replaced by a more macroscopically sized particle of the same material as to which macroscopic bacterial adhesion is studied. In this way, the interaction forces between more macroscopic surfaces and bacteria, immobilized for instance in membrane filters [19], can be measured. Recently, we have applied this methodology to assess the interaction forces between oral bacteria and salivacoated enamel particles [20]. Clearly, this would be an ideal approach to study the interaction between bacteria and activated carbon particles, although both the numbers of carbon particles and bacteria that can be realistically involved in an experiment are always low compared with the numbers involved in true, macroscopic adhesion. The aim of this paper is to quantify the interactions forces operative between two waterborne bacterial strains, R. terrigena and E. coli, and different activated wood- and coconut-based carbon particles using AFM. The role of EPS in establishing interaction forces between R. terrigena and a basic, wood-based carbon prior to and after application of a positive charge will be determined for R. terrigena treated with N -acetylcysteine (NAC) to remove EPS. NAC is a non-antibiotic mucolytic agent with antibacterial properties that disrupts disulfide bonds in mucus [21].

2. Materials and methods 2.1. Strains and culture conditions R. terrigena ATCC 33257 was cultured in nutrient broth (NB, OXOID, Basingstoke, Great Britain) and E. coli ATCC 25922 in tryptone soya broth (TSB, OXOID). For each experiment, a preculture was inoculated from agar into broth and cultured for 24 h. A second culture was inoculated and grown for 16 h, yielding late stationary state cells. Bacteria were harvested by centrifugation (5 min at 10,000g), washed twice with ultrapure water (i.e., tap water filtered through a ultrapure cartridge kit, Arium 611 D1, Sartorius AG, Goettingen, Germany, specific conductivity better than 0.05 µS) and re-suspended in stabilized water to a concentration of approximately 108 bacteria per liter for further experiments. For stabilization, the water was weakly buffered with 0.00025 mol L−1 KH2 PO4 /K2 HPO4 , yielding an ionic strength of 0.001 mol L−1 and pH 6.8. Extracellular slime [21] was removed by vortex mixing 5 mL bacterial suspension (108 bacteria per mL) in stabilized water with a NAC solution (4 mg mL−1 ) and left for 5 min at room temperature. After 5 min, the bacterial suspension was centrifuged at 10,000g and bacteria were resuspended in stabilized water. 2.2. Bacterial cell surface characterization Bacterial contact angles were determined with water, formamide, methylene iodide and α-bromonaphthalene using the sessile drop technique for R. terrigena prior [22] to and after NAC treatment as well as for E. coli [22]. Briefly, bacteria were resuspended in ultrapure water and deposited onto a 0.45 µm pore size filter (Millipore) using negative pressure. A lawn of approximately 50 stacked layers of bacteria was produced on the filter. The filters were left to air dry at room temperature and humidity until so-called “plateau water contact angles” could be measured (approximately 30 min) using an automated contour monitor. For each strain, contact angles were measured in triplicate with separately cultured bacteria. Average zeta potentials of the bacterial strains in stabilized water (pH 6.8) were determined by particulate microelectrophoresis, using a Lazer Zee Meter 501 (PenKem), discarding heterogeneous subpopulations, as found for R. terrigena and E. coli [22]. 2.3. Carbon particles A basic wood-based activated carbon, RGC (MeadWestvaco Corp., Carbon Department, Covington, VA), an acidic wood-based activated carbon, CA-10 (Carbochem, Inc., Ardmore, PA), and a coconut carbon (Calgon Carbon Corp., Pittsburgh, PA) were used as received. RGC was furthermore coated with polyvinyl amine (PVAM) to a level of about 4–5% (w/w) as measured by thermogravimetric analysis. CA-10 and RGC are carbon particles with a total pore volume of around 1.26 mL g−1 and a volume of pores with diameter larger than 2 nm of about 0.64 mL g−1 . Coconut carbon (total pore volume 0.77 mL g−1 ) had a volume of pores with diameter larger

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than 2 nm of about 0.15 mL g−1 . Based on the pore volumes, coconut carbon can be classified as microporous and all other carbons used are mesoporous. Coating the RGC carbon with PVAM did not affect the mesoporous character of the carbon particles and yielded a reversal of charge from a zeta potential of −43 to +33 mV. Carbon particles were sieved upon receipt to obtain a fraction with size between 25 and 50 µm. 2.4. Atomic force microscopy A Nanoscope III AFM (Veeco Instruments, Santa Barbara, CA) operating in the contact-mode was used to measure interaction forces between bacteria and carbon particles. To this end, an activated carbon particle was fixed to the cantilever (a silicon nitride V-shaped Si3 N4 tipless cantilever, Veeco Instruments Inc., Woodbury, NY) using a micromanipulator (Narishige, Narishige International USA, Inc., East Meadow, NY) with Pattex Super Mix glue. Use of excessive amounts of glue and attaching the particle while the glue is still liquid-like should be avoided, as this leads to covering of the particle, which can be easily assessed using scanning electron microscopy. As can be seen in Fig. 1, however, use of the proper amount and right timing of particle attachment preserves the fine surface structures on the upper part of the carbon particles, indicating that no glue migrated through the pores to contaminate the area involved in contact with the bacteria. Care was taken to always fix a particle at the same distance from the free end of the cantilever. Only large (well above 1 µm in diameter) carbon particles were employed for AFM as selected using the light microscope of the micromanipulator, because if the particle is considerably larger than the bacterial diameter, the bacterial radius of curvature determines the actual contact area and not the size of the particle selected. Since the spring constant denotes the proportionality constant in Hooke’s law between deflection and applied force, the spring constant is not affected by the weight of the carbon particle which merely yields an additional deflection accounted for during AFM measurements as an off-set with no influence on the forces measured. Spring constants were experimentally determined by measuring the resonance frequency of each tip used, from which the spring constant can be calculated according to k = af 3 ,

(1)

where k is the spring constant, f the true resonance frequency and a is a proportionality constant provided by Veeco. Spring constants measured were similar to those provided by the manufacturer (0.06 N m−1 ). AFM was done on bacteria immobilized in an isopore polycarbonate membrane [23], as shown in Fig. 2. Contact-mode, topographic images were taken with an applied force maintained below 1 nN at a scan rate of ∼2 Hz, and the tip was positioned over an immobilized bacterium after progressive zooms, after which scanning was stopped and force measurements were taken at random positions on the bacterium. Single contact between the carbon particles and the bacterial cell surfaces was ensured by examining the topographic images for double con-

Fig. 1. Scanning electron micrograph of activated carbon particles RGC (top) and RGC + PVAM (bottom) attached to the AFM cantilever. Bar marker indicates 10 µm.

tour lines, which would indicate double contacts, and eliminating such cases from the analysis (which seldom occurred). Force–distance curves were taken with a z-displacement of 300 nm and found independent of the scan rate over the frequency range 0.5 to 18.9 Hz, which demonstrates the absence of hydrodynamic artifacts. A scan rate of 1.99 Hz, corresponding with a loading rate of about 72 nN s−1 , was preferred, however, since this yielded the smallest SD in the parameters measured. Integral and proportional gains of the feedback loop were about 2 and 3, respectively and the ramp size was set at 100 V. In the region of the force vs. distance curves, where probe and sample are in contact, the slope of the retraction curve was used to convert the position sensitive detector voltage into a cantilever deflection. By multiplying the deflection (d) of the cantilever with its spring constant (k) and subtracting the deflection (d) from the position of the piezo-tube (z), a force–distance curve is obtained. The approach curve was fitted to a negative exponential in which F is the measured force at distance d, Λ is the characteristic decay length (separation distance over which F decays from F0 to F0 /e) and F0 is the force at 0 nm separation

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Table 1 Contact angles (degrees) with various liquids of the two bacterial strains involved in this study, including NAC-treated R. terrigena ATCC 33257 (all measurements were done in triplicate with separately cultured bacteria) Strain

Water

Formamide

Methylene iodide

α-Bromonaphthalene

R. terrigena ATCC 33257a NAC-treated R. terrigena ATCC 33257 E. coli ATCC 25922a

32 ± 1

33 ± 2

46 ± 3

43 ± 1

32 ± 2

36 ± 2

58 ± 4

53 ± 3

25 ± 1

37 ± 1

46 ± 3

40 ± 2

a These contact angles have been published previously in [22].

than 250 nm were discarded as being instrumental instabilities, due to lack of actual contact between the carbon particle and the bacterial cell surface. The force curves were measured for ten immobilized bacteria on ten randomly chosen positions on each bacterium and for each type of carbon particle at least 4 different cantilevers with attached carbon particles were used. As a control, the adhesion forces between the membrane filter and the carbon particle tip were measured before and after bacterial force measurements. Since the quantitative features of the approach and retraction force–distance curves show a broad, non-Gaussian distribution of values for F0 , Λ, and Fadh , simple calculation of average values with standard deviations for the relevant parameters is not realistic. For this reason, we present their median and range and apply non-parametric statistical analyses to the AFM data (Sign test for the median). 3. Results and discussion

Fig. 2. AFM height (top) and deflection (bottom) images of R. terrigena obtained by AFM, imaged with an activated carbon particle (RCG55m) attached to the cantilever.

distance: F = F0 e−d/Λ .

(2)

The retracting curves sometimes showed adhesion peaks, but were perfectly non-interactive in others. The percentage of curves showing adhesion forces was therefore determined. Retracting curves with adhesion peaks were analyzed for the maximum adhesion force observed in a retracting force distance curve Fmax and the maximum range over which adhesion forces were observed, dmax . Adhesion forces less than 0.1 nN were considered noise, while those extending over a longer range

Table 1 shows that both strains involved in this study, including R. terrigena after NAC treatment are hydrophilic by their low water and formamide contact angles, while their apolar surface properties are nearly equal, as judged from the methylene iodide and α-bromonaphthalene contact angles [24]. Applying the acid–base approach of van Oss et al. [24] to convert measured contact angles into surface free energies, it can be calculated that the Lifshitz–van der Waals surface free energy components are 35 ± 1 mJ m−2 for R. terrigena and 36 ± 2 mJ m−2 for E. coli, respectively, but NAC treatment of R. terrigena yields a major and significant (p < 0.01, Student t -test) decrease in the Lifshitz–van der Waals free energy component to 29 ± 2 mJ m−2 . Alternatively, NAC-treatment of R. terrigena shows a non-significant increase of the electrondonating and electron-accepting surface free energy parameters from 46.2 ± 2.1 and 1.0 ± 0.4 mJ m−2 to 49.4 ± 2.4 and 1.6 ± 0.6 mJ m−2 , respectively. E. coli has the strongest electron-donating surface (60.2 ± 3.8 mJ m−2 ), and is weakly electron-accepting (0.3 ± 0.3 mJ m−2 ) as compared with R. terrigena. R. terrigena and E. coli are both negatively charged under the ionic strength conditions of the experiments, with population-averaged zeta potentials of −41.8 ± 7.5 mV and

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to six-fold longer distances. The repulsive force (F0 at contact) amounts 1–4 nN upon contact, and is generally smaller for the E. coli strain than for R. terrigena. Previously, the nature of this repulsive force has been described by several authors as being electrosteric [17,25]. The relatively long range of the repulsive force measured necessitates the same interpretation for the present strains. Upon retraction, not all curves measured show adhesion forces, and the percentage of curves showing adhesion (see Table 3) is highest for the combinations of R. terrigena and coconut carbon and PVAM. E. coli shows the highest percentage of curves with adhesion in combination with RGC. Median values for the adhesive forces amount up to 1 nN, which is the same order of magnitude as found for bacteria in general with original AFM tips [12,17]. Interestingly, upon retraction, the forces extend over two-fold larger distances than upon approach (Table 2, dmax ), demonstrating that outer cell surface structures stretch until failure upon retraction of the carbon particles. A similar stretching of cell surface structures has been described by van der Mei et al. [26] for the retraction of an AFM tip from fibrillated oral streptococci and by Yongsunthon et al. [27] for Stapylococcus aureus from adsorbed fibronectin coatings. Fig. 4 shows the distribution of Fadh calculated from the retracting force–distance curves between activated carbon particles with a negatively (RGC) and a positively charge (PVAM) activated carbon and R. terrigena prior to and after treatment of the EPS layer with NAC. The median and range values of these distributions prior to and after NAC-treatment as well as of F0 , Λ and dmax are presented in Table 2, while Table 3 summarizes the percentage of curves demonstrating adhesion in the retracting mode. NAC treatment has no effect on the median adhesion force upon retraction for the negatively charged, wood-based RGC carbon particle, although it can be seen from Fig. 3 and Table 3 that the percentage of force–distance curves revealing adhesion forces decreases from 21% to 13% upon treatment of the EPS layer. With respect to the organisms interaction with the positively charged PVAM carbon particle, NAC treatment

Fig. 3. Force–distance curves between activated carbon particles with a negative (RGC 55) and positive charge (RGC 55 + PVAM) and R. terrigena ATCC 33257 by AFM. The dotted line is the approach curve, whereas the solid line is the retract curve.

−25.5±9.5 mV, respectively [22]. NAC-treatment did not have a significant effect on the zeta potential of R. terrigena. Fig. 3 shows examples of the approach and retraction curves between bacteria trapped in a membrane filter and activated carbon particles, while Table 2 summarizes the median and range values of the interaction curve characteristics. Upon approach, a repulsive force is measured that extends over 20–40 nm (median), with no difference between both strains and the carbon particle involved, although it can be seen from the range values presented that these repulsive forces can extend over five

Table 2 Characteristics of the approach and retraction curves between activated carbon particlesa and R. terrigena ATCC 33257 and E. coli ATCC 25922 Parameter

F0 at contact [nN]

Repulsive force range Λ [nm]

Fmax upon retraction [nN]

CA-10

RGC

PVAM

Cnut

CA-10

RGC

CA-10

Median Range N

2.9 13.0 107

1.6 6.2 82

3.3 10.5 111

3.1 6 102

22 126 107

17 106 82

Median Range N

nd nd nd

2.1 5.8 100

1.1 4.8 100

nd nd nd

Median Range N

2.1 5.0 99

1.0 4.9 102

3.6 5.3 96

1.9 3.6 98

PVAM

Cnut

dmax [nm]

RGC

PVAM

Cnut

CA-10

RGC

PVAM

Cnut

0 −2.7 82

−0.7 −4.2 111

−0.8 −8.8 102

0 238 107

0 165 82

57 231 111

25 226 102

nd nd nd

NAC-treated R. terrigena ATCC 33257 20 22 nd nd 0 22 16 nd nd −8.3 100 100 nd nd 100

0 −2.7 100

nd nd nd

nd nd nd

0 0 100

0 0 100

nd nd nd

32 69 99

37 107 102

E. coli ATCC 25922 39 34 0 82 98 −4.8 96 98 99

0 −3.8 96

0 −6.2 98

0 393 99

15 138 102

0 152 96

0 262 98

R. terrigena ATCC 33257 33 26 0 94 71 −8.8 111 102 107

−0.1 −3.0 102

a CA-10, acidic wood-based activated carbon; RGC, basic wood-based activated carbon; PVAM, RGC carbon particle coated with polyvinyl amine; Cnut, coconut carbon particle; NAC, N -acetylcysteine. Range denotes the maximal value in the non-parametric distribution of values for each parameter.

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Table 3 The percentage of retraction curves between activated carbon particles and R. terrigena ATCC 33257 (prior to and after NAC treatment) and E. coli ATCC 25922, showing adhesion (total number of curves for each combination evaluated amounts approximately 100) Carbon type

R. terrigena ATCC 33257

NAC-treated R. terrigena ATCC 33257

E. coli ATCC 25922

CA-10a RGC PVAM Coconut

22 21 66 69

nd 13 11 nd

36 53 44 28

a CA-10, acidic wood-based activated carbon; RGC, basic wood-based acti-

vated carbon; PVAM, RGC carbon particle coated with polyvinyl amine; Coconut carbon particle; NAC, N -acetylcysteine.

causes a decrease in the median adhesion force upon retraction from −0.7 to 0 nN (p < 0.001, Sign test for the median). NAC clearly affects the EPS, which is generally considered as the glue for biofilms, either for gluing bacteria to a substratum

or gluing biofilm organisms together, as demonstrated by Chaw et al. [28], showing a reduction in internal strength of Staphylococcus epidermidis biofilm after deterioration of its EPS by silver ions. The percentage of curves with adhesion (see Table 3), goes down from 66% to 11%. This suggests that there is a distribution of both positively and negatively charged attractive interaction sites throughout the EPS layer. Negatively charged attractive sites, however, must be in excess of the positively charged sites, because a positively charged PVAM particle encounters more attractive sites than a negatively charged RGC particle. This is in line with the overall negative charge on the bacterial cell surface. In essence, a particle attached to the AFM cantilever has traversed the entire EPS layer until contact with the cell wall upon approach and the percentage of curves showing adhesion upon retraction thus reflects the attractive sites that the particles has encountered. Mendez-Vilas et al. [31] has suggested that a penetrating AFM tip may cause irreversible damage to the inner cell surface including the bacterial membrane upon traversing the EPS layer, as concluded from saw-tooth patterns in the force–distance curves at close ap-

Fig. 4. Distribution of the adhesion force (Fadh ) calculated from force–distance curves between R. terrigena prior to and after NAC-treatment and negatively and positively charged, wood-based carbon particles in stabilized water. Top: Negatively charged carbon particle (gray bar, prior to, and black bar after NAC treatment). Bottom: Positively charged carbon particle (gray bar, prior to, and black bar after NAC treatment).

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Fig. 5. The log-reduction of R. terrigena and E. coli established by activated, mesoporous carbon particles (CA-10, acidic wood-based activated carbon; RGC, basic wood-based activated carbon; PVAM, RGC carbon particle coated with polyvinyl amine) in a batch test [22] as a function of the percentage of retraction curves, showing adhesion between bacteria and activated carbon particles by the atomic force microscope. Linear and rank correlation coefficients amount both 0.72. (Q) E. coli ATCC 25922; (2) R. terrigena ATCC 33257.

proach. As we observed no such patterns in our force–distance curves, it is considered unlikely that the AFM tip has caused cell surface damage. In Fig. 5, we relate the percentage retraction curves revealed by traversing an activated carbon particle through the outer bacterial surface layer with the previously determined, macroscopic bacterial removal in a batch assay [22]. In Fig. 5, only data for mesoporous carbon particles have been included and not for microscopic coconut carbon particles, since it is known that molecular scale roughness has a major impact on bacterial deposition and adhesion [29,30]. From Fig. 5, it is evident that macroscopic bacterial removal by mesoporous carbon particles with a similar roughness positively correlates well with the number of attractive sites in the outer bacterial surface layer. In conclusion, waterborne R. terrigena and E. coli adhere only weakly to different activated carbon particles, and the main difference between different types of activated carbons is the number of attractive sites revealed upon traversing of a carbon particle through the outer bacterial surface layer. The number of these attractive sites seems determinant for the ultimate removal of R. terrigena and E. coli by mesoporous carbon particles. References [1] J.M. Bifulco, J.J. Shirey, G.K. Bissonnette, Appl. Environ. Microbiol. 55 (1989) 2214–2219.

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