Bacterial Accumulation at the Air−Water Interface - Environmental

Influence of Growth Phase on Bacterial Deposition: Interaction Mechanisms in Packed-Bed Column and Radial Stagnation Point Flow Systems. Sharon L. Wal...
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Environ. Sci. Technol. 1998, 32, 3704-3712

Bacterial Accumulation at the Air-Water Interface A N K E S C H A¨ F E R , † H A U K E H A R M S , * A N D ALEXANDER J. B. ZEHNDER Swiss Federal Institute for Environmental Science and Technology (EAWAG) and Swiss Federal Institute of Technology (ETH), U ¨ berlandstrasse 133, CH-8600 Du ¨ bendorf, Switzerland

Accumulation at the air-water interface of four bacterial strains with different hydrophobicities was studied. To exclude system influences, both a static batch system and a dynamic system involving rising bubbles were used. All bacteria accumulated at the air-water interface in both systems. Generally, attachment to the air-water interface increased with bacterial surface hydrophobicity. Accumulation of two hydrophilic bacteria decreased with decreasing ionic strength, indicating that electrostatic repulsion between the negatively charged bacteria and the negatively charged air-water interface influenced the attachment. No such influence was found for hydrophobic bacteria. The calculation of DLVO interaction energies revealed that additional attractive forces were needed to explain the observed attachment. Hydrophobic interactions were calculated on the basis of contact angles and were shown to promote the accumulation. A colloidchemical approach combining DLVO interactions and hydrophobic interactions seems to be appropriate to explain the accumulation at the air-water interface of all investigated strains.

When rising air bubbles burst at the air-water interface, bacteria are ejected into the air (12). Because only material from the former bubble surface gets into the ejected water drops (13), the transport of bacteria into air is a consequence of their enrichment at the air-water interface of the rising bubbles (12, 14). It is thought that microbial aerosols are thus created by bubbles rising in the sea (15), and pathogens are distributed by air-conditioning systems (16) and sewage plants (17). The accumulation of bacteria at the air-water interface also influences their transport in unsaturated porous media, e.g., the vadose soil zone (18, 19). The data indicate that the retention of bacteria in unsaturated soil is more efficient than in saturated soil of the same type. Colloids appear to have a general inclination to attach to air-water interfaces, as observations with inorganic particles have shown. The preferential attachment of hydrophobic minerals to rising air bubbles has been used for many decades for the separation of complex ores in a process known as froth flotation (20, 21). The attraction of hydrophobic and hydrophilic silica particles to air bubbles was quantified by atomic force microscopy (22). Nevertheless, the mechanism of particle attachment to the air-water interface is still insufficiently understood (23). Recently, it was visualized in glass micromodels that bacteria accumulate at clean air-water interfaces (24). Bacterial attachment was hindered by the addition of surfactants (25), implying that the hydrophobicity of the airwater interface may be crucial for the attachment process. However, the exact mechanisms of bacterial adhesion to the air-water interface and their relation to cell surface properties still remain to be solved. Therefore, we chose a number of bacteria with different surface properties and studied their enrichment at the air-water interface. Two well-defined experimental systems were used that allow for the quantification of bacterial enrichment at the air-water interface. Concepts derived from colloid chemistry were tested for their applicability to our observations.

Experimental Section Introduction In nature, most microorganisms live in contact with interfaces (1). The initial adhesion of bacteria to solids has been investigated intensively in the past decade (2-6). Although it has been known for a long time that bacteria adhere to the air-water interface (7), the underlying mechanism has not been studied. The accumulation of bacteria at the air-water interface plays a role in many natural environments. Bacteria accumulate in the surface microlayers of aquatic systems (8, 9) where also organic molecules such as lipids, proteins, and polysaccharides concentrate. The bacterial community in this microlayer is different from the one in the bulk water (10), and it has been suggested that the cell surface hydrophobicity promotes the initial adhesion of bacteria to the air-water interface (10, 11). However, these findings related to air-water interfaces covered with lipid surface films. To be able to make mechanistic statements about bacterial attachment to the air-water interface, one has to distinguish between influences of the air-water interface as such and those of interface-associated compounds. * Corresponding author phone: +41-1-823 5519; fax:+41-1-823 5547; e-mail:[email protected]. † Present address: Department of Geography and Environmental Engineering, The Johns Hopkins University, Baltimore, MD 212182686. 3704

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Buffers. All media were prepared with deionized water (Nano pure cartridge system, SKAN AG, Basel, Switzerland). Phosphate-buffered saline (PBS) solutions (pH 7.2) of various ionic strengths were used. PBS with an ionic strength of 100 mM contained 4.93 g of NaCl, 0.29 g of KH2PO4, and 1.18 g of K2HPO4 L-1 and was diluted to the lower ionic strengths. Bacteria. The behavior of four different strains was studied. Corynebacterium sp. strain DSM 44016 (C6) was routinely cultivated on brain heart broth (Merck, Dietikon, Switzerland), Rhodococcus sp. C125 (C3) (26) and Pseudomonas putida mt2 (P4) (27) were cultivated on nutrient broth (Labo-Life, Pully, Switzerland), and Pseudomonas oleovorans ATCC 29347 (P1) was cultivated on a mineral medium (28) supplemented with 2.88 mL of ethanol L-1 as the sole source of carbon and energy. Bacteria were grown at 30 °C, harvested in the early stationary phase, washed twice with 1 mM PBS, and resuspended in PBS of the desired ionic strength. To avoid cell aggregates, the bacterial suspension was repeatedly pressed through a syringe with a very fine needle and was controlled microscopically afterward. For enumeration on solid medium, C6 and P1 were grown on brain heart agar, and C3 and P4 were grown on nutrient agar. Physicochemical Characterization of Bacteria. The hydrophobicity of lawns of bacteria on cellulose acetate filters was determined by measuring the static water contact angles after 2 h as described previously (29). The electrophoretic 10.1021/es980191u CCC: $15.00

 1998 American Chemical Society Published on Web 10/03/1998

FIGURE 1. Experimental setup for the bubble experiments. mobilities (u) of the bacteria were measured by dynamic light scattering (Zetamaster, Malvern Instruments Ltd., Worcestershire, U.K.) in 1, 10, and 100 mM PBS at pH 7.2. ζ-potentials were calculated from u by the HelmholtzSmoluchowski equation (30), which was valid in the range of Debye lengths and particle sizes investigated here. The effective cell radii (Re) of the bacteria were determined microscopically from the average cell widths (w) and lengths (l) of 50 cells as Re ) 0.5(wwl)1/3. Diffusion coefficients were calculated with the Stokes-Einstein equation De ) kT/6πηRe, where k is the Boltzmann constant (1.380658 × 10-23 J K-1), T is the absolute temperature (K), and η is the dynamic viscosity of water (1.002 × 10-3 N s m-2 at 20 °C). Bubble Experiments. Experiments were conducted in a glass apparatus (Figure 1) adapted from Blanchard and Syzdek (12). Rising air bubbles in a bacterial suspension enriched bacteria at their surfaces. At the water surface, the air bubbles burst and produced a set of four or five droplets, which were ejected into the air. Sizes and ejection heights of the jet drops that are formed from the jet rising from the collapsing bubble cavity depend on the bubble diameter (14). When a bubble has risen in water for more than 20 cm, over 85% of the bacteria that have been captured by the bubble are transferred into the top jet drop (31). The top drops of several bubbles were caught on an agar plate, and the mean bacterial numbers in the droplets were calculated after plating. The volumes of the drops were determined with magnesium oxide-covered slides (32). The numbers of bacteria transferred to the top drops with the droplet volume of bulk suspension were subtracted from the total bacterial numbers in these droplets to obtain the numbers of bacteria originating from the air-water interface. These excess numbers of bacteria in the droplets were then converted to final cell densities on the bubble surfaces, assuming transfer of all bacteria from the bubble surface into the top jet drop. The main section of the two-branched glass device was a tube of 3 cm i.d. and a length of 30 cm (Figure 1). Air bubbles were generated by pumping air through a piece of silica capillary tubing (i.d. 5 µm) that was span into a Svagelock column fitting. The air bubbles had diameters of 600 ( 15 µm as was inferred from their terminal rise velocities (33). Experiments were conducted with mixed suspensions of either C6 and P1 or C3 and P4 in 1, 10, or 100 mM PBS buffers, which were prepared from individual cultures after harvesting the cells. Mixtures of a hydrophilic and a hydrophobic bacterium were used to ensure that different enrichment at the air-water interface was due to different surface properties and was not caused by experimental artifacts or bacterial surfactant production. Concentrations of around 104 bacteria mL-1 per species were used except for P1 at 1 mM, where a concentration of >106 cells mL-1 was

chosen because of its low surface enrichment. During the experiments, the bacterial suspension was pumped slowly into the right-hand branch of the glass device to achieve a steady overflow of the surface where the bubbles burst. This served to maintain a clean air-water interface. The total bubble rise distance of 27 cm was passed in ca. 3 s. The average top drop ejection height was 6 cm. Forty top drops per agar plate were collected and, subsequently, were spread on it evenly with 0.1 mL of sterile 0.9% NaCl. At least four agar plates per experiment were used. The average volume per top drop was 2.3 × 10-7 ( 2.3 × 10-8 mL. The two bacterial species used in each experiment were distinguished by their colony morphologies. A sample of the bulk suspension was taken after the experiment from 2 to 3 cm below the surface to quantify the bacterial bulk concentration on solid medium. Filter Experiments. Suspensions of 1 × 107-2 × 108 bacteria mL-1 in 1, 10, and 100 mM PBS were prepared and filled into small beakers (i.d. 3.5 cm) with a pipet. During filling and incubation, turbulence in the suspensions was avoided. After 115 min, black Nuclepore polycarbonate filters (0.2 µm in pore size, 25 mm in diameter; Costar, Cambridge, MA) were used to strip off the liquid surface layer for bacterial enumeration. The long incubation time was chosen to ensure that maximal bacterial concentrations at the air-water interface were reached. Independent experiments had indicated that the bacterial enrichment at the air-water interface leveled off after about 20 min due to sedimentation (data not shown). A filter was placed flat on the air-water interface with a forceps and removed after 5 s together with the adhering water layer. The filter was transferred, the water layer side up, to a closed, preweighed Petri dish, which was immediately weighed again to determine the volume of suspension transferred with the filter. Three air-water interfaces were sampled for each experimental condition. The number of bulk bacteria was determined by filtering an appropriate volume of the initial suspension through the same type of polycarbonate filters. Bacteria on all filters were enumerated by acridin-orange direct counting (34). Bacterial densities (bacteria cm-2) in the collected microlayers were corrected for those bacteria transferred with the bulk suspension. Bacterial densities were normalized to a bulk concentration of 107 bacteria mL-1 assuming proportionality between the concentration of bacteria and their enrichment at the air-water interface. The diffusion of particles to a surface is proportional to the bulk particle concentration (35). For comparison between bubble and filter experiments, cell densities on the bubble were also normalized to a bulk concentration of 107 bacteria mL-1. Determination of Bacterial Sedimentation Velocities. Because of the long duration of the filter experiments, bacterial sedimentation had to be considered. The sedimentation of the bacteria was determined with a spectrophotometer (Hitachi, U-1100) at 546 nm according to the method of Shonnard et al. (36). A polystyrene spectrophotometer cuvette (base: 0.4 × 1 cm) was masked with dark tape to give a 1-mm horizontal slit 9 mm above the cuvette base. Cells were suspended in 1, 10, or 100 mM PBS to an OD546 of about 0.8, corresponding to cell concentrations between 9 × 107 and 8 × 108 cells mL-1. A total of 550 µL of bacterial suspension was filled in the cuvette, which was subsequently sealed and placed in the spectrophotometer. Cell settling was recorded as the change in absorbance when the settling front passed the slit positioned 2.7 mm below the suspension surface. The decrease in OD546 was monitored for 8-24 h, and the sedimentation velocity was determined by fitting the movement of the settling front to the convective-dispersive equation with the help of the AQUASIM software (37). The sedimentation could be determined for all strains and conditions except for P4 and P1 in 100 mM VOL. 32, NO. 23, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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PBS, where the cells apparently adhered to the vertical cuvette walls and thus disturbed the measurement. Transport Calculations. An important mechanism for collision of bacteria with rising air bubbles is interception (38). The interception efficiency is a function of the bubble surface properties. When rising and picking up surface-active solutes, the bubble changes gradually from a sphere with a mobile surface to one with a quasi-rigid surface (38). Trace impurities, which solidify the air-water interface, are assumed to be present in adequate quantities even in triply distilled water (39). Even after a rise distance of more than 20 cm, it is likely that the air-water interface is partially mobile. Equations described elsewhere (38) were used to calculate the interception efficiencies between bacteria and bubbles with either rigid or mobile surfaces (see Appendix). These interception efficiencies were used to calculate the ratio between the number of bacteria collected experimentally, BE, and the number of bacteria collected theoretically, BT, due to interception by bubbles exposing either fully rigid or purely mobile surfaces. Transport of bacteria to the water surface in the filter experiments was numerically simulated with the AQUASIM software (37) taking into account diffusion and simultaneous sedimentation (see Appendix). The attachment of colloids to an air-water interface can be assumed to be irreversible, leading to an effective trapping of all arriving particles (35). Experimental cell densities on the filters were also expressed as the layer thickness of bulk suspension that theoretically had been cleared by bacterial accumulation at the air-water interface. Calculation of Interaction Energies. The DLVO theory of colloidal stability (40, 41) describes the interaction energy between two surfaces as a sum of the van der Waals and electrostatic interaction energies. DLVO interaction energies, GDLVO (h) (J), between bacteria and an air-water interface as a function of the separation distance h (nm) were calculated using equations described elsewhere (42). The Hamaker constants used for the calculations of van der Waals interactions were 15.3 kT for bacteria (43), 9.34 kT for water (44), and 0 kT for air (45)(1 kT ) 4.0 × 10-21 J). Hamaker constants varying between 9.09 and 9.58 for water (44) and between 12.0 and 18.6 for the bacterial surface (43) were tested and were found to influence quantitative interaction energies just marginally (data not shown). ζ-potentials used for the calculation of the electrostatic interactions are listed in Table 1. Air bubbles are generally negatively charged in solutions of inorganic electrolytes at neutral pH. The different hydration enthalpies of H+ and OH- ions lead to the exclusion of protons from the interface (46). ζ of the air bubbles were taken from Li and Somasundaran (47) and Graciaa et al. (48) and are listed in Table 1. Additional to the DLVO interactions, hydrophobic forces act between particles and air-water interfaces (22). Yoon et al. proposed to calculate asymmetric hydrophobic interactions between two surfaces based on the respective water contact angles (23). Hydrophobic interactions between a small particle and a flat surface can thus be described by

therefore

-K123r

∫

h

2

)-

K123r h

(1)

where Fh is the interactive force (N), r is the particle radius (nm), and Gh is the hydrophobic interaction energy (J). The force constant K123 for asymmetric interactions between macroscopic bodies of 1 and 2 in a medium 3 can be described 3706

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ionic strength (mM)

particle air bubblea

bacteriab Corynebacterium sp. DSM 44016 (C6)

Rhodococcus sp. C125 (C3)

Pseudomonas putida mt2 (P4)

Pseudomonas oleovorans ATCC 29347 (P1)

ζ (mV)

1 10 100

-60 -50 -25

1 10 100 1 10 100 1 10 100 1 10 100

-40.2 -39.1 -34.2 -48.0 -45.3 -40.0 -26.8 -12.5 -11.1 -34.2 -30.4 -21.9

a ζ of the air bubbles were taken from refs 43 and 44. b Each value represents the mean of at least two independent measurements.

by the following relationship:

log K123 ) a

(

)

cosθ1 + cosθ2 +b 2

(2)

θ1 and θ2 are the water contact angles of the two surfaces, in our case bacterium and air-water interface, and a and b are probably system-specific constants. Because water droplets have an ideal spherical shape in air, its water contact angle is 180°. For a system of silica surfaces with differing contact angles, a was -7 and b was -18 (23). We adjusted a and b to match the observation that attachment of P1 is unfavorable in 1 mM but not in 10 mM or 100 mM PBS. This assumption resulted in a ) -5 and b ) -20. With these parameters, total interaction energies, Gsum (h) (J), representing the sum of van der Waals, electrostatic, and hydrophobic interactions were calculated. We felt it was reasonable to use the same parameters for all bacterial strains, although differences in the macromolecular composition of the outer cell surfaces exist. The bubble radius in our experiments was sufficiently large in comparison to the bacterial radius to treat the bubble surface as a flat surface. An alternative model for the calculation of hydrophobic interactions has been proposed by van Oss (49). However, his calculation of interaction energies as a function of the separation distance, which is based on purely mechanistical concepts, is much better defined for hydrophilic surfaces than for hydrophobic surfaces (49, 50). Because disagreement about the origin of hydrophobic interactions prevails (49, 51-54), we chose to use a model based on empirical measurements over an entirely theoretical approach.

Results

Fh K123 )- 2 r h

Gh(h) ) -

TABLE 1. ζ-Potentials of Bacteria and Air Bubbles

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Physicochemical characteristics of bacteria. Physicochemical properties of the bacteria are listed in Tables 1 and 2. C6 and C3 were very hydrophobic and highly negatively charged but differed in size. P4 was mesohydrophilic and slightly negatively charged, whereas P1 was hydrophilic with a higher negative surface charge. The ζ-potentials of bacteria and air bubbles always decreased with increasing ionic strength due to shielding effects of the diffusive ion layer. Bubble Experiments. The results of the bubble experiments are summarized in Figure 2. Each data point is the mean value of 3-5 independent experiments. The bubble

TABLE 2. Bacterial Properties bacterial strain

water contact angle (deg)

Re (µm)

De (10-13 m2 s-1)

C6 C3 P4 P1

102.8 ( 2.5 91.8 ( 1.2 38.9 ( 1.5 21.8 ( 1.4

0.55 0.74 0.61 0.56

3.89 2.89 3.51 3.82

FIGURE 3. Bacterial accumulation at the air-water interface in the filter experiments at various ionic strengths for Corynebacterium sp. DSM 44016 (C6) (1), Rhodococcus sp. C125 (C3) (b), Pseudomonas putida mt2 (P4) ((), and Pseudomonas oleovorans ATCC 29347 (P1) (2). Error bars given are one standard deviation. Normalized net bacterial concentrations for strain P1 are 0 cells cm-2 in 1 mM PBS, 1.5 × 104 cells cm-2 in 10 mM PBS, and 2.8 × 104 cells cm-2 in 100 mM PBS.

FIGURE 2. Bacterial accumulation at the air-water interface in the bubble experiments at various ionic strengths for Corynebacterium sp. DSM 44016 (C6) (1), Rhodococcus sp. C125 (C3) (b), Pseudomonas putida mt2 (P4) ((), and Pseudomonas oleovorans ATCC 29347 (P1) (2). Error bars given are one standard deviation. size and its rise distance influence the absolute numbers of ejected bacteria to a considerable extent, as has been discussed extensively elsewhere (15). Therefore, the bubble experiments were not designed to express absolute bacterial concentrations at the air-water interface but to have a measure to compare (i) the enrichment of different strains and (ii) the accumulation rates of individual strains with theoretical transport rates. All strains enriched at the airwater interface. With exception of C6 at high ionic strength, accumulation was directly correlated to cell surface hydrophobicity. P1 and P4 accumulated only slightly in 1 mM PBS but significantly more in 10 mM PBS. No additional increase was found in 100 mM PBS. This indicated that electrostatic repulsion controlled the attachment at low ionic strength but was neutralized at high ionic strength. C3 accumulated equally well at all ionic strengths. C6 accumulated preferably at low ionic strength. Filter Experiments. The relative accumulation of all strains in the filter experiments was in reasonable agreement with the bubble experiments (Figure 3). Each data point presents the mean value of at least three beakers. P1 did not accumulate at the air-water interface in 1 mM PBS but accumulated in 10 and in 100 mM PBS. Attachment of P4 was slightly higher and increased between 1 and 10 mM PBS. Unfortunately, P4 could not be counted on the filters obtained at 100 mM PBS because apparently an excretion product of this strain interfered with the acridin-orange stain at high ionic strength. Accumulation of C3 at the air-water interface was independent of the ionic strength, whereas C6 accumulated less with increasing ionic strength. The average thickness of the water layer collected with the filters was found to be 17 ( 4 µm. In our experimental setup, sedimentation of bacteria or bacterial aggregates could possibly influence the outcome of the filter experiments. Therefore, sedimentation velocities were determined (Table 3). Increased sedimentation at high ionic strength due to cell aggregation was small and occurred for all strains but P1. Hence, aggregation of C6 at high ionic strength did not

TABLE 3. Sedimentation Velocities of the Bacteria bacterial strain

ionic strength (mM)

νsed (mm d-1)

C6

1 10 100 1 10 100 1 10 1 10

17.8 19.2 24.7 25.4 28.6 30.7 5.3 7.7 8.4 7.0

C3

P4 P1

explain its peculiar ionic strength dependence in the filter experiments. Accumulation of C3 was relatively larger in the filter experiments than in the bubble experiments or attachment of C6 was relatively smaller. The fact that the accumulation of bacteria was in the same order of magnitude in the bubble as in the filter experiments despite the very short bubble rising time is probably due to the presence of convection as an additional transport mechanism toward the bubbles. Transport Calculations for Bubble Experiments. Table 4 shows the ratio between the number of bacteria collected experimentally (BE) and the number of bacteria collected theoretically (BT) due to interception by bubbles with rigid or mobile surfaces. For most strains and conditions, the number of accumulated bacteria in the experiments exceeded transport calculations based on air bubbles with fully rigid surfaces and fell behind calculations for air bubbles with purely mobile surfaces. The difference between interceptional collection by rigid and mobile surfaces was 2 orders of magnitude. Unfortunately, there is no method available to calculate the relative mobile and rigid contributions to bubble surfaces. However, accumulation of C6 at low ionic strength and C3 at high ionic strength even exceeded the interception by bubbles with mobile surfaces, suggesting the presence of an attractive force. Less P1 adhered in 1 mM PBS than would have been collected by interception with bubbles with rigid surfaces. This indicates the existence of an energy barrier between bacteria and the interface that surpassed the kinetic energy of the bacteria. VOL. 32, NO. 23, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 4. Ratios between the Number of Experimentally Collected Bacteria in the Bubble Experiments (BE) and the Number of Theoretically Collected Bacteria (BT) Due to Interception with Rigid and Mobile Bubbles bacterial strain

ionic strength (mM)

BE/BT for rigid bubbles

BE/BT for mobile bubbles

C6

1 10 100 1 10 100 1 10 100 1 10 100

397.9 256.7 115.2 144.4 169.4 196.6 36.8 116.5 110.2 0.6 59.8 64.1

2.0 1.3 0.6 1.0 1.2 1.3 0.2 0.7 0.6 0.0 0.3 0.3

C3

P4

P1

TABLE 5. Calculated Diffusive Transport of Bacteria to the Air-Water Interface in the Filter Experiments

bacterial strain C6

C3

P4

P1

ionic strength (mM) 1 10 100 1 10 100 1 10 100 1 10 100

diffusive transport including sedimentation (bacteria cm-2) for 107 bacteria mL-1

layer thickness cleared from bacteria (µm)

3502 3256 2559 1878 1687 1572 9340 6832

266 127 92 666 551 603 79 123

6782 8005

0 15 28

Transport Calculations for Filter Experiments. In Table 5, the calculated diffusional transport of bacteria to the airwater interface during the experiment and the theoretical layer thickness of the bulk suspension completely cleared of bacteria are given. Sedimentation was accounted for and led to a leveling off of the diffusional transport to the interface after 10-20 min, which confirmed experimental results (data not shown). Comparison with Figure 3 shows that only for P1 in 1 mM PBS the diffusive transport alone could explain the enrichment of bacteria at the air-water interface. Here, the calculated diffusive transport exceeded the actual enrichment at the interface, which again indicates the existence of an energy barrier between bacteria and the interface. All other strains, however, enriched more than could be explained by diffusion only: for example, C3 from a layer thickness of more than 500 µm was concentrated at the air-water interface. Forces ranging over such a long distance are hardly conceivable, implying that the beakers were probably not perfectly quiescent. Calculation of Interaction Energies. An example for calculated DLVO interaction energies can be seen in Figure 4 for P4 in 10 mM PBS. Positive interaction energies between bacterium and air-water interface are thermodynamically unfavorable, whereas negative energies are favorable. Due to unfavorable van der Waals interactions between all bacteria and the air-water interface, not even P4, which has the lowest negative charge of all investigated strains, ought to be attracted by an air bubble according to the DLVO theory. Since this strain accumulates, it is evident that an attractive 3708

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FIGURE 4. van der Waals interactions (- -), electrostatic interactions (-‚-), and total DLVO interactions (s) calculated for Pseudomonas putida mt2 (P4) in 10 mM PBS. force between bacteria and air-water interface must account for the accumulation. Yoon and co-workers postulated that the major driving force for bubble-particle adhesion is the hydrophobic force (23). We first modified their relationship to match the observation of an energy barrier between P1 and the airwater interface in 1 mM PBS. Then we calculated the hydrophobic interaction energies for all bacteria. Figure 5, panels a-d, shows the total interaction energies (the sum of DLVO and hydrophobic interactions) of bacteria approaching the air-water interface for all bacterium-ionic strength combinations as a function of their separation distance. P1 encounters an energy barrier in 1 mM PBS that cannot be overcome by the kinetic energy of the cells and prevents to a certain extent enrichment at the air-water interface. In 10 and 100 mM PBS, electrostatic repulsion is reduced by a shielding effect of the ions and compensated by hydrophobic attraction, allowing for attachment to the air-water interface. For P4, a smaller energy barrier present in 1 mM PBS disappears in 10 and 100 mM PBS, where the interaction energies are equally favorable. This could explain why the accumulation of P4 at the air-water interface increased between 1 and 10 mM PBS but not between 10 and 100 mM PBS. For the hydrophobic strains C3 and C6, hydrophobic attraction dominates the total interaction energy at all ionic strengths. This is in agreement with the constantly high enrichment for C3 at all ionic strengths in the bubble and the filter experiments but cannot explain the peculiar influence of the ionic strength on the enrichment of C6.

Discussion In this study, the accumulation of bacteria at the air-water interface as a function of their macroscopic surface properties was quantified. Theories based on colloid chemistry were tested for their suitability to explain bacterial interactions with the air-water interface. Two different experimental systems were used, a static filter system and a dynamic rising bubble system, in which convection could be studied as an additional transport mechanism. All investigated bacteria accumulated at the air-water interface in both systems. We have shown that bacterial attachment to the airwater interface increases with increasing surface hydrophobicity (Figures 2 and 3). It had been suggested before that hydrophobicity may play a role in the initial adhesion of bacteria to air-water interfaces covered with lipid films (10). Although many natural interfaces are indeed covered with films of organic molecules, the nature of these compounds

FIGURE 5. Complete interaction energies (DLVO plus hydrophobic interactions) at 1 mM (s), 10 mM (- -), and 100 mM PBS (-‚-) as calculated for Pseudomonas oleovorans ATCC 29347 (P1) (A), Pseudomonas putida mt2 (P4) (B), Rhodococcus sp. C125 (C3) (C), and Corynebacterium sp. DSM 44016 (C6) (D). may vary from location to location. To make mechanistic statements about bacterial attachment to the air-water interface, one has to work in well-defined experimental systems where influences of the air-water interface as such and influences of interface-associated compounds can be separated. The attachment of hydrophilic bacteria to the air-water interface is reduced at low ionic strength, indicating the significance of electrostatic repulsion between the negatively charged interaction partners. For at least one hydrophobic bacterium, in contrast, electrostatic repulsion seems to be compensated by a strong attractive force so that attachment to the air-water interface is equally attractive at all ionic strengths. To identify interactions between bacteria and solid surfaces, it is a common procedure to calculate their interaction energies according to the DLVO theory of colloidal stability (5, 55, 56), which explains the total energy as a sum of van der Waals and electrostatic interactions. Discrepancies between experimental observations and calculated DLVO energies must be due to additional types of interaction. This is the first study using an DLVO approach to explain interactions between bacteria and the air-water interface. Although, strictly speaking, the DLVO theory holds only for the equilibrium situation between particle and interface, which is neither reached in the bubble experiments due to the limited contact time between bacterium and bubble nor in the filter experiments due to sedimentation, it can be used to get a general idea about the nature of interactions between small particles and the air-water interface under nonequilibrium conditions (57). Derjaguin and Dukhin (57) pos-

tulated that, in case of repulsive van der Waals and electrostatic interactions, no particle attachment to the airwater interface can occur. Since DLVO energies between bacteria and the air-water interface over a thin water film actually comprise repulsive van der Waals and electrostatic forces, they are insufficient to explain bacterial attachment. The hydrophobic force has repeatedly been suggested to be responsible for such deviations from DLVO principles. Pashley et al. (58) were the first to show that the attractive forces measured between hydrophobic surfaces are much stronger than van der Waals forces and operate at significantly longer distances. Since then, it has been verified that the hydrophobic force is decaying exponentially with distance (59), consists of a short range and a long-range part (54), and also exists when only one of the two partner surfaces is hydrophobic (22, 51). However, no consensus has been reached regarding the origin of hydrophobic forces. Opinions range from the correlation of large dipole moments associated with molecular domains (51, 53) and electron donor-electron acceptor interactions (49) over cavitation due to capillary forces (54) to subcritical density fluctuations due to enhanced thermal fluctuation (52). The lack of a unifying concept about the origin of these forces has in most cases prevented the development of models for calculating them. Total interaction was usually measured by atomic force microscopy and hydrophobic contributions calculated by subtraction of DLVO interactions. Recently, Yoon et al. (23) developed an empirical model to calculate hydrophobic interactions between silica particles on the basis of their contact angles, a concept we have tested for its applicability to biological particles. Our VOL. 32, NO. 23, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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calculations show that the attachment of many bacteria to the air-water interface can be explained by combining DLVO-type interactions and hydrophobic interactions. The large energy barrier for P1 in 1 mM PBS and the smaller one for P4 prevented both strains from high enrichment at the air-water interface in our experiments. However, for all other strains and conditions, the primary energy minimum near the interface was easily accessible, leading to high enrichment. The magnitude of enrichment was probably controlled by the interactive forces, which are expressed as the negative slopes of the interaction energy curves. With increasing bacterial hydrophobicity, the interactive forces increased, and therefore, more bacteria were dragged to the air-water interface. Although the application of colloid chemical concepts can help our understanding of the mechanisms governing bacterial attachment to interfaces, the state has not been reached where a priori predictions can be made. The origin of hydrophobic interactions is still insufficiently understood to give us more than a rough measure of their exact quantity between different bacteria and the air-water interface. Additionally, bacteria are no smooth particles but possess structural and chemical surface heterogeneities that makes it hard to characterize them sufficiently by macroscopic properties. Especially at small separation distances between bacterium and air-water interface, probably also steric interactions contributed to the attachment, as has been shown for bacterial adhesion to solid surfaces (60). The accumulation of bacteria at the air-water interface exceeded the calculated transport in some cases in the bubble experiments and in all cases in the filter experiments. Deviations of experimental from predicted transport in the bubble experiments were rather small and can either be explained by hydrophobic attraction or by the rodlike shape of bacteria, which, assuming rotating cells, possibly leads to a larger effective radius than the calculated one. Deviations in the filter experiments were several orders of magnitude and cannot solely be explained by attractive forces of molecular origin since the range of those had to be much higher than sensibly conceivable. Calculations showed that water evaporation from the beakers in the course of the experiment probably led to a temperature gradient in the suspension that was sufficient to cause considerable microconvection as an additional transport mechanism (data not shown) (61). Secondary minimum adhesion could have played a role for P1 and P4 at low ionic strength (compare Figure 5). However, visual observations had shown irreversible attachment of hydrophilic bacteria to the air-water interface at low ionic strength with respect to mild shear stress (24). Therefore, we felt justified to assume irreversible bacterial attachment to the air-water interface in our transport equations. Two experimental observations could not be explained, of which the first one was the peculiar ionic strength dependence of C6. We could rule out several possible effects of increased ionic strength: (i) increased negative charge by ζ-measurements, (ii) increased aggregation at the air-water interface by microscopic enumeration of bacteria in the filter experiments, and (iii) increased aggregation in the bulk phase causing increased sedimentation by determination of sedimentation velocities. Hence, besides the types of interactions we have already considered, additional factors probably play a role for some bacteria. The second observation was the relatively lower attachment of C6 to the air-water interface in filter experiments as compared to C3. C6 is more hydrophobic, less negatively charged, and has a lower sedimentation velocity than C3, properties that all should promote its attachment to the air-water interface. Our investigations have shown that bacteria with different surface properties accumulate at the air-water interface to 3710

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different degrees and that their attachment can be influenced by environmental factors such as ionic strength. Air-water interfaces do not only play an important role in aquatic systems such as seas and lakes but also in subsurface environments. The accumulation of bacteria at the airwater interface can govern bacterial retention in unsaturated porous media. Since rain and groundwater are very low in ionic strength, electrostatic interactions have to be considered for the attachment of hydrophilic bacteria to the air-water interface in natural systems, but probably not for that of hydrophobic bacteria. The transport of bacteria in the vadose soil zone plays an important role for the success of bioaugmentation. Their enrichment at the air-water interface brings them in closer contact to pollutants mainly transported by the gas phase and probably allows for their more efficient degradation. An understanding of the mechanisms governing bacterial attachment to the air-water interface will help the development of sophisticated models dealing with bacterial and pollutant transport in unsaturated soil.

Acknowledgments This research has been conducted as part of the OPUS-IA project and has been supported by the Board of the Swiss Federal Institutes of Technology. We thank J. Lyklema, J. Morgan, W. Stumm, N. Brooks, and C. O’Melia for helpful discussions. We acknowledge A. Wu ¨ est for his help about microconvection and P. Reichert for his assistance with the model calculations.

Appendix: Transport Calculations Bubble Experiments. If a bacterium is to be collected by a rising air bubble, it first has to collide with the bubble and attach to it. The major mechanism causing collisions between bacteria and rising bubbles is interception (38). If a bacterium moves along a streamline that approaches the bubble surface more closely than one bacterial radius, it will collide with the bubble. The rate of collection of bacteria, however, decreases with height of bubble rise because the bubble surface loses mobility as it picks up surface-active materials and, consequently, changes from a sphere with a mobile surface to a sphere with a quasi-rigid surface. The collision efficiency by interception, Ei, describes the fraction of bacteria in the cylindrical volume swept out by the bubble that collides with it. We calculated Ei for bubbles with a rigid surface with the following equation, which represents a good approximation for bubble Reynolds numbers Re < 300 (38):

[

]( )

3 Re Re 16 3 Ei ) 1 + 0.56 2 Rb 1 + 0.249 Re

2

(A1)

Re is the effective bacterial radius (µm) and Rb is the bubble radius (µm). The Reynolds number (Re) can be calculated by the following equation:

Re )

2 RbFwν η

(A2)

Fw is the buoyant density of water (0.9982 g cm-3 at 20 °C), ν is the bubble rise velocity (7.3 cm s-1), and η is the dynamic viscosity of water (1.002 × 10-2 g cm-1 s-1 at 20 °C). For our experiments, Re was 44. The following equation approximates the collision efficiency for bubbles with a mobile surface for all bubble Reynolds numbers (38):

[

Ei ) 1 +

2 37 1+ Re

( )

0.85

]( ) Re Rb

(A3)

The number of bacteria theoretically colliding with the rising air bubble was calculated for mobile and rigid bubbles as BT ) EiCπRb2l. CπRb2l describes the number of bacteria swept out by the rising bubble with C as the bacterial concentration (cells cm-3) and l as the bubble rise distance () 27 cm). BT was compared with BE, the number of bacteria that were collected by the bubble experimentally and subsequently ejected into the air with the top jet drop. Filter Experiments. Bacterial transport processes in the beaker can be described by the following equation when diffusion and sedimentation are taken into account:

∂C ∂2C ∂C ) De 2 - νsed ∂t ∂x ∂x

(A4)

C is the bacterial concentration in the liquid phase (cells cm-3), De is the bacterial diffusion coefficient (cm2 s-1), νsed is the sedimentation velocity (cm s-1), x is the axial coordinate (cm), and t is time (s). It was assumed that cells that were transported to the water surface (x ) 0) were removed from the bulk liquid and instantaneously transferred into the airwater interface as a second compartment. All bacteria located in the air-water interface were irreversibly trapped by it. The bacterial concentration at the interface (cells cm-2) after 115 min was numerically simulated with the AQUASIM software (37) for a bulk concentration C of 107 cells cm-3. The following boundary conditions were used:

∂C νsedC - De ) -kC ∂x ∂C )0 ∂x

at x ) 0

at x ) 5

(A5a) (A5b)

where k (min-1) is the rate constant for bacterial transfer from the bulk liquid phase into the air-water interface. k was set to 100 to represent instantaneous transfer of bacteria from the water surface into the air-water interface. The second boundary condition approximates an infinitely deep reaction vessel, stating that the bottom of the beaker does nor exert any influence on the processes at the air-water interface.

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Received for review February 24, 1998. Revised manuscript received July 13, 1998. Accepted August 17, 1998. ES980191U