Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite ...

Vol. 60, No. 9

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3349-3357

0099-2240/94/$04.00+0

Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur ROBERT C. BLAKE II,* ELIZABETH A. SHUTE, AND GARY T. HOWARDt Department of Biochemistry, Mehany Medical College, Nashville, Tennessee 37208 Received 2 May 1994/Accepted 17 June 1994

ThiobaciUus ferrooxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacilusferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed.

Thiobacillus ferrooxidans is the most extensively characterized member of a group of chemolithotrophic bacteria that inhabit ore-bearing geological formations exposed to the atmosphere and obtain all of their energy for growth from the dissolution and oxidation of minerals within the ore. The possibility of exploiting this activity to extract metals for commercial gain, a process known as bacterial leaching, has gained attention in recent years. A widespread application of bioleaching is its use in the pretreatment of refractory goldbearing ores (16, 19). It is estimated that $10 to $50 million worth of gold was recovered by biooxidation in 1988 and that this value would rise to $2 to $3 billion by 1998 (22). Furthermore, the global market for the bioleaching of base metals (mainly copper) and uranium amounted to $2 billion in 1988 and should reach $8 billion by 1998. Despite the environmental and possible economic importance of these bacterial activities, very little fundamental information concerning chemical and biochemical events at the mineral surface during dissolution in the presence of bacteria is available. Although there is electron microscopic (1, 2, 20, 23) and physical (7, 9, 18, 21, 33) evidence for the adsorption of T. ferrooxidans onto mineral surfaces, the relative contribution to the leaching process of sessile [by a direct contact mechanism(s)] versus planktonic (by indirect chemical reactions with soluble ferric ions) organisms remains unclear. Major obstacles include difficulties in enumerating particlebound bacteria (autotrophic bacteria that are notoriously difficult to culture on solid media) and the microheterogeneity of natural mineral samples. The present paper describes electrophoretic mobility mea-

surements of the interaction of T. ferrooxidans with ferric ions, pyrite (FeS2), and colloidal sulfur by laser Doppler velocimetry. Surfaces of metal sulfide minerals react with anions and cations in solution to acquire a net charge. Bacteria are also amphoteric by virtue of the ionogenic residues on their cell surfaces and the preferential adsorption of ions. Aqueous suspensions containing both types of amphoteric particles may be viewed, in principle, as a mixture of colloids. The goal of these experiments was to investigate whether techniques commonly employed to study the interactions and stability of colloidal systems could be applied to the interactions of chemolithotrophic bacteria with their insoluble mineral substrates. The binding of ferric ions, pyrite, and sulfur to T. ferrooxidans was observed under physiological conditions for this acidophilic bacterium. The net surface charge on T. ferrooxidans was such as to minimize charge repulsive forces in its interactions with any mineral surface.

MATERIALS AND METHODS Cell culture. T. ferrooxidans ATCC 23270 was grown autotrophically on soluble ferrous ions in an apparatus that permitted in situ electrochemical reduction of the product ferric ions to achieve enhanced yields of the bacteria (3). The ferrous sulfate medium (28) was adjusted to pH 1.8 and amended with 1.6 mM cupric sulfate. The bacterial growth chamber was a 12-gallon (ca. 45-liter) glass carboy filled to capacity. Iron-grown cells were harvested by removal and centrifugation of small portions (1 to 2 liters) of the culture, typically containing 1 to 2 g of wet cell paste per liter. The soluble iron in the medium was electrochemically reduced and maintained in a completely reduced state 1 h prior to harvesting. The volume in the growth chamber was maintained at a constant level by the addition of fresh sterile medium. Cultivation of T. ferrooxidans on either pyrite (catalog no. 8455; National Institute of Standards and Technology, Gaith-

* Corresponding author. Present address: College of Pharmacy, Xavier University, New Orleans, LA 70125. Phone: (504) 483-7489. Fax: (504) 488-3108. t Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA 70402.

3349

3350

BLAKE ET AL.

ersburg, Md.) or elemental sublimed sulfur (Fisher Scientific, Norcross, Ga.) was accomplished in the liquid medium described previously (15) containing 0.8 g of monobasic potassium phosphate per liter and a 1% (wt/vol) amendment of the insoluble inorganic substrate. The elemental sulfur was sterilized by heating the dry powder for 1 h in a boiling-water bath for each of 3 successive days. The pyrite was autoclaved as the dry powder. Bacterial growth on pyrite or sulfur was monitored by the solubilization of iron or the acidification of the medium, respectively. Pyrite- or sulfur-grown cells were harvested in an identical manner. The inorganic solids remaining in a 2- to 4-week-old 19-liter culture were permitted to settle by gravity, and the bulk of the clarified liquid was decanted. The supernatant was then concentrated by tangential flow filtration on a Millipore Pellicon (Millipore Corporation, Bedford, Mass.) equipped with a 100,000-molecular-weight cutoff membrane. Centrifugation of the retentate in a Sorvall Refrigerated Superspeed centrifuge at 10,000 x g for 15 min yielded only traces of cell mass regardless of whether the growth substrate was pyrite or sulfur, indicating that the majority of the cells remained associated with the insoluble substrate. Substrate-associated cells were subsequently recovered by bringing the filtrate obtained above to 0.01% (wt/vol) in Triton X-100 (Sigma Chemical Co., St. Louis, Mo.), adding the amended filtrate back to the particulate substrate, and repeating the steps described above. Typical yields varied from 1 to 3 g of wet cell paste per 19 liters. Harvested cells were routinely washed three times with 0.001 N sulfuric acid and stored (1 g of wet cell paste per 4 ml of 0.001 N sulfuric acid) at 4°C. Electrokinetic measurements. Electrophoretic mobility was determined with a DELSA 440 instrument (Coulter Scientific Instruments, Inc., Hialeah, Fla.). Electrophoretic motion was detected by the Doppler shift in frequency of scattered coherent light by a heterodyne method. Doppler frequency spectra were obtained simultaneously at four different scattering angles. Values for the corresponding mobilities and zeta potentials were determined from the Doppler frequency spectra by using operating and analysis software provided by Coulter. Instrument control and data analysis were accomplished with a Gateway 2000 486DX-33 computer (North Sioux City, S. Dak.) interfaced to the DELSA 440. Samples for analysis were diluted at least 100-fold into the desired solution and placed in the observation cell so that no air bubbles were visible. The observation cell was positioned in the instrument so that all measurements were conducted at the upper stationary layer. The frequency shift range was 250 Hz in all experiments. The instrument was operated in the constantcurrent mode with field strengths, indicated below, that varied with the conductivity of the analyte. All measurements were performed at 25°C. Bacterial suspensions (108 cells per ml, final concentration) were prepared in 0.01 N solutions of either sulfuric, perchloric, hydrochloric, or nitric acid immediately prior to the measurement. Ferric ions, when present, were added as the salt of the corresponding conjugate base so that each solution contained only one major species of anion. Colloidal sulfur (Ruger Chemical Co., Irvington, N.J.), when present, was added from a 1% (wt/vol) suspension in deionized water to a final concentration of 0.4 g of sulfur per ml. Ground pyrite (NIST catalog no. 8455) was further reduced by amalgamation on a Silamet dental amalgamator (Vivadent, Schaan, Austria) and grinding with a mortar and pestle. The resulting powder was suspended in distilled water, and colloidal pyrite was taken as the fraction of suspended fine particles that were decanted after the suspension had settled for 2 h. The suspension of colloidal

APPL. ENVIRON. MICROBIOL.

pyrite was brought to pH 2.0 with sulfuric acid for measurements that involved pyrite in acid. Gel electrophoresis. The cell wall and outer membrane components of T. ferrooxidans grown on each of the three substrates were prepared as described elsewhere (4). The components of each preparation were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a PhastSystem with PhastGel Gradient 8 to 25% polyacrylamide gels and PhastGel SDS Buffer Strips (Pharmacia LKB Biotechnology, Piscataway, N.J.). The molecular weight standards were the Dalton Mark VII-L kit from Sigma Chemical Co. The gels were double-stained according to a silver-Coomassie brilliant blue R-250 staining strategy that permitted color-coded differentiation of proteinaceous and sugar-containing components (8). Silver staining was performed on the PhastSystem by adapting the manual protocol for the Bio-Rad silver stain kit (catalog no. 161-0443; Bio-Rad Laboratories, Hercules, Calif.), while Coomassie staining was conducted by the standard automated protocol suggested by Pharmacia. Iron determinations. The total iron associated with washed cells of T. ferrooxidans was determined on whole-cell lysates by atomic adsorption spectrometry on a Perkin Elmer 2100 (Norwalk, Conn.) operated in the flame mode. Cell numbers were obtained with a Multisizer Ile (Coulter Scientific Instruments, Inc.) fitted with a 30-,um aperture. The instrument was programmed to siphon 100 ,ul of sample volume containing 0.8% (wt/vol) NaCl as the electrolyte. Cells for iron analysis were boiled in concentrated nitric acid (10 ml of HNO3 per g of wet cell paste). The resulting solutions were diluted with 1.0 N sulfuric acid for iron analyses by atomic adsorption. RESULTS AND DISCUSSION

Zeta potential. An electrical potential due to charge separation frequently develops at the interface where two dissimilar materials are in contact. Particles immersed in aqueous solutions acquire a surface charge for a variety of reasons, the most prominent being the ionization of surface molecular groups and the preferential adsorption of ions. Ions of opposite charge to the charge on the particle surface (counterions) are attracted to the surface, while ions of like charge to those on the surface (co-ions) are repelled. The surface charge plus the excess of counterions over co-ions near the surface constitutes the electrical double layer. Figure 1 illustrates those features of the electrical double layer of immediate interest (14). Most of the surface charge is neutralized by tightly bound counterions in the Stern layer; the remaining charge is balanced by the diffuse or Gouy layer of counterions whose concentration decreases as a function of the distance from the surface. When the charged particle is set into motion under the influence of an applied electric field, ions in the Stern layer move with the particle, while ions in the diffuse layer are constantly changing as the particle moves through the continuous phase. The imaginary plane inside which the tightly associated ions migrate with the particle surface, the shear boundary, lies just outside the Stern layer. The electrical potential determined at the shear boundary with respect to the bulk liquid is the zeta (t) potential. A negative ; potential indicates that the particle is negatively charged and migrates toward the anode in an electric field, while a positive ; potential indicates just the opposite. The greater the absolute value of the t potential, the greater is the charge density on the surface. Electrophoretic mobility. Zeta potential is measured by particle electrophoresis methods. In the laser Doppler veloci-

VOL. 60, 1994

ELECTROPHORETIC MOBILITY OF T. FERROOXIDANS

-

A

*@ (3E03 (3E03'2(

(0

3351

1.00 N c

Charged surface-

0 10

0.50

.-

I!

(i) (3 (315:

*(3

0.00 -20

(i

-15

-10

-5

0

Sin(e/2)

Frequency, Hz

Mobility, pm-cm/V-s

N

Diffuse layer

Stern layer

,
,A

._

0

1.00 -2

0 *0

0

0

0.50

(a

-6

0

-8

._

m

0.00 -20

N

0

-10

-4

0. co

10

0

5

10

15

20

25

30

Fe(lIl), mM

Zeta Potential, mV

FIG. 3. Effect of ferric ions on the electrokinetic properties of ferrous ion-grown T. ferrooxidans. (A) Effect of ferric ions on the 17.40 mobility spectrum of T. ferrooxidans in 0.01 N perchloric acid. The concentration of ferric ions was 0, 2.8, 9.5, and 25.2 mM in experiments a, b, c, and d, respectively. The conductivity ranged from 4.26 to 10.58 mS/cm in experiments a through d; the current was held constant at 2.0 mA. (B) Dependence of the zeta potential of T. ferrooxidans on the concentration of ferric ions. Each datum point and error bar represents the mean and standard deviation, respectively, for 12 determinations, comprising values extracted from the 17.40, 26.0°, and 34.70 mobility spectra of four individual measurements. (Inset) Linear plot of the reciprocal of At, the difference between the value of t in the presence of ferric ions minus that

in their absence, versus the reciprocal of the concentration of ferric ions. The ordinate intercept and slope were determined by a linear regression analysis that omitted the lowest iron concentration.

causes of frequency spectrum broadening are sample heterogeneity and Brownian motion of the particles. Line width broadening due to sample heterogeneity is directly proportional to the magnitude of the scattering vector K, where

K

=

4-rrn sin(0/2)/10

(2)

dependence of the relative intensity of scattered light on the 4 potential, calculated from the Smoluchowski equation,

rl'/(EcO) (4) where -q and £ are the viscosity and dielectric constant, respectively, of the medium and is the permittivity of free space. Each 4 potential spectrum in Fig. 2D yielded a mean value for the 4 potential that represented the entire spectrum. T. ferrooxidans and ferric ions. Ferric ions in the growth medium had an effect on the apparent 4 potential of cells harvested from that medium. The t potential of cells grown to stationary phase on soluble ferrous ions and harvested from an environment high in ferric ions varied from culture to culture, ranging from -5.0 to + 1.5 mV. Other laboratories have reported t potentials for ferrous-grown T. ferrooxidans ranging from -4.6 mV (21) to 0 mV (6). Efforts to devise a washing routine to reduce the t potential of such cells to a common reproducible value were unsuccessful. However, when we exploited our in situ electrochemical apparatus to maintain the soluble iron in the growth medium in the ferrous state, the t potential of cells harvested under such reducing conditions was reproducibly between -7.0 and -8.0 mV. Analytical analyses C

=

e0

while that due to Brownian motion is related to K by line width

=

DK2/I7

(3)

where D is the diffusion coefficient. The shape of a line width versus the scattering angle plot will thus be linear if heterogeneity is the major source of broadening but quadratic [dependence on sin2(0/2)] if Brownian motion is the major source. Figure 2C shows the dependence of the half-width at halfheight for peaks of the frequency spectra in Fig. 2A on the sine of the bisected scattering angle. The linear dependence indicated that the line width broadening observed with T. ferrooxidans was due primarily to heterogeneity in the mobility of the population of bacterial cells and that Brownian motion of the cells was negligible relative to that of smaller particles within the analytical range of the instrument. Third, values for the electrophoretic mobility and 4 potential were calculated from the frequency spectrum at each angle. Electrophoretic mobility, ,u, is defined as the linear velocity (centimeters per second) per unit of electric field gradient E (volts per micrometer) and is given as ,u = Aw/EK. Figure 2D shows mobility spectra of the relative intensity of scattered light as a function of the electrophoretic mobility of T. ferrooxidans corresponding to each of the frequency spectra in Fig. 2A. As the electrophoretic mobility of a particle under a given set of solution conditions is an inherent physical property, the mobility spectra obtained at different detection angles overlay as shown. Figure 2D also shows the corresponding

revealed that cells harvested in the presence of ferric ions had much as sevenfold more iron associated with them than did cells harvested in the presence of ferrous ions (1.0 x 1012 versus 1.4 x 10-13 g of iron per cell for T. ferrooxidans harvested under oxidizing and reducing conditions, respectively). All subsequent experiments that involved cells cultured on soluble iron were done with cells harvested under reducing conditions to ensure that the t potential of the starting material was reproducible. The t potential of T. ferrooxidans harvested from ferrous media was dependent on the concentration of ferric ions added. Figure 3A shows examples of the mobility spectra of T. as


VOL. 60, 1994

3353

TABLE 1. Electrokinetic properties of T. ferrooxidans >

E

2

0 -2

0.

-4

Growth substrate

Principal anion

Soluble iron

C104S042Cl-

/

-6/

NO3-

N

-8 0

ClO4-

Pyrite

5 10 15 20 25 30

S042-

Cl-

Fe(III), mM FIG. 4. Effect of different anions on the electrokinetic properties of ferrous ion-grown T. ferrooxidans. The dependence of the zeta potential of T. ferrooxidans on the concentration of ferric ions was determined in the presence of nitrate (A), chloride (0), sulfate (0), and perchlorate (- - -, representing the line and data in Fig. 3B). The current was 2.0 mA in all experiments; the conductivity range for each anion was: sulfate, 3.54 to 8.65 mS/cm; nitrate, 3.34 to 10.72 mS/cm; chloride, 3.07 to 10.33 mS/cm; and perchlorate, as given in the legend to Fig. 3. The curves drawn through the datum points were generated by assuming the applicability of equation 5 and using the values for the constants listed in Table 1.

ferrooxidans at different concentrations of ferric ions in 0.01 N perchloric acid. The data in the figure were limited to those of the 17.40 angle for clarity of presentation. The addition of ferric ions lowered the electrophoretic mobility of T. ferrooxidans, making the organism behave as though it were less negative (or more electropositive). Values for the mean 4 potential were determined at different iron concentrations for each of three detector angles in four separate determinations, and the results are plotted in Fig. 3B. The dependence of the t potential on the concentration of Fe(III) was fit to the following rectangular hyperbola:

tmax[Fe(III) KFe(III) + [Fe(III)]

+

(5)

where tbobs iS the observed t potential, to and tmax are the t potentials at ferric concentrations of zero and infinity, respectively, and KF,(tII) is the concentration of ferric ions required to achieve half of the Fe(III)-dependent change in the t potential. Values of tma. and KFe(III) were obtained from the ordinate and abscissa intercepts, respectively, of the doublereciprocal plot shown in the inset of Fig. 3B. These data indicated that ferric ions adsorbed preferentially to T. ferrooxidans and increased the positive charge on the organism until a maximum t potential of 2.1 mV was achieved. The apparent dissociation constant for this binding was 11 mM. The t potential of T. ferrooxidans was also influenced by the identity of the principal anion present. Figure 4 shows the t potential of ferrous iron-grown T. ferrooxidans as a function of the ferric concentration in the presence of each of four anions, perchlorate, sulfate, chloride, and nitrate. The t potential of the bacterium in the absence of ferric ions varied from -7.4 mV (perchlorate) to -1.8 mV (nitrate). These data indicated that the t potential of T. ferrooxidans at pH 2.0 resulted from a complex combination of the ionization of surface molecular groups and the preferential adsorption of anions. The dependence of the r potential on the concentration of the anion was not investigated. The t potential of the bacterium was a rectangular hyperbolic function of the ferric concentration regardless of the identity of the anion. Values of to, tmax and

NO3Cl04S042-

Sulfur

)max

KFe(mMl)

-7.4 -6.0 -4.7 -1.8

(mV) 2.1 3.1 4.1 2.2

-2.3 -1.4 -1.3 -1.7

2.1 2.9 3.7 3.0

11 11 9 13 40

(mV)

Cl-

NO3-

(mM) 11 12 10 8

-0.34

5.2

-1.1

6.5

36

-0.8 -0.9

5.7 7.7

54 45

KFe(III) obtained in the presence of different anions are

summarized in Table 1. The t potential of T. ferrooxidans was influenced by the growth history of the organism. Figure 5 shows the r potential of T. ferrooxidans in 0.01 N perchloric acid as a function of the concentration of ferric ions for bacteria that were cultured on colloidal sulfur, pyrite, or ferrous ions. The experiments shown in Fig. 5 were also conducted in sulfate, chloride, and nitrate (primary data not shown). Values of to, ;mav and KFe(III) obtained in the presence of each of the four anions for bacteria grown in the presence of each of three substrates are summarized in Table 1. The values of tm. and KFe(III) were relatively constant for ferrous iron- and pyrite-grown ceils regardless of the identity of the principal anion present. The higher values of to in pyrite-grown cells relative to those in ferrous-grown cells

E

4._

o Na

2 0 -2 -4 -6 -8

0

5

10

15

20

25

30

Fe(III), mM FIG. 5. Effect of the growth history of T. ferrooxidans on its electrokinetic properties in 0.01 N perchloric acid. The dependence of the zeta potential of T ferrooxidans on the concentration of ferric ions was determined by using washed cells that were grown autotrophically on elemental sulfur (a), pyrite (b), and soluble ferrous ions (c). Each datum point and error bar represents the mean and standard deviation, respectively, for 12 determinations. The curves drawn through the datum points were generated by assuming the applicability of equation 5 and using the values of the relevant constants in Table 1. (Inset) Linear plot of the reciprocal of A4, as defined in the legend to Fig. 3, versus the reciprocal of the concentration of ferric ions. The ordinate intercept and slope of each line were determined by linear regression

analysis.

3354


BLAKE ET AL.

Mobility, pm-cm/V-sec

ig, 66,000 45,000 36,000 29,000 24 000 -

-1

v*k4..

20,100

1

1.00

j

0.50

14,200

1

0

2 3

FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the outer membrane and cell wall components of T. ferrooxidans grown autotrophically on ferrous ions (lane 1), elemental sulfur (lane 2), or pyrite (lane 3). The molecular mass standards (in daltons, from high to low) were bovine albumin, egg albumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsinogen, trypsin inhibitor, and ot-lactalbumin. Arrowheads identify the locations of distinct yellow bands that faded after 24 h.

could be due, in part, to the residual iron present in cells harvested from pyrite, where microbial growth resulted in the accumulation of significant levels of soluble ferric ions. Iron analyses indicated that pyrite-grown cells contained 8 x 10-13 g of iron per cell, a value close to that obtained for ferrousgrown cells harvested under oxidizing conditions. Sulfur-grown cells had values of O much nearer to the isoelectric point of the cells and bound added ferric ions much more weakly than cells cultured in the presence of soluble iron. The influence of growth history on the number and nature of outer membrane and cell wall components was also investigated by gel electrophoresis under reducing conditions. Outer membrane preparations of T. ferrooxidans grown on ferrous ions, elemental sulfur, and pyrite were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained sequentially with silver and Coomassie blue to permit the color-coded differentiation of glyco-containing and proteinaceous species. Figure 6 compares gel patterns for T. ferrooxidans grown on each of the three substrates. Bands that stained yellow contained carbohydrate, while bands that stained blue were predominantly proteinaceous. Other laboratories have reported that the lipopolysaccharides of T. ferrooxidans grown on soluble ferrous ions or sulfur are chemically different (13, 31) or identical (34) for different strains. The difference in banding patterns in Fig. 6 indicated that the growth history of strain 23270 had a demonstrable influence on the structure of the outside of the cell. Carbohydrate-containing bands present in cells grown on ferrous ions were absent in cells grown on sulfur and vice versa. The presence of all the carbohydratecontaining bands in the pyrite-grown cells suggested that such cells were exposed to and respond to both soluble iron and elemental sulfur during the microbe-dependent dissolution of pyrite. T. ferrooxidans and pyrite. The C potential of pyrite was a complex function of the ions present in the suspending solution. Mobility spectra for pyrite alone are shown in Fig. 7. Each panel in Fig. 7 shows spectra from two determinations (a minimum of four were determined for each panel); the spectra were limited to those derived from the 260 angle for clarity of presentation. Figure 7A shows the fine particles from a suspension of ground pyrite in distilled water. The unfractionated pyrite comprised a mix of many particle sizes that was reflected in the heterogeneity apparent in the complex spectra. The lack of a discernible trend toward positive or negative mobility in distilled water indicated that this pyrite exhibited no apparent preference for protons or hydroxide ions. Figure 7B shows the

.

4)0

0.00 1.00

)

0.50

co

0.00 1.00 0.50 0.00 1 -20

-10

0

10

20

Zeta Potential, mV FIG. 7. Effect of sulfate and ferric ions on the electrokinetic properties of finely ground pyrite. Each panel shows the 26.0° mobility spectrum for two determinations. (A) Pyrite in water at neutral pH. (B) Pyrite in 0.01 N sulfuric acid. (C) Pyrite in 0.01 N sulfuric acid containing 12.5 mM ferric ions. The values of the conductivity and corresponding currents were: (A) 0.008 mS/cm and 0.004 mA; (B) 2.69 mS/cm and 1.6 mA; and (C) 5.96 mS/cm and 3.0 mA.

same pyrite in 0.01 N sulfuric acid. Even though the proton concentration increased nearly 100,000-fold, the C potential of the pyrite shifted to a negative value. These data indicated that sulfate was a potential-determining ion for pyrite; that is, sulfate bound to the pyrite and made it negatively charged. Figure 7C shows pyrite in sulfuric acid containing 12.5 mM Fe(III). The shift from a negative to a more positive potential indicated that ferric ions bound to the pyrite-sulfate complex. Detailed studies of the dependence of the 4 potential of pyrite on the concentration of individual ions were not attempted because of the tendency of the pyrite to adhere to the glass surface of the observation chamber in the DELSA 440, necessitating tedious disassembly, thorough scrubbing, and recalibration of the chamber after each measurement. The 4 of T. ferrooxidans was influenced by pyrite, as illustrated in Fig. 8. Figure 8A shows mobility spectra for pyrite in 0.01 N sulfuric acid. The inset shows a nonlinear plot of the half-width at half-height for the peak of the frequency spectrum versus the sine of the bisected scattering angle. The same data showed a linear dependence on sin2(0/2) according to equation 3 (plot not shown). These observations indicated that the suspended pyrite was sufficiently small to be subject to considerable Brownian motion. Estimates of the size of this pyrite by elastic light scattering indicated that the average size was less than 200 nm (data not shown). Figure 8B shows mobility spectra for pyrite-grown T. ferrooxidans. As no purposeful effort was made to remove residual iron bound to the cell during dissolution of the growth substrate, the cells as harvested exhibited a slightly positive C potential. Figure 8C shows mobility spectra obtained when the two samples were


VOL. 60, 1994

Mobility, pm-cm/V-sec

Mobility, pm-cm/V-sec -4

0

-1

3355

-2

0

1 0.50

1.00

0.00 1 .00

0.50

>b

>._ 0

c

S

0.00 1.00

0.50 0.00 1 .00

C NW

S

0.50

'U .0

0.00 1 .00

0.50

0.50

0 0

0.00 1 .00

0.00 1.00

0.50 0.00 -60

0.50s 0.00 L -20

-40

-20

0

20

Zeta Potential, mV

0-

-10

0

Zeta Potential,

I * * 10 20

FIG. 9. Effect of colloidal sulfur on the electrokinetic properties of sulfur-grown T. ferrooxidans. Each panel shows the 26.00 mobility spectrum for two determinations in 0.01 N sulfuric acid. The conductivity in samples containing colloidal sulfur was 5.21 mS/cm; the

mV

current was 2.0 mA in all(Cexperiments. Colloidal sulfur (B) T ferrooxidans T. ferrooxidans colloidal alone; through E) (A) plusalone; sulfur at 5 min (C), 10 min (D), and 20 min (E) after mixing.

FIG. 8. Effect of pyrite on the electrokinetic properties of pyriteT. ferrooxidans. Each panel shows the 26.00 mobility spectrum for two determinations in 0.01 N sulfuric acid. The current was 2.0 mA in all experiments. (A) Pyrite alone. Inset, nonlinear dependence of the half-width at half-height for the peak of the frequency spectrum on the sine of the bisected scattering angle. (B) T. ferrooxidans alone. (C) T. ferrooxidans plus pyrite 5 min after mixing. Inset, linear dependence of the half-width at half-height for the peak of the frequency spectrum on the sine of the bisected scattering angle. Each datum point and error bar in the insets represent the mean and standard deviation, respectively, for 18 determinations. grown

mixed and incubated for 5 min. The pyrite- and cell-dependent mobility spectra disappeared and were replaced with new, broad mobility spectra that corresponded to a colloidal particle with an electrophoretic mobility intermediate between those of the free components. These new spectra exhibited the heterogeneity of the free pyrite but the size of the free bacteria, as indicated by the angular dependence shown in the inset of Fig. 8C. This intermediate-mobility particle thus exhibited the properties anticipated for a complex between the bacterial cells and the particulate pyrite. T. ferrooxidans and sulfur. The 4 potential of T. ferrooxidans was influenced by colloidal sulfur. Mobility spectra for the interaction between T. ferrooxidans and colloidal sulfur are presented in Fig. 9. Figure 9A shows mobility spectra for colloidal sulfur alone in 0.01 N sulfuric acid. The average 4 potential of -40 mV indicated that there was significant charge repulsion among the individual sulfur particles, contributing to the stability of the colloidal suspension. Figure 9B shows mobility spectra for sulfur-grown T. ferrooxidans. Cells

cultured autotrophically on elemental sulfur were slightly negative. Figures 9C through E show mobility spectra obtained when the two samples were mixed and incubated for 5, 10, and 20 min, respectively. The mobility spectra of colloidal sulfur gradually diminished, while those of T ferrooxidans became progressively more negative. After 20 min, only one spectrum remained, with an electrophoretic mobility intermediate between those of the free starting components. Once again, this intermediate-mobility particle exhibited the properties anticipated for a complex between the bacterial cells and the colloidal sulfur. T. ferrooxidans double layer. The long-range forces between particles in solution are an important subject in colloid and surface chemistry. Particles in suspension experience both van der Waals' forces and electrostatic interactions. The van der Waals' interaction is attractive until the particles are sufficiently close for overlap of their atomic orbitals to create substantial repulsive forces. The electrostatic interaction is usually repulsive because both bacteria and surfaces are predominantly negatively charged in nature. The balance between these attractive and repulsive forces determines the ease and hence the rate with which the particles can approach sufficiently closely for possible short-range forces (hydrophobic and hydrogen bonds, etc.) to contribute to adherence. The adsorptive interactions between particles of the same charge have frequently been considered in terms of the Derjaguin-Landau-Verwey-Overbeck (DLVO) theory of colloid stability (24). Although the DLVO theory has been applied to

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BLAKE ET AL.


studies of viral, bacterial, and eukaryotic cell adhesion (5, 14, 17, 29, 30), numerous complications arise because cells are not nonpermeable objects of well-defined structure and surface topography. One must know the value of the surface potential to calculate the electrostatic repulsion forces. The surface potential is usually assumed to be equal to the t potential for colloidal systems in which t is less than 50 mV (11), but with cells, such an assumption is doubtful. The fixed ionogenic groups that are distributed at different levels throughout the gel-like structure of outer layers of the cell all contribute to the total effective surface charge. Ionic groups far from the shear boundary may not possess counterions that move with the cell as a unit. Cells may not be uniformly charged on their surface but may possess regions of higher and lower charge density. Also, the transformation of electrophoretic mobility to t potential by the Smoluchowski equation is strictly applicable only to impenetrable spherical particles. The more penetrable is a particle by counterions, the more inaccurate is the calculation of t from ,u (12). These and other factors make the DLVO theory at best a qualitative guide for predicting cell deposition onto surfaces. Washed cells of T. ferrooxidans grown in the presence of reduced iron were negatively charged. The thickness of the diffuse layer of counterions (protons in the absence of added ferric ions) around T ferrooxidans was estimated from the relationship

1/K =

[(4re21/ckT) E CiZ2

I

where e is the charge on an electron, k is Boltzmann's constant, T is absolute temperature, and c and z are the concentration and charge, respectively, of the ith ion. The value of 1I/K, technically the distance from the shear boundary where the potential has reached 1/exp of the t potential, was about 30 A (ca. 3 nm) for iron-grown T. ferrooxidans in sulfate at pH 2.0. Calculations of charge density in the diffuse layer by integrating curves such as those shown in Fig. 1C gave an excess of 1.0 x 10-12 mol of protons in a column of solution of 1 cm2 cross section and a deficiency of 0.6 x 10-12 mol of anions for a t potential of -7 mV. There was, correspondingly, a compensating negative surface charge of 1.6 X 10-12 mol of electric charge per cm2. Similar diffuse layers of even greater charge density existed around particulate pyrite and sulfur under the same conditions. The initial long-range force between T. ferrooxidans and its insoluble substrates in the absence of iron would thus be repulsive. The addition of Fe(III) to the Stern layer neutralized the net charge on the surface of T. ferrooxidans and removed any double-layer repulsive barrier to the cell's interaction with negatively (or positively) charged particles. High concentrations of iron (7 to 200 mM) are typical of acid mine drainage and mining environments (10, 32). The net charge on T. ferrooxidans under physiological conditions is thus neutral or slightly positive. Indeed, the t potential before washing of bacteria harvested from pyrite or oxidized soluble iron was zero or slightly positive. Similarly, when T. ferrooxidans was grown on elemental sulfur, the structure of the bacterium was altered so as to impart a net charge of about zero on the surface. T. ferrooxidans thus appears to regulate the net charge on its surface to minimize double-layer repulsive forces with its particulate, charged substrates. The binding of T. ferrooxidans with pyrite and sulfur was observed instrumentally. Since the immediate output of these Doppler velocimetry experiments is relative, not absolute, light-scattering intensities, the binding results must be interpreted qualitatively. That is, one cannot use this method to

quantitatively characterize the binding of T. ferrooxidans to its particulate substrates in terms of dissociation constants and binding stoichiometries. One can only conclude that binding is evident. Other investigators have studied the sorption between bacterial cells and clay particles by changes in particle size and distribution using an electrical impedance method commonly employed in particle characterization (25, 26). It is anticipated that these and other physical measurements used to characterize colloidal systems may be exploited to quantify the physicochemical characteristics of the binding of T. ferrooxidans and related organisms to minerals. Once quantitative assays for the binding of bacteria to mineral surfaces have been developed, fundamental questions concerning the interaction can be addressed. ACKNOWLEDGMENTS We thank Stephen McGinness for preliminary experiments on the DELSA 440. This research was supported by grants DE-FG05-85ER13339 and DE-FG05-92ER20087 from the United States Department of Energy. REFERENCES 1. Bennet, J. C., and H. Tributsch. 1978. Bacterial leaching patterns on pyrite crystal surfaces. J. Bacteriol. 134:310-317. 2. Berry, V. K., and L. E. Murr. 1978. Direct observations of bacteria and quantitative studies of their catalytic role in the leaching of low-grade, copper-bearing waste, p. 103-136. In L. E. Murr, A. Torma, and J. A. Brierley (ed.), Metallurgical applications of bacterial leaching and related microbiological phenomena. Academic Press, New York. 3. Blake, R. C., II, G. T. Howard, and S. McGinness. Enhanced yields of iron-oxidizing bacteria by in situ electrochemical reduction of soluble iron in the growth medium. AppI. Environ. Microbiol. 60:2704-2710. 4. Clark, J. M., Jr., and R. L. Switzer. 1977. Transport studies with bacterial membrane vesicle preparations, p. 195-198. In Experimental biochemistry, 2nd ed. W. H. Freeman and Co., New York. 5. Collins, Y. E., and G. Stotzky. 1992. Heavy metals alter the electrokinetic properties of bacteria, yeasts, and clay minerals.

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