Vol. 58, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, p. 1592-16000099-2240/92/051592-09$02.00/0Copyright X) 1992, American Society for Microbiology

Heavy Metals Alter the Electrokinetic Properties ofBacteria, Yeasts, and Clay Minerals

Y. E. COLLINS AND G. STOTZKY*

Laboratory of Microbial Ecology, Department of Biology,New York University, New York New York 10003

Received 24 June 1991/Accepted 11 November 1991

The electrokinetic patterns of four bacterial species (Bacillus subtilis, BaciUlus megaterium, Pseudomonasaeruginosa, and Agrobacterium radiobacter), two yeasts (Saccharomyces cerevisiae and Candida albicans), andtwo clay minerals (montmorillonite and kaolinite) in the presence of the chloride salts of the heavy metals, Cd,Cr, Cu, Hg, Ni, Pb, and Zn, and of Na and Mg were determined by microelectrophoresis. The cells andkaolinite were net negatively charged at pH values above their isoelectric points (pI) in the presence of Na, Mg,Hg, and Pb at an ionic strength (p,) of 3 x 10-i; montmorillonite has no pI and was net negatively charged atall pH values in the presence of these metals. However, the charge of some bacteria, S. cerevisiae, and kaolinitechanged to a net positive charge (charge reversal) in the presence of Cd, Cr, Cu, Ni, and Zn at pH values above5.0 (the pH at which charge reversal occurred differed with the metal) and then, at higher pH values, againbecame negative. The charge of the bacteria and S. cerevisiae also reversed in solutions of Cu and Ni with a p,of >3 x 10-4, whereas there was no reversal in solutions with a ,u of <3 x 10-4. The clays became netpositively charged when the ,u of Cu was >3 x 1O-4 and that of Ni was >1.5 x 1O-4. The charge of the cellsand clays also reversed in solutions containing both Mg and Ni or both Cu and Ni (except montmorillonite) butnot in solutions containing both Mg and Cu (except kaolinite) (p. = 3 x 10-4). The pIs of the cells in thepresence of the heavy metals were at either higher or lower pH values than in the presence of Na and Mg.Exposure of the cells to the various metals at pH values from 2 to 9 for the short times (ca. 10 min) requiredto measure the electrophoretic mobility did not affect their viability. The specific adsorption on the cells andclays of the hydrolyzed species of some of the heavy metals that formed at higher pH values was probablyresponsible for the charge reversal. These results suggest that the toxicity of some heavy metals tomicroorganisms varies with pH because the hydrolyzed speciation forms of these metals, which occur at higherpH values, bind on the cell surface and alter the net charge of the cell. This change in charge could affectvarious physiological functions of the cell, as well as its interactions with other cells and inanimate particulatesin the environment.

Metals are introduced into the environment from varioussources, e.g., from industrial processes and during themining and refining of metal ores. The rapid expansion ofindustry and increases in domestic activities in the pastcentury have caused a concomitant increase in the quantitiesof metals that are being released to the environment. Thenatural recycling of some metals that generally occurs inbiogeochemical cycles has been disrupted as a result of thelarge quantities of metals and pollutants that are currentlyentering the environment from various sources (5, 6, 10, 14,44).

Metals can be classified according to the principle of"hard" and "soft" acids and bases (36) or as class A, classB, and borderline ions (35). Class A cations (hard acids)preferentially bind to oxygen-containing ligands rather thanto nitrogen- and sulfur-containing ligands, whereas the se-quence for class B cations (soft acids) is sulfur-containing,nitrogen-containing, and then oxygen-containing ligands.Borderline ions have both class A and class B properties anda high affinity for both oxygen- and nitrogen-containingligands, and they can also bind to sulfur-containing ligands,especially to sulfhydryl (-SH) groups. Hard acids usuallybind to hard bases, and soft acids usually bind to soft bases.Some metals that are classified as class A (e.g., Ca, K,

Mg, and Na) are essential for microorganisms. For example,

* Corresponding author.

Mg is required for the assembly and stability of the plasmamembrane of bacteria (16), for the integrity of the outermembrane of the cell wall of gram-negative bacteria (23), andfor the stabilization of intracellular structures, especiallyRNA and DNA, and macromolecules involved in the pro-duction and use ofATP (35). Some metals (e.g., Cu, Fe, Mn,and Zn) that are required in trace concentrations (micronu-trients) are classified as borderline ions. Some metals (e.g.,Hg and Pb) that are considered to be pollutants, as they arenot necessary for biological functions and are toxic, areclassified as class B.The chemical reactivity of a heavy metal, as measured by

the physicochemical parameter of softness, correlates wellwith the metal's lethal dose that kills 50% of a population (1,4, 22, 37, 46, 47). Jones and Vaughn (28) suggested that thehard and soft acid and base theory can be used to correlatemetal ion toxicity and the relative effectiveness of therapeu-tic chelating agents with hardness and softness.The biochemical and physiological mechanisms whereby

metals exert their effects on microorganisms have beenreviewed (2, 5, 7, 15, 18, 27, 45). The toxicity of heavy metalpollutants to microbes is affected by biotic and abioticenvironmental factors (8, 9, 14, 43, 44), as are their mutage-nicity and clastogenicity (3). Microbes can also be used toassay the toxicity and mutagenicity of heavy metals (10).The surface of a cell has an important role in the relation-

ship between the cell and its environment, as the surface is

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HEAVY METALS AND ELECTROKINETIC PROPERTIES 1593

0.3 mM NoCI A Candida albicons= Agrobacterium radiobactera Pseudomonos coruginosa* Bacillus megaterlumo BacIllus subtilisIUSaccharomyces cerevisio

0 1 2 3 4 5 6 7 8 9 10

pHFIG. 1. Influence of NaCl, MgCl2, HgCl2, and PbCl2 (pu = 3 x 10-4) on the EPMs of four species of bacteria and two species of yeasts:

x standard error of the mean, which is within the dimensions of the symbols. 0 = isoelectric point; + indicates movement towards thecathode; - indicates movement towards the anode.

in direct contact with the ambient environment of the cell,and both essential (i.e., nontoxic) and nonessential (i.e.,toxic) metal ions are transported across the surface into thecell. When heavy metals are deposited into an environment,they may bind on the surface of microorganisms, which isprobably the initial step in the uptake and concentration ofthe metals by the microbes and in the toxic effects of thesemetals (12). The cell surface is important in microbialecology, in the adhesion of microbes on surfaces, and ininteractions between microorganisms and clay minerals andother particulates (34, 42).

Studies of the electrophoretic mobility (EPM) of microor-ganisms have provided information on the effects of physi-cochemical characteristics of the environment (e.g., pH andions) on the cell surface, on the relationships betweenmicroorganisms and their environment (e.g., adhesion), andon how changes in the cell (e.g., acquisition of antibioticresistance) can affect the surface properties of the cell (24,25, 38, 41, 42). Metal ions have been shown to affect the

electrokinetic properties of cells of mammals and amphibi-ans and of inanimate particulates, such as clay minerals (29,30, 39, 40, 42).

This study examined the changes in the electrokineticpatterns of bacteria, yeasts, and clay minerals in the pres-ence of some heavy metal ions at different pH values, todetermine whether changes in the surface charge of the cellswere related to the toxicity of these ions.

MATERUILS AND METHODS

Organisms. The bacteria (Bacillus subtilis, Bacillus mega-terium, Pseudomonas aeruginosa, and Agrobacterium ra-

diobacter) and the yeasts (Saccharomyces cerevisiae andCandida albicans) were maintained at 4°C on slants ofnutrient agar (Difco) and Sabouraud dextrose agar (Difco),respectively. They were grown in shaken broth cultures(nutrient broth for the bacteria and Sabouraud dextrosebroth for the yeasts): 100 ml of broth was inoculated with 0.1

+3.0

+2.0 _

+1.0.

0

-1.0o-

-20 _

-3D0

O mM MgCl2

O Agrobacterium radiobactera Pseudomonas acrugonos5a Bacillus meogateriumO Bacillus subtilisU Saceharomyces cer*visiatA Candido albicans

E0

0

a)0

m

Co

0

U

FLLr00H0

wF-L)

-4.0

+3.0

+2.0 -

+1.01

a Candido albicons* Sacchoromyces cerOvisioeaAgrobacterlum radloboctera Pseudomonas aeruglnosa. Bacillus megaterlum

. Bacillus subtills

-1.0 F

-2.01

-4.0

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1594 COLLINS AND STOTZKY

-a301

0.1 mM CuCI2

= Agrobacterlum radiobocter

A Pseudomonas aeruginosoA Condido albicans

0 2 3

Bacillus megaterlum. Bacillus subtilis

* Socchoromvceg cerevlsiae4 5 6 7 8 9 10

Agrobocterium radlobacter

£ Pseudomonas aeruglnosa* SaccharomyS, cerevisioe

0 2 3 4 5

. Bacillus megaterium

. Bacillus subtills

£ Condido albicans

6 7 8 9 10

pHFIG. 2. Influence of ZnCl2, NiC12, CuCl2, and CdCl2 (pL = 3 x 10-') on the EPMs of four species of bacteria and two species of yeasts.

(See legend to Fig. 1 for details.)

ml of an overnight (18-h) broth culture (ca. 2 x 107 cellsml-') and incubated for 18 h with shaking (100 rpm) at either25°C (A. radiobacter and the yeasts) or 37°C. For themeasurement of EPM, 2 ml of the culture (ca. 2 x 108 cellsml-') was centrifuged (12,000 x g for 10 min), washed (onetime with 2 ml of quadruple-glass-distilled water), and resus-pended in 2 ml of the chloride salt of each heavy metal, Na,or Mg in quadruple-glass-distilled water at an ionic strength(p.) of 3 x 10-' and at the desired pH. The effects of werestudied by resuspending the washed cells in 2 ml of thechloride salts of Cu or Ni at a of 3 x 10-5, 1.5 x 10-4, or3 x 10-'. The effects of combinations of two metals (Cu andNi, Mg and Cu, and Mg and Ni) were studied, because heavymetals usually exist in combinations and because Mg ispresent in all microbial habitats, by resuspending the washedcells in 2 ml of a solution containing equal amounts of eachmetal to yield a total p. of 3 x 10-4. The pH of the solutionswas adjusted with 1 N HCl or 1 N NaOH from pH 1.5 to 9.5immediately before use. All reagents were of analyticalgrade.Homoionic clays. Homoionic clays (i.e., clay minerals with

only one type of cation saturating the exchange complex)were prepared by washing the 2-p.m fraction three times withthe appropriate chloride salt and then with distilled wateruntil the supernatants produced no precipitate with AgNO3(21).EPM measurements. EPM was determined with a Zeta-

Meter apparatus (Zeta-Meter, Inc., Long Island City, N.Y.).The suspension, consisting of cells or clay particles and thesuspending solution, was placed in the electrophoresis cell(10 cm long; 4.4-cm inside diameter), and a molybdenumrod-type anode and a platinum-iridium strip-type cathodewere inserted into the opposite ends of the cell and con-nected to the Zeta-Meter power unit. The EPM was deter-mined by timing, with a stereoscopic microscope, the move-ment of cells or clays over a distance of 160 p.m with a directcurrent of known voltage. The reliability of the instrumentwas periodically verified by calibration against a standardtest colloid (Min-U-Sil [Zeta-Meter, Inc.], a colloidal silica,manufactured by Pennsylvania Glass Sand Corp., with anaverage diameter of 1.1 p.m), which maintained a constant

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+3D0

+ 20 F

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-40

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HEAVY METALS AND ELECTROKINETIC PROPERTIES

+301

+ 2.0 [

E

a -3.0 suooe euioa~

- 1.0

0

ECandida albicans

a Bacillus megateriumo 0 Bacillus subtilis

*30 Pseudomonas aeruginlosa0 Agrobacterium radiobacter

w

-4.0 _ Sacchoromyces cerevisioe

0 ~~~23 4 5 6 7 8 9 10

pH

FIG. 3. Influence of CrCl3 (p. = 3 x 10-4) on the EPMs of four species of bacteria and two species of yeasts. (See legend to Fig. 1 fordetails.)

zeta (4) potential of -29 + 1 or 2 mV over a wide range ofconcentrations when suspended in distilled water.The EPM of at least 20 cells or clay particles was deter-

mined in each experimental solution, and the mean + thestandard error of the mean was calculated. The temperatureof the sample was measured after each EPM measurement,and the EPM was corrected to a standard temperature of25°C. The rate of movement of the cells or clays was timedwith an Elapsed Timer (model 750C; Royson EngineeringCo., Hatboro, Pa.).The absolute EPM was determined by the formula EPM =

(160 p.m/t) x (10 cm/I), where EPM is the electrophoreticmobility in micrometers second` volt-' centimeter-'; t isthe time, in seconds, to traverse 160 p.m; V is the appliedvoltage; and 10 cm is the length of the electrophoresis cell.

Survival studies. To determine the effects of the combina-tions of heavy metals and pH on the survival of the micro-organisms, ca. 105 cells ofA. radiobacter and B. subtilis, asrepresentatives of gram-negative and gram-positive organ-isms, ml-' were shaken at 25 and 37°C, respectively, in250-ml Erlenmeyer flasks at 100 rpm in 100 ml of filter-sterilized (0.45-p.m pore size; Millipore Corp., Bedford,Mass.) saline containing 1 x 10-' M NiCl2 or MgCl2 or 3 x10-4 M NaCl (the last two solutions were controls), adjustedto different pH values with 1 N HCl or 1 N NaOH. Aliquotsfrom three replicate flasks of each metal-pH combinationwere serially diluted and spread plated in duplicate onnutrient agar after 0, 1, 2, and 6 h of exposure. Eachorganism was evaluated at least twice.

RESULTSEffects of the metals on EPM. The EPMs of the four species

of bacteria and two species of yeasts in the presence of Na,Mg, Hg, and Pb, at a p. of 3 x 10-', are shown in Fig. 1. Allorganisms were net negatively charged at pH values abovetheir isoelectric points (pIs) (when present) in the presenceof these metals.

In the presence of Zn, the bacteria and S. cerevisiaeremained net negatively charged at pH values between theirpIs and ca. pH 7.5 (Fig. 2). At pH values between ca. 7.5 and8.5, the cells became net positively charged, i.e., there wasa reversal of charge on the cells, and at pH values above ca.8.5, the cells again became net negatively charged. Therewas no charge reversal of C. albicans, which remained netnegatively charged at pH values above its pI.

In the presence of Ni, the cells were net negativelycharged at pH values between their pIs and ca. pH 7.0 (Fig.2). At pH values above 7.0, all cells became net positivelycharged, and most remained positively charged to pH valuesabove 9.0.

In the presence of Cu, the cells remained net negativelycharged at pH values between their pls and ca. pH 6.0 (Fig.2). At pH values above 6.0, the bacteria and S. cerevisiaebecame net positively charged and then again net negativelycharged at pH values above ca. 8.0. Charge reversal in thepresence of Cu occurred over a broader pH range (6.0 to 9.5)than in the presence of Zn and Ni. There was no chargereversal of C. albicans above its pI.

In the presence of Cd, the cells were net negativelycharged at pH values between their pIs and ca. pH 8.0 (Fig.2). At pH 8.0 and above, the cells assumed and maintained anet positive charge, except P. aeruginosa, S. cerevisiae, andC. albicans, which showed no reversal of charge in thepresence of Cd.The effects of Cr on the EPMs of the bacteria and yeasts

are shown in Fig. 3. Charge reversal of B. subtilis, A.radiobacter, and S. cerevisiae occurred between ca. pH 5.0and 8.0, but there was no charge reversal of B. megaterium,P. aeruginosa, and C. albicans.

Charge reversal of montmorillonite and kaolinite (Fig. 4)occurred in the presence of Cu and Ni but not in thepresence of Zn, Na, and Mg. The pH values at whichreversal of charge occurred were similar to those observedwith the bacteria and yeasts.

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1596 COLLINS AND STOTZKY

.+ 3.0-MontmorilloniteKaint

EC4

+ 2.0

+ 1.0-

C)~~~~~~~~~~~~~~c

0

E

-1.0

-2.0-

Na

3 4 5 6 7 8 9 10 3 4 5 6 7 8 9 10

pH

FIG. 4. EPMs of montmorillonite and kaolinite in the presence of the chloride salts of some heavy metals, Mg, and Na (p. = 3 x10)

(See legend to Fig. 1 for details).

Effects of the metals on the pL. The pIs of the bacteria andyeasts in the presence of the heavy metals are summarized inTable 1. In the presence of Cd, Cr, Cu, Mg, and Ni, the pI ofB. subtilis was shifted to pH values that were higher than inthe presence of Na. The pI of B. megaterium was shifted tohigher pH values in the presence of Cr and Cu, but it waslower in the presence of Cd, Hg, Mg, Ni, Pb, and Zn than inthe presence of Na. The pl of P. aeruginosa was lower thanin Na in the presence of Cd, Cu, Mg, Ni, and Pb, but it wasshifted to higher pH values in the presence of Cr, Hg, andZn. The pI of A. radiobacter was lower than in Na in thepresence of Cd, Cu, Ni, Pb, and Zn, but it was shifted tohigher pH values in the presence of Cr, Hg, and Mg. The pIof C. albicans was equal to that in Na with Cu, Mg, Pb, andZn, but it was lower in the presence of Cd and Hg and higherin the presence of Cr and Ni. No pI of S. cerevisiae wasobserved in the presence of any of the metals.

Effects of combinations of metals on EPM. The effect of acombination of Mg plus Cu, at the same total p. (3 x 10-') asthat of Mg or Cu alone, on the EPMs of bacteria and yeastsis shown in Fig. 5. Only B. subtilis in the presence of Mg plusCu showed a reversal of charge, which occurred over a

narrower pH range (8.1 to 8.7) than in Cu alone (6.2 to 9.3)(Fig. 2).Charge reversal of all species of bacteria and yeasts

occurred in the presence of Cu plus Ni (Fig. 5). However,charge reversal of C. albicans did not occur in the presenceof Cu (Fig. 2).Charge reversal of all species of bacteria and yeasts

occurred in the presence of Mg plus Ni (pH 6.0 to 9.5) (Fig.5). Charge reversal also occurred in the presence of Ni alone(Fig. 2) but not in the presence of Mg alone (Fig. 1).Charge reversal of kaolinite occurred in the presence of

Cu plus Ni, Mg plus Ni, and Mg plus Cu. However, chargereversal of montmorillonite occurred only in the presence ofMg plus Ni (Fig. 6).

Effects of , on EPM. Increasing the of Cu or Ni from 3X 10-4 to 3 x 10-3 also caused charge reversal in B. subtilis,A. radiobacter, S. cerevisiae, and the clay minerals (Tables2 and 3). However, decreasing the of Cu or Ni to 1.5 x10-4 and 3 x 10-5 did not cause charge reversal in B.subtilis, A. radiobacter, and S. cerevisiae. Charge reversalof kaolinite occurred in the presence of Cu or Ni at a of 1.5X 10-4 and above but not at a of 3 x 10-5. With

TABLE 1. Isoelectric points of four species of bacteria and two species of yeasts in the presence of the chloride salts of the heavymetals, magnesium, and sodium (p. = 3 x 10-4)

pla in presence of:Organism

NaCI MgCl2 HgC12 CdCl2 CuCI2 NiCI2 ZnCI2 PbCI2 CrCl3B. subtilis <1.0 3.6 <1.6 1.5 1.3 1.9 <1.2 <1.8 2.7B. megatenum 2.4 1.9 2.2 2.0 2.5 2.3 2.0 2.0 3.1P. aeruginosa 2.7 2.0 3.3 1.7 2.3 2.5 3.0 2.5 3.9A. radiobacter 3.2 3.5 3.6 2.0 3.1 2.6 3.1 3.0 4.0S. cerevisiae <2.3 <3.4 <2.9 <1.9 <2.1 <2.0 <3.0 <2.2 <1.9C. albicans 2.3 2.3 2.0 2.2 2.3 2.8 2.3 2.3 3.6

a <, isoelectric point was not observed at indicated pH, which was the lowest pH measured.

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HEAVY METALS AND ELECTROKINETIC PROPERTIES 1597

ITI montmorillonite, charge reversal did not occur in the pres-<y; ,71 ELECTROPHORETIC MOBILITY (um sec volt-' cm-') ence of Cu at a pI below 3 x 10-4 but did occur in Ni at a p.____________*r > X v of 1.5 x10

The pH at which charge reversal occurred was affected bythe p, of the suspension. Charge reversal generally occurredat lower pH values when the p. of Ni and Cu was increasedfrom 3 x 10-4 to 3 x 10-3 (Tables 2 and 3).

Effects of metals and pH on viability. In the presence ofo w - /10' M NiCl2, the viability of B. subtilis and A. radiobacter

decreased by 0.5 to 1 order of magnitude after 6 h at pH 3 to9. Decreases after 1 and 2 h were considerably less. At pH 2,no viable cells were detected after 1 h, the shortest interval

O evaluated. Similar reductions in viability at the different pHKI+ values occurred in the presence of 1 x i0 M MgCl2 and 3r0sjD

O O xXl10-4 M NaCl. Inasmuch as the cells were added to thec-|1Alvarious metal solutions at the different pH values just beforen<_3t 21/ l ~~~~~~measurement of the EPM, which required at most 10 min, itis doubtful that the differences in EPM observed were the

cn 00 3 - |\ _result of cell death and lysis, especially in the pH range inwhich charge reversal occurred and in which there were nosignificant differences in viability after 1 h of exposure.

______________0_________lDISCUSSION0.

3: W r- l The net surface charge of some bacteria, yeasts, and clayminerals reversed from negative to positive and then back tonegative in the presence of some heavy metals at elevatedpH values. The ability of a metal to cause charge reversalappeared to be related to the speciation of the metal thatoccurs at different pH values and to the ability of somespeciation forms to be specifically adsorbed on the cellsurface. The bioavailability and toxicity of a heavy metal isdependent on various factors, such as the pH; the type andxconcentration of adsorbents, cations, and anions; and the

laD o n ySs tspeciation form of the metal (6, 14, 36). As the pH of ao > ,ratsolution increases, a hydrated metal ion will hydrolyzeaccording to the following general equation (6, 7, 19, 31, 43):

5[0H [OH-] [OH-] [OH-||MOMOH+ [M(OH)2 M(OH)3- I2[2 M(OH)42-

Charge reversal of the cells, as well as of the clays, usuallyoccurred in the pH range in which the concentration of the

0 W ~ ,divalent cation (M2+) was decreasing and the concentrationof the monovalent hydroxylated cation (MOH+) was in-

g___________________ creasing.The extent of hydrolysis is dependent on the metal (11),

o - * * * and the adsorbability of different speciation forms of thesame metal [e.g., Cu2 (aq) compared with CuOH+] varieswidely. There is a critical pH range, usually of less than 1 pHunit, for each metal wherein the amount of metal adsorbedincreases from almost 0 to 100%; e.g., the adsorption of Feand Pb on quartz increased abruptly when hydrolysis oc-

g | 1< curred (17), as did the adsoTtion of Co on silica (26). TheKt̂[g 1\l Ftl 1 3 free hydrated ion [e.g., Cu +(aq)J does not cause charge

reversal. However, if the adsorbent has a net negative+ charge and a negative surface potential, the sign of thez° D c ° / N ^ | potential is reversed when the metal ion hydrolyzes and

metal hydroxides are precipitated.Charge reversal occurred with Cd, Cr, Cu, Ni, and Zn,

which are classified as borderline metals by Nieboer andRichardson (35). Charge reversal occurred in the pH rangewherein the metal changes from the divalent to the mono-valent hydroxylated cation (with Cu, from pH 5.5 to 8.0;with Zn, from pH 6.0 to 8.0; with Ni, from pH 8.0 to 10.0;and with Cd, from pH 8.0 to 10.0 [11, 19]). Charge reversal

_ I l in the presence of Cr occurred in the pH range (5 to 8)

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1598 COLLINS AND STOTZKY

+3.00

o +2.0

04)El + 1.0S

C,.

c o_O -1.0

H -2.0

0x -3.00

U -4.0W_W

5 6 7 8 9 5 6 7 8 9

pH

FIG. 6. Influence of mixtures of Mg plus Ni, Mg plus Cu, and Cu plus Ni (total = 3 x 10-') on the EPMs of montmorillonite andkaolinite. (See legend to Fig. 1 for details.)

wherein Cr changes from the divalent hydroxylated cation(CrOH2") to the monovalent hydroxylated cation [Cr(OH)2+] (20).

In aqueous solutions, Mg and Na hydrolyze only at highpH values (>9) and exist as the free hydrated ion at the pHvalues used in these studies (11); therefore, Mg and Na didnot cause charge reversal. Hg and Pb also did not causecharge reversal, as Hg and Pb form negatively charged anduncharged chlorinated species in the presence of C1- at thepH values used in these studies (19). These chloride speciesare not adsorbed on the cell wall and therefore do not causecharge reversal. In contrast, Cd, Cr, Cu, Ni, and Zn have alow affinity for Cl- in aqueous solutions and preferentiallyform hydroxylated species that are specifically adsorbed onthe cell wall.At higher pH values, the cations become neutrally

[M(OH)2] or negatively [M(OH)2- and M(OH)22-] charged,which accounts for the reversal of the cells and clay mineralsback to a net negative charge. These neutral or negatively

TABLE 2. pH (above the isoelectric points of the cells) at whichthe net negative charge of some bacteria, yeasts, and clay

minerals reversed to a net positive chargein the presence of CuCl2

Organism pH in presence of CuCl2 concn (M) ofa:or clay 1 x 10-3 1X 10-4 5 x 10-5

B. subtilis 5.9 6.2A. radiobacter 5.7 6.9S. cerevisiae 5.7 6.8Montmorillonite 6.4 6.8Kaolinite 5.4 6.3 6.2

a _ no charge reversal occurred. For 1 x 10-3 M, 3 x 10-3; for 1 x0-4 M, p. = 3 x 10-4; for 5 x 10-5 M, ,u = 1.5 x 10-4. In the presence of10-5 M CuCl2 (p = 3 x 10-5), no charge reversal occurred.

charged species are not adsorbed on the net negativelycharged cells and clays.Although reversal of charge of the bacteria and S. cerevi-

siae occurred in the presence of Cu at high pH values,charge reversal did not occur in the presence of Mg plus Cu(except with B. subtilis). Charge reversal of kaolinite oc-curred at high pH values in the presence of both Cu and Mgplus Cu but not of Mg alone. However, no charge reversal ofC. albicans occurred in the presence of Cu, Mg, or Mg plusCu. Inasmuch as charge reversal of the cells did not occur inthe presence of Mg plus Cu, the ability of the hydrolysisproduct of Cu to bind on the cells appeared to be affected bythe presence of Mg.Charge reversal of all species of bacteria, yeasts, and clay

minerals occurred in the presence of both Ni and Mg plus Nibut not of Mg alone. Apparently, the ability of the hydrolysisproduct of Ni to bind on the cells and clay minerals was notaffected by the presence of Mg. This suggests either that thehydrolysis products of Ni and Cu bind on different sites in

TABLE 3. pH (above the isoelectric point of the cells) at whichthe net negative charge of some bacteria, yeasts, and clay

minerals reversed to a net positive chargein the presence of NiCI2

Organism pH in presence of NiCl2 concn (M) of:or clay 1 x 10-3 1 X 10-4 5 X 10-5

B. subtilis 6.1 8.0A. radiobacter 7.2 7.1S. cerevisiae 5.8 7.8Montmorillonite 8.2 8.2 6.5Kaolinite 6.7 7.4 6.5

, no charge reversal occurred. No reversal occurred in the presence of10-5 M NiCl2. p. values for each concentration are as listed in Table 2,footnote a.

Kaolinite

'fMontmorillonite

* Cu + Nio Mg + Ni* Mg + Cu

I

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HEAVY METALS AND ELECTROKINETIC PROPERTIES 1599

the cell wall or that the relative affinities of these productsand of Mg for the cells and clays differ.Charge reversal of the bacteria and S. cerevisiae occurred

in the presence of Cu, Ni, and Cu plus Ni, further indicatingthat Cu and Ni bind on different sites in the cell wall. Chargereversal of kaolinite occurred in the presence of Cu, Ni, andCu plus Ni. However, there was no charge reversal ofmontmorillonite in the presence of Cu plus Ni, althoughcharge reversal of montmorillonite occurred in the presence

of Cu or Ni. These differences may have been the result ofdifferences in the structure and composition of these clayminerals, as kaolinite is a 1:1 (Si-Al) nonexpanding clay witha low cation-exchange capacity and surface area, whereasmontmorillonite is a 2:1 expanding clay with a high cation-exchange capacity and surface area (42).The results of these studies indicated that the adsorption

of hydrolyzable metal ions on microbial cells may be similarto the behavior of these metal ions at mineral oxide surfaces(e.g., the adsorption of Fe, Ni, and Co on quartz [32, 33])and that theories of the mechanisms of adsorption of metalson inorganic colloidal particles can be extended to cellularsurfaces. Charge reversal of the cells and clays occurred as

the result of the specific adsorption of the first hydrolysisproducts of Cd, Cr, Cu, Ni, and Zn on the cells or clays,indicating close binding of these metals on the surface (i.e.,in the Stern layer).

Different hydroxylated forms of the same metal havedifferent toxicities to microbes (6, 7, 43). Inasmuch as thespeciation form of a metal in an aqueous solution affects itsadsorption on a charged surface, the effects of pH on thetoxicity of metals probably result from the effects of pH on

speciation and the relative ability of these different specia-tion forms to bind on the cell surface. The close binding ofcertain metals on sites in the cell wall may lead to perturba-tions of the cell surface, disrupt normal functioning of thecell, and cause toxic effects. Inasmuch as different metalsmay bind on different sites on the cell (13), strong specificbinding could, in some cases, enhance toxicity at a particularpH, e.g., as a result of the replacement of essential metals(e.g., Mg) by toxic metals (e.g., Ni), and decrease toxicity inother cases.

Although there were some differences in the electrokineticpatterns among and between the species of bacteria, yeasts,and clays, the differences between the different metals were

more pronounced. Consequently, the speciation form of themetal, rather than the type of cell or particle, appeared to bethe important factor in determining adsorption and, hence,changes in charge.

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