The binding of heavy metals to algal surfaces

War. Res. Vol. 22, No. 7, pp. 917-926, 1988 Printed in Great Britain.All rights reserved

0043-1354/88 $3.00 + 0.00

Copyright © 1988PergamonPress pie

THE BINDING OF HEAVY METALS TO ALGAL SURFACES HAN-BIN XUE*, WERNER STUMMand LAURA SIGG Institute for Water Resources and Water Pollution Control (EAWAG), Swiss Federal Institute of Technology (ETH), Ziirich, Switzerland

(Received April 1987; accepted m revised form January 1988) Abstract--Biological particles can profoundly influence the distribution of heavy metals in natural waters because the functional groups on the cell surfaces are able to bind metal ions. The interaction of Cu(III) and Cd(II) was evaluated from titration of algal suspensions (Chlamydomonas rheinhardii) (i) at a constant pH with increments of metal ions and (ii) in the presence of the metal ions with increments of acid. A voltammetric methodology was developed to measure (without prior separation of the solid phase) the metal ions in solution in the presence of algae and to assess the binding of metals to the surfaces of algae. The surfaces of algal cells have a high affinity for Cu(II) and Cd(II), even in the presence of 10-3 M Ca2+; their functional group ligands can compete with soluble complex formers typically present in natural waters. The adsorption of metals is readily interpreted in terms of surface complex formation equilibria or--mathematically equivalent--Langmuirtype adsorption equilibria. The "average" conditional equilibrium constant extracted from the experimental data permits a generalization valid for a certain range of metal surface loading and can be used in multi-metal multi-ligand speciation calculations. Understandably, the data cannot be fitted over a large range of free metal ion concentrations or metal loading of the surface because the tendency to form surface complexes decreases with increasing metal loading, because there are a variety of surface ligands, and the metal ions bind first to the surface groups with highest affinity and subsequently to groups with lower affinity. A much better fit of the data is obtained if models are used with more than one adjustable constant such as the constant capacitance or the Fowler Guggenbeim Frumkin model or a two-site Langmuir isotherm. The kinetics of adsorption and uptake of Cu(II) to Chlamydomonas is characterized by the establishment of a relatively fast pseudo-adsorption equilibrium with the surface that is followed by slow diffusion-controlled uptake into the inside of the cell.

Key words--heavy metals, adsorption algae, pH dependence, metal adsorption, aquatic chemistry, biological particles

INTRODUCTION The interaction of heavy metals with particulate materials is important in regulating the concentration of metal ions in water and soil systems. Phytoplankton cells exhibit in these systems relatively large surface areas containing various functional groups, such as carboxylic, amino, thio, hydroxo and hydroxy-carboxylic, that can interact coordinatively with heavy metal ions. The extent of surface binding on biological cell surfaces may also be generalized by mass law equations, e.g. ---SH+Cu2+~-=SCu ++H+;

gscu

(1)

where - S designates a (deprotonated) bidentate surface group or chelating site, such as

#0 R/c "OH " NH2

,~0 R"c'OH "r..~,O - ~' OH

¢0 R..~HOH

H/H R~"C-SH NH2

(2) *Visiting scholar from the Chinese Academy of Sciences, Research Center for Leo-Environmental Sciences, Academia Sinica, P.O. Box 934, Beijing, People's Republic of China.

In lakes, it has been shown (Sigg, 1985, 1987) that biological surfaces (algae, biological debris and biologically coated surfaces) of settling materials play a dominating role in binding heavy metals, thereby regulating the residual concentrations of dissolved metals. In oceans, where most particulate material produced in situ is of biological origin, the conveyor belt of continuously settling biological particles and their partial dissolution in the deeper parts of the ocean influence profoundly the distribution of most of the trace elements in seawater and the composition of any individual water sample (Whitfield and Turner, 1987). The objectives of the present work are to examine the binding of heavy metals to the surface of living algae, specifically to develop a model for the binding of metals based on the concept of surface complex formation. Special efforts were made to develop a careful methodology to assess the binding of metals to surfaces of algae and their exudates; voltammetric measurements (differential pulse anodic stripping voltammetry) were used to measure directly (without prior separaiton of the solid phase) the metal ions in solution in the presence of algae (Gon~alves et aL, 1986). This paper is organized along the following lines.

917

918

HAN-BIN Xu~ et al.

First we review the models of surface complex formation that are applicable to data interpretation and that permit a generalization on the affinity of algal surfaces as a function of pH and solution variables; second, we describe the experimental methodology that was used after careful evaluation, to measure soluble metal ions in the presence of algae as a function of pH and solution variables. It is illustrated that the voltammetric assessment of metal ions (differential pulse anodic stripping voltammetry) permits to draw conclusions on the extent of metal ion binding to the surface and to exudates; third, the experimental data are interpreted in terms of the simple surface complex formation models available. Finally some consideration is given to the kinetics of adsorption. It is shown that a fast surface binding is followed by a slow and probably diffusion controlled metal ion uptake.

MODELS OF SURFACE COMPLEX FORMATION TO GENERALIZE BINDING OF METALS TO ALGAL SURFACT~

Our model has been patterned according to 'the complex formation of metal ions by hydrous oxide surfaces. The customary way to represent reactions at these surfaces has been through equilibria of the type (Stumm and Morgan, 1981): - S O H + M e 2+~--=SOMe + + H + ;

*K]

2-SOH+Me2+~(-SO)2Me+2H+;

*/~.

(3) (4)

The degree of surface protonation depends on the acid-base equilibria = SOH~ ~ -- SOH + H+;

K~,

(5)

-= SOH ~ - SO- + H +; K:2.

(6)

The experimental constants, usually defined for a constant ionic medium reference state, may have to be corrected for electrostatic interaction of the charged surface with the ions. Thus, intrinsic equilibrium constants can be specified for the proton reactions KS,(intr.) = K:, e x p ( - - F T d R T )

(7)

K~:(intr.) = KS2 exp(--bWJRT)

(8)

where ~ is the inner potential of the surface. The intrinsic equilibrium constants are postulated to be independent of the composition of the solid phase. Therefore the surface complex formation constant with metal ions -: SO}I + Me 2+ ~ --SOMe + + H + may also be expressed as the following type with a correction of surface charge: { ~ SOMe +} [H +1 *K](intr.) ffi ~ _ ~ exp(--F~dRT)

(9)

surface potential, ~[V], and surface charge, a[C m-2], are related to specific capacitance (C[F m-2]). In the most simple Helmholtz Model or

the constant capacitance model (Schindler and Stumm, 1987) = ~/c.

(lO)

The algal cell surface contains various functional groups [equations (1) and (2)]; their acid-base properties may be characterized by RH~ ~ R H + H+;

Ka,

R H ~ - R - + H+:

Ka2.

(ll)

The complex formation of metals with the algal surface may be approximated similarly to that with polyfunctional macromolecules: R~Hx + M e 2+~R~Me a - x ~ + x H +;

K~

(12)

where Ri designates the deprotonated surface site. Because the surface groups are nonidentical, it is not possible to determine all complexation constants. However, a general macroscopical adsorption equilibrium involving various adsorption mechanisms will be assumed, giving an average equilibrium constant. RH, + Me 2+ ~- RMe ~2-~ + n H+;

Kh

(13)

In equations (9) and (11)-(13), the charges given to the surface groups are arbitrary; the equations simply reflect the stoichiometry of changes in the charge. In the subsequent equilibrium expressions, "species" charges are omitted; i.e. the equilibrium constant for equation (13) may be defined as Kh =

{RMe} [H+]" { g a , } [ M e 2+]

(14)

where {RMe} and {RH} are, respectively, the concentrations of metal ions and hydrogen ions bound to the surface expressed as mol g-~ (algal dry wt). For any pH we can define equation (14) in terms of a conditional constant, Kh, valid for a given pH: {RMe} Kh = {RH,} [Me 2+]'

(15)

An equilibrium quotient, such as that given in equations (14) or (15), has been shown (Stumm and Morgan, 1981; Sposito, 1983; Buffle, 1984)to depend somewhat on the charge of the surface which in turn depends on the extent of surface binding of metal ions and protons. As given in equation (7)-(9), zF~s is the energy required to bring an ion from the bulk of the solution to a surface site at a potential T,, where z is the charge number of the ion under consideration. Thus intrinsic constants can be defined for equilibria (12)-(14) analogue to those given by equations (7)-(9). For equilibrium (13), we can write

zF logKh --iogKh~oL,.~ RTIn 1-------6Ts.

(16)

Unfortunately, values for ~fs are not accessible experimentally. Equation (16)can be formulated--in

The binding of heavy metals to algal surfaces accordance with the constant capacitance modelm semi-empirically in terms of the surface charge or the surface coverage with metal ions logK h = loggh~,~.~ - ~ {RMe}.

TM0 =

(18a)

Fm~ [Me2+] (Kh)_ l + [Me2+],

or {RMe} =

{RT}[Me2+]

(18b)

• (K[I) -I + [Me 2+] where {RMe} and {RT} correspond to FM. and Fm~, respectively, are the amount of metal ion adsorbed and the maximum value of metal ion adsorption capacity (complexing capacity) [tool g-l] (dry wt). Values for Kh can be readily obtained by using typical Scatchard or Langmuir plots. By comparing values obtained at different surface coverage, {RMe}, an intrinsic constant in accordance with the constant capacitance model, equation (17), can be obtained. Wilson and Kinney (1977) have applied an electrostatic correction to the interaction of a mono-functional humate with metal ions. As the surface complex formation equilibrium expression (15) is the constant capacitance model [equations (16) and (17)] equivalent to the Langmuir isotherm (18a, 18b), so is the Fowler Guggenheim Frumkin expression (Frumkin, 1925). 0 1 - 0 = Bh[Me2+]exp(2a0)

(19a)

or {RMe} Bh = {RH} [Me 2+1 e x p ( - 2aO)

interpret the surface as being composed of two different ligands with two different binding capacities, Fm~,and Fro2and to use a two-site Langmuir isotherm (Stroes-Cascoyne et al., 1986):

(17)

The intrinsic constant and x are obtained from a plot of loggh vs {RMe}. As a first and useful approximation we may treat the surface as if it were uncharged (qJs = 0) and neglect the second term in equations (16) and 07). Kh values obtained this way are a semi-empirical expression for the average effect of metal ion binding to a polyfunctional surface, valid for certain condi"tions of pH and ranges of relative surface coverage, 0 = {RMe}/{RT}. It may be noted that equilibrium (15) can be interpreted also in terms of a (mathematically and conceptually equivalen0 Langmuir isotherm: 0 1 - 0 = Kh[Me2+]

919

(19b)

where a is a constant representative of the interaction energy at the surface. The coefficient a acquires a negative value if the interaction between surface sites is repulsive, i.e. the binding intensity of the metal ion to the cell surface decreases with increasing surface coverage. Another way to interpret the data on the interaction of metal ions with the surface ligands, is to

{RMe} =

/'m, [Me2+] /'m2 [Me2+] (K~)_l + [Me2+] t- (K[)_l + [Me2+].

(20)

There is no simple, fully rational model available to account for the surface complex formation on an algal surface. K h and Bh values are equilibrium quotients capable of expressing the average effect of metal ion binding to a mixture of algal surface ligands and valid to certain generalizations within certain conditions. The fitting of the data does not prove the validity of the model used.

EXPERIMENTAL METHODS The algae used were Chlamydomonas rheinhardii; the strains were isolated from a nearby lake (Greifensee). The nutritive solution was continuously fed to keep a continuous culture, using a standard nutrient medium (Staub, 1961). Under the microscope the algae appear to be unicellular spheres. The diameter ranges from 4.2 to 7/zm. A cell number of 1.35 × 106cellsml-I is suspended in the medium to give a solution of 0.22gl -~ (dry wt). An approximate specific surface area of approx. 0.62 m2g- ~can be estimated from the apparent geometry. The culture medium of algae was centrifuged at 3000 rpm for 15 rain, then washed 4 times with a solution of 0.01 M KNO3. The sample was then kept in 0.01 M KNO3 and stored in the refrigerator until used. The concentration is determined by dry weight after filtering and drying the residue for 2 h. All experiments were carried out in the above ionic medium. When a constant pH of the solution was needed, 5x 10-4M hydroxymethyl aminomethane (Tris) or H3BOa was added as a buffer. The pH was adjusted by adding 0.1 M HNO3 or 0.1 M NaOH to the suspension of algae. The concentration of algae in the suspension was 12.8-17.4mgl -l for the experiments with Cu and 45-90 mgl -* for the experiments with Cd. After ceutrifugation, resuspension and storage in the refrigerator for 24 h, the major fraction of the Chlamydomonas algae is still alive, as checked by microscopic examination; the fraction of motile cells (about 17% in the fresh cell culture) decreased somewhat after the treatment.

Hydrogen ion binding of algae Alkalimetric (acidimetric) titrations (glass electrode) were used to characterize the proton-binding capacity of the algae. This capacity reflects approximately the number of functional groups available at the surface. The titration was carried out in 0.01 M KNO3 with NaOH or HNO3. The titration curve (Fig. I) reflects the sequential binding of protons (and the variations in that portion of the charge which is caused by H+-binding) by the various functional groups of the algal surface. In a simplifiedway, the titration curve could be interpreted as if the surface contained primarily carboxylic acid groups that protolyze around pH 4-5 (comparable with the second acidity constant of phtalic acid PKa~~ 5) and amino groups that undergo proton transfer around pH 8-9.5 (comparable to the pK value of giycine pK,2 ~ 9.5).

Equilibration time, storage time of algae The uptake of heavy metals was found to occur in two steps: first a relatively fast adsorption step (pseudoequilibrium) and second, a relatively slow, probably diffusion-controlled uptake into the inside of the cell. Experiments on the adsorption of metal ions on the surface of

HAN-BIN XUE et al.

920 11

kinds of information can be gained from the voltammetric measurement:

10 With algae

(1) The presence of labile complexes causes a shift in the peak potential which can be expressed quantitatively by the equation according to DeFord and Hume (1951):

9 8

aEp[mV] =

6 5 4 3 2

E p M c 2 + - - Epcotnpte x

59.15 = -log(l + Xfl°rt[OH-]'+ Eft,L,) 2

7

f 0.6

(21)

where EpM~ = peak potential in absence of ligands, Epco~,p,ox= peak potential in presence of ligands L~, 3 = stability constant for complex with OHand ligand L, respectively. 0.2

I I 0 0.2

HN03 odded mL

o 3 4 5 6 7 8 9 ~- j ~ pH I 0.6 1.0 1.4

I 1.S

NaOH added mL

Fig. l. Titration curves of algae with acid or base; pH as a function of acid or base added. 43.5 mg (dry wt) algae suspended in 50 ml of 0.01 M KNO 3 solution were titrated with 0.I M HNO 3 or 0.1 M NaOH. Insert: protons consumed g-* dry wt of algae as a function of pH.

algae were carried out to attain the pseudo-equlibrium with a waiting period of 10 min. Since the extent of metal binding was found to depend also somewhat on the time the stock algal suspensions were stored in the refrigerator (tendency of increase in metal binding capacity with increased storage time), the storage time was kept constant at 24 h.

Polarographic measurements Voltammetric measurements were carried out as differential pulse anodic stripping voltammetry (DPASV) with a hanging mercury drop electrode (Metrohm VA 663). We used an Ag/AgCI reference electrode and a counter electrode of graphite combined with a Metrohm E506 polarecord. The scan rate was 4 mV s -~, the pulse height 20mV, the deposition time for DPASV was 120 s.

(2) The presence of inert complexes (slowly dissociating) or other electroinactive species causes a decrease of the peak current in comparison with a simple, non-complexing medium. In the absence of other ligands, hydroxo species dominate the speciation at pH values above pH 7, e.g. for Cu: [CU]di~so,,ed= [Cu 2+] + [CuOH +] + [Cu(OH)zl. Such hydroxo species should be labile in the voltammetric measurements. Potential shifts corresponding to those calculated for hydrolysis were observed in acid-base titrations of Cu-solutions. In order to avoid the complications of additional complexes, the experiments were conducted in a borate buffer which does not form complexes with Cu. In addition to hydrolysis (change in Ep) a decrease in peak current was observed in neutral and alkaline Cu(II) solutions, even in the absence of algae, thus indicating a loss of dissolved labile Cu from the solution. This loss starts around pH 6 and increases progressively at higher pH

080

0 70

~:E 060

~

Q

METHODOLOGY, VOLTAMMETRIC ASSESSMENT

Distinction between dissolved and particulate species The assessment of metal ion binding to algae makes use of the ability of voltammetric methods to distinguish between dissolved and particulate species without phase separation. This distinction between dissolved and particulate species relies upon the effects of slow mass transfer of particles to the electrode and often of slow dissociation of surface complexes that make the contribution of particulatebound species to the voltammetric response negligible (Whitfield and Turner, 1979). The validity of this approach has been tested in previous work (Gongalves et al., 1985, 1987) in different systems including inorganic particles and bacteria. The voltammetric assessment (without separation of the solid phase) of metal ion binding to biological surfaces is especially valuable because any filtration or centrifugation of algae is fraught with complications and possibilities of artifacts (possibilities for adsorption loss or contamination; changes in the partition between liquid or charged surface phases during the separation step; lysis of algal cells). pH-dependence of the speciation of Cu e+, Cd 2÷ and o f the vohammetric response In order to use voltammetric methods for the measurement of metal ions at different pH, the various effects of pH-changes on the speciation and on the voltammetric response have to be considered carefully. Two different

050

040

" 0 30

E 0 ao

~_ 0 10

O.C

I i .2 .4 .6 .8 I 1.2 ].4 added {Cu](1), residual dissolved [Cu1(2),{3} [pM}

Fig. 2. Titration curves of algae with Cu(II) at pH 7. Concentration of algae 12.8 mg (dry wt) 1-~. Curve 1, labile Cu(II) as a function of added C'u(II); curve 2, free [Cu 2+] as a function of total dissolved residual Cu(II) (corrected for loss); curve 3, labile Cu(II) as a function of dissolved residual Cu. Curve 1 is not parallel to the calibration even at high concentration, but curve 3 is. This indicates that it is necessary to correct added Cu(II) to total dissolved residual Cu(II) in order to evaluate binding. The concentrations of free Cu in curve 2 was obtained by correcting for hydrolysis [equation (27)].

The binding of heavy metals to algal surfaces values. It is most likely due to the formation of multinuclear hydroxo Cu(II) species and to the adsorption of Cu on the glass wall (Gongalves et al., 1987; Mfiller, personal communication). In order to calculate the Cu concentrations bound to algae at different pH values, a correction for these effects has to be made. For this purpose, a calibration of labile Cu is necessary for each of the pH values. The calibration of peak currents vs added Cu is linear at different pH values and can be expressed as: i4 = k 4 [ C u ] ~

(22)

ij = kj[CU],d,~

(23)

where i4, ij are the peak currents at pH 4 and p H j and k4, kj the slope of the calibration at pH 4 and pH j. The total "residual" dissolved Cu is as follows:

= ~ [CU],dd~.

(24)

[CU]T

The labile Cu (unbound) and the Cu bound to algae (CU,d,), respectively, are expressed: i~

(25)

[CuII.b = kj

200-

921

where i~ is the current measured in the presence of algae; CU~d,= [Cu]T-- [CULb. (26) In order to evaluate the binding capacities and stability constants based on Cu 2+, the free Cu ion is calculated from this measured labile Cu: [Cu]lab = [Cu2+](I + flOH[OH-] + fl°S[OH-]2) (27) where flOH= 2 x l06 and flon = 6.3 x l012 are the stability constants of CuOH + and Cu(OH)° (aq.), respectively. The titration curves [addition of Cu(II) to an algal suspension at constant pH] in Fig. 2 show the difference between total residual dissolved Cu and added Cu as well as labile Cu and frec Cu ion at pH 7. Only the curve oflabile Cu as a function of residual dissolved Cu is parallel to the calibration for high concentration of Cu(II). The same method is applied in order to evaluate the total effective Cd; the labile Cd can be taken as free Cd ion, since hydrolysis of Cd occurs only at higher pH values. Figure 3 illustrates an evaluation of Cu and Cd binding to algae [acidimetdc titration of algae in the presence of Cd(II)]. The decrease in the concentration of labile Cu(II) or Cd(II) in the absence of algae at higher pH values is caused by adsorption to the glass wall. From the difference of the measurements made in presence and absence of algae, the percentage of the "residual" Cd(II) bound to the surface of algae can be estimated.

without algae

180-

m

160-

-- ~

RESULTS

Cu (11) m

t40120<: ¢ 100.o. 806040200 pH

80-

{RMe} = Kh {RT} -- Kh {RMe} [Me 2+]

Cd (II)

70-

Experimental data obtained from the titration of algal suspensions at constant p H with increments of metal ions (as illustrated by Fig. 2) and of algal suspensions in presence of metal ions with increments of acid (as illustrated by Fig. 3) can be used to evaluate the affinity of metal ions to the functional groups of the algae surface. As a first approximation, we may derive from the experimental data equilibrium constants, K H, as defined by equations (15) or (18). This is conventionally done from Scatchard or Langmuir linear plots, respectively, by transforming equation (15) into

(28)

or equation (18) into 80-

1 50
1

{RME} = {RT}

40-

._o-

3020-

®

~00 4

pH

Fig. 3. Titration curves of Cu(II) (3.14 x 10-6 moll -I) (A) and Cd(II) (9.6 x 10-Tmoil -I) (B) as a function of pH in 0.01 M KNO3 and in presence of algae (68 mg dry wt 1-~ for Cd; 22.8 mg dry wt 1-1 for Cu). The decrease of Cu and Cd at high pH in the absence of algae is due to the adsorption on the glass wall and to the formation of polymeric hydroxo species (Cu). The difference between the two curves gives the Cu, respectively Cd, bound to algae.

-+

1 Kh{RT}[Me 2+]

(29)

and by plotting {RMe}/[Me 2+] vs {RMe} and {RMe}-J vs [Me2+] - l, respectively. Figure 4(A) and (B) illustrate such plots of the same data for the Cu(II) binding to algae at p H 6.5. Obviously the data cannot be fitted readily; at high coverage of the surface with Cu(II) or at high [Cu 2+] considerable deviation from a linear relationship is observed. The tendency of Cu(II) binding decreases with increased Cu(II) loading of the algal surface. This indicates that Cu(II) ions become b o u n d first to the highest affinity surface ligands and subsequently to those of lesser activity. Furthermore, increased surface coverage and increased surface charge may induce some electrostatic repulsion. This effect on surface coverage is less pronounced with Cd(II).

HAN-BIN XuE et al.

922

! I000

2oo ~'= 180 800

(A)

16o

700

m,t,*

t~

E

500

~,.., ,.E. L

g

.o:.

300

7

200

~I

t00

o

~oo

o~

400

It-

140

7-~ 1 2 0

600

~ I

I

I

I

5

10

15

I

I

I

80

6o 40

20

I

20 2,5:30:55 40

0

{R-Cu} r/J.rnot (;0(11") bound

I

I

I

I

1

25

5o

75

loo

125

I 150

[du~*] -~ [ U - ~ ] xlO e

g-lalgoe]

Fig. 4. Scatchard plot (A) [equation (28)] and Langmuir plot (B) [equation (29)] for the binding of Cu(II) to the algal surface at a constant pH of 6.5. The non-linearity is due to a decrease in binding tendency with increasing surface coverage. Thus, the equilibrium constants, K h, obtained from the experimental data show some tendency to decrease with increased surface coverage. Constants evaluated from low surface coverage or from low [Me2+] are of more general interest with regard to natural waters. Such equilibrium constants for Cu(II) and Cd(II) are given in Table 1. In fresh natural waters the affinity of heavy metals to algal surfaces is somewhat reduced because of the presence of Ca 2÷ and Mg 2+ in large excess. The tendency to repress the complex formation with Cd(II) and Cu(II) by 10 -3 M Ca 2+ is illustrated in Fig. 5; complexing constants valid in the presence of 10 -3 M Ca 2+ are also given in Table I. The data can be fitted better by interpreting them with a constant capacitance model or with the Fowler Guggenheim Frumkin equation. Figure 6 plots the data for Cu(II) binding in the logarithmic form of equation (19a) In

0 1 [(--i-Z-~_O).([---~e2+])]=lnB~+2aO.

(30,

From such a plot, values of the equilibrium constant, Bh and of the surface interference factor a are obtained. These constants are given in Table 1. They

show, in agreement with equation (14), a pH dependence according to 6 IogBh/6 p H = 0 . 6 . The negative values for a indicate that the interaction between sites is repulsive or, more generally, that the affinity of the sites for Cu(II) or Cd(II) decrease with increasing surface coverage. Finally, the data can be fitted fairly well with a two-site Langrnuir model as shown in Fig. 7. The good fit reflects that, as shown in Table 1, two capacities, Fro, and Fm: and two binding constants have to be considered (or adjusted) to fit the data. A computer program adapted to include surface chemical species (Coves and Sposito, 1986) was used. Figure 8 summarizes the pH and concentration dependence of Cu(II) to the surfaces of Chlamydomonas. The tendency to bind Cu(II) reaches an optimum around pH = 7. At higher pH values, this tendency decreases primarily because Cu(II) hydrolizes to hydroxo species which are not bound to the functional surface ligands.

Exudates Algae typically release some soluble exudates, Measurements of the filtrates of the algal suspensions used in our studies gave for the exudates concen-

Table 1. Bindingconstantsto the surface of Chlamydomonasalgae logK~ IogB~ a IogK~ IogK~ F~ Metal pH [equation(5)] [equation(19)] [equation(20)] 10-5 Cu(II) 6.0 7.3 6.4 -5.8 8.0 6.3 0.7 6.5 7.5 6.8 - 5.7 8.0 6.45 1.0 7.0 7.6 7.0 -6.1 8.0 6.5 1.3 7.5 7.1 Cu/Ca* 6.0 6.7 Cd(n) 6.4 5.8 7.0 5.8 Cd/Ca* 7.0 6.1 *In presence of 10-3 M Ca2..

F~2 molg-~ 4.1 4.0 3.9

The binding of heavy metals to algal surfaces

2.0 1.e

(A)

~ ~,9

923

1.0 - (B)

/

0.9

W

, e,

j/ , + f =,//_oo

, o.

.,y

i o.=

1.o o=

¢

.J

0.6

,&"

04

.J 0.3

0.4

0.2

0.2

0.1 I

0

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

iv/+o

x~o~' l j J

0

1

I

I

I

I

I

I

I

I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

" ReslduoL dissoLved Cu(n') [p.M]

ReslduoL dissoLved Cd(Tr) [p.M]

Fig. 5. Binding of Cu (pH = 6.0) (A) and of Cd (pH = 7.0) (B) to the surface of algae in presence and in absence of Ca at constant pH. The algal surface is titrated with increasing concentrations of Cu, respectively Cd. The concentrations of Cu and Cd bound to algae are decreased in the presence of 10-3 M Ca 2+ due to the competition by Ca 2+ for the surface sites. trations of TOC in the concentration range of 10--20 mg C g-~ (dry wt) of algae. The complexing capacity of the exudate is smaller by at least one order of magnitude than that of the algae in the concentration range used; thus, although some of the metal ions in solution become b o u n d to the exudates, their effect on the evaluation of metal binding to the surfaces is small. Our studies have shown that exudates form non-labile complexes with Cu(lI) and Cd(II). Conditional stability constants of Cu(II) in accordance with equation ( 1 5 ) a t pH6--7 are logK u ~, 6.7; i.e. they are slightly lower than those of the surface ]igands.

equilibrium with the algal surface seems to be attained. Subsequently, its concentration decreases more slowly. After 2 or 3 h one typically observes a transient increase in peak current. The labile Cu(II) concentration decreases with the square root of time [Fig. 9(B)], thus indicating that this secondary slower

13,

~',- ~ ,,

Uptake kinetics of Cu(H)

1.4"

Figure 9 shows a plot of peak current as a function of reaction time. The u n b o u n d Cu 2+ initially decreases rapidly for a few minutes; a pseudo-

"

i

ol= .7-

15.5

14.5 "

~

A

g .5-

d

0

t4.0

~

13.5

.4.S-

.

,,

~ o

.2

.,

[Cu

,>

.

.

.

.

Cu-ads

"

Total Co

12.5

°.050 O.lOO o.15o

L|

bM]

.2-

13.0

12.0

"

,,

.9.

0 0

20

,.a. ~.a. ~,.,.

t6.o

15.0

pHT.0/

..... ~ ,0 ~'~

o. oo

G

Fig. 6. Cu(II) binding to algal surface plotted in terms of Fowler Guggenheim Frumkin equations [equation (30)]. Points are experimental data for different pH values.

•

•

116

l , SI"

[}JMI

Fig. 7. Speciation of Cu(II) with two-site Langmuir model, calculated by MICROQL-UCR program with two-site model, logKI , 8.0; logK2, 6.45; Fro, 1.02 x 10-Stool g-I; F,= 3.98 x 10-5 tool g- l, at pH 6.5. The calculated curves fit well to the titration data (Cu-ads = Cu bound to algae surface). Insert: Two-site Langmuir isotherm for Cu(II)-binding to algae. The curves are calculated according to equation (20), with the parameters given in Table 1.

HAlq-BIN XI.~ et al.

924

uptake is a diffusion controlled process, most likely the diffusion into the inside of the cell. The transient peak in ip is not--as we thought initially--caused by a Cu release of the ceil, but by the release of some organic compounds which are reducible at the electrode. This was shown by making additional measurements on the residual Cu(II) by atomic absorption and by comparing voltammetrically the filtrate with reducible organic model substances that have a halfwave potential similar to that of Cu(II).

9O

~z o ~ ao

DISCUSSION 6

As shown in this study on the interaction of Cu(II) and Cd(II) with Chlamydomonas, the functional groups on the surface of algae have a rather large affinity for metal ions. Many of the features observed here are similar to those observed earlier by Gon~alves et al. on the interaction with bacterial cell suspensions (Klebsiella pneumonia). The conditional surface complex formation constants at comparable pH values are of the same order of magnitude. The functional groups of biological cells bind metal ions in a similar way as soluble ligands. The cell surface has a remarkably high affinity for Cu 2+ and appears to outweigh that observed for colloidal iron(III) oxide. The tendency to form surface complexes decreases with increasing metal loading of the surface because the metal ions bind first to the surface groups with highest affinity and subsequently to groups with lower affinity. The metal binding tendency of cell surfaces can be readily interpreted in terms of surface complex for-

,

,

7

8

pH Fig. 8. Bound Cu as a function of pH at constant residual dissolved Cu. % bound Cu is calculated as: [Cur- ~ C u T ] x 100, wber¢ CuT = total residual dissolved Cu at the pH given. The data are obtained by transformation of titration curves series of constant pH values. The concentration of algal suspension is 12.8mg 1-I. 1, residual dissolved Cu(II), 0.16 #M; 2, residual dissolved Cu(II), 0.31 #M; 3, residual dissolved Cu(lI), 0.63 #M.

mation equilibria or--as has been shown to be equivalent--in terms of Langmuir type adsorption equilibria. Both equilibrium concepts imply, in a simplifying way, that the surface consists of a uniform polymeric network of repeating, energetically equivalent, surface functional groups. Even if this assumption were true, one would need to correct the experimental constants for electrostatic interaction. The assumption of uniform functional groups is of

(B) 6 -o 60

50

f

(A) E 4 o qr-

3

52 '<

I

4O

30

1

I

I

t

I

6

8

10

12

14

V~" ( mln )I/2

"~ 2o

(L

10

I

I

I

30

60

90

I

I

I

I

1

I

I

120 150 180 210 2 4 0 2 7 0 3 0 0 Time (rain)

Fig. 9. (A) Kinetics of Cu(II) binding and uptake to algae. The peak current of Cu(II) is plotted as a function of time (A). The kinetic process can be divided into two steps: a first fast decrease of the labile Cu occurs within a few minutes; in a second step, a slow decrease during 2 or 3 h is observed. [:], Storage time 4.5 h; O, storage time Z6 h; A, storage time 48 h; ~, storage time 90 h. (B) Labile Cu during the second step is plotted as a function of the square root of reaction time; a finear function is obtained which is characteristic of a diffusion process.

The binding of heavy metals to algal surfaces course not fulfilled with biological cell surfaces. Similar as with humic acids (Buflte, 1984), heavy metal ions interact with a mixture of functional surface sites. The representation of the extent of metal interaction with a cell surface in terms of one single equilibrium constant is a construct, similar as if we tried to express the interaction of a metal ion with a variety of soluble complex formers in terms of one constant only. Nevertheless such a constant is an average equilibrium quotient, obtained by applying the law of mass action to a mixture of functional groups; it permits a generalization for a given range of coverage of the surface by metal ions. The experimental data can be fitted much better by introducing factors that diminish the binding tendency with increasing surface coverage (electrostatic correction or repulsive interaction). Although the use of equations such as the constant capacitance or the Fowler Guggenheim Frumkin adsorption model may provide a better generalization to wider ranges of concentrations of metal and biological cells, one must be aware that the better fit of the data is essentially due to the use of two adjustable constants and does not "prove" the validity of the model or of its underlying assumptions (Westall and Hohl, 1980). Although we are aware that the binding of metal ions to the surface is characterized by a multi-site adsorption system, it is interesting to note that a two-site Langmuir isotherm--depending in principle on four adjustable constants--can reproduce the experimental data very well (Hunston, 1975). Of course, one can go a step further and interpet the experimental data in terms of an observed distribution of equilibrium quotients. Such an approach has been illustrated for humic acids, for example, by Buffle and Altmann (1987). However, one should consider that the transformation of experimental data to a continuous set of equilibrium quotients provides no marked gain in insight that is not already available from the titration curve, e.g. free [Me :+] vs [Me(II) added]. Dzombak et al. (1986) have shown with a critical examination of metal-humate interaction that an affinity spectrum does not correspond to an actual distribution of ligands. Furthermore, one often depends in the chemistry of natural waters on quantitative speciation models, on the complicated network of equilibria between various metals and H + and a variety of soluble and insoluble ligands in order to be able to circumscribe the relative importance of different variables. For these usually computerprogrammed models, one depends on the input of simple equlibrium quotients for the interaction of metal ions with the surfaces present. That equilibrium "constants" extracted from the experimental data are data fitting quotients, valid for certain conditions, and should, however, not detract from the fact that the surfaces of the algae and of other biological ceils--often as or more important than the presence of humic or fulvic acids--have a great effect on metal ion speciation, metal ion

925

buffering and the residual concentrations in natural waters. There is little question that Cu(II) and other heavy metals "adsorbed" on the surface of biological cells is bound by inner sphere type of coordination. Recent studies by M6hl et al. (1988) using a combination of magnetic resonance methods (electron spin resonance, ESR; electron spin echo-envelope modulations, ESEEM; and electron nuclear double resonance spectroscopy, ENDOR) have presented evidence that on the surface of the bacterium Klebsiella, Cu(II) coordinates to the bidentaMigand histidin¢. Application of these data to natural water systems must be done with caution. Experimentally in the laboratory we use concentrations of metal ions (10-s-10 -7 M) and of algal cells (20-100 mg dry wt 1-t) that are 103-105 times larger than the concentrations typically encountered in natural waters. We do not know to what extent the data and the constants obtained in such laboratory studies can be extrapolated to the lower concentations. Nevertheless, relative affinities may be compared in a semiquantitative way. One major result is that biological surfaces or particles coated with biogenic substances can exhibit more than inorganic surfaces the affinity required for the efficient metal scavenging that is observed in oceans, lakes and rivers. The adsorption and uptake kinetics of Cu(II) reported here is consistent with a model in which the metal ions become in a first fast step surface coordinated to the algal surface and subsequently become transferred in a slow step into the inside of the cell (Davis, 1978; Williams, 1981; Harvey and Leckie, 1985). Such uptake kinetics is consistent with the established observation that physiologic or toxicologic effects of heavy metals depend usually on their free metal ion concentrations (and not on that of their complexes). Acknowledgements--Valuable advice was given by Beat

Miiller on the adsorption of metal ions on the glass wall. We also thank Heinz Bachmann and Max Reutlinger for experimental help and Laurent Charlet for considerable assistance on the use of the MICROQL-UCR computer program. Discussion with R, J. P. Williams is gratefully acknowledged. REFERENCES

Buffle J. (1984) Electroanalytical measurements of trace metals complexation in natural aquatic conditions. IUPAC Commission. Buffle J. and Altmann R. S. (1987) Interpretation of metal complexation by heterogeneous complexants. In Aquatic Surface Chemistry. Chemical Processes at the ParticleWater Interface (Edited by Stumm W.). Wiley-Inter-

science, New York. Coves J. and Sposito G. (1986) MICROQL-UCR, a surface chemical adaption of the speciation program MICROQL. User's Manual. Department of Soil and Environmental Science, University of California, Riverside, Calif. Davis A. G. (1978) Pollution studies with marine plankton. Part II. Heavy metals. Adv. mar. Biol. 15, 381-508. DeFord D. D. and Hume D. N. (1951) The determination

926

HAN-BIN XUE et al.

of consecutive formation constants of complex ions from polarographic data. J. Am. chem. Soc. 73, 5321-5322. Dzombak D, A., Fish W. and Morel F. M. M. (1986) Metal-humate interactions. 1. Discrete ligand and continuous distribution. Envir. Sci. Technol. 20, 669-683, Frumkin A. N. (1925) Die Kapillarkurve der h6heren Fetts/iuren und die Zustandsgleichung der Oberfl~ichenschicht. Z. phys. Chem. 116, 466--484. Gonqalves M. L. S., Sigg L. and Stumm W. (1985) Voltammetric metl.,ods for distinguishing between dissolved and particul~Lte r,aetal ion concentrations in presence of hydrous oxides. A case study on lead(II). Envir. Sci. Technol. 19, 14!-146. Goncalves M. L. S., Sigg L., Reutlinger M. and Stumm W. (1987) Metal ion binding by biological surfaces: voltammetric assessment in presence of bacteria. Sci. Total Envir. 60, 105-119 (1987). Harvey R. W. and Leckie J. O. (1985) Sorption of lead onto two gram-negative marine bacteria in seawater. Mar. Chem. 15, 333-344. Hunston D. L. (1975) Two techniques for evaluating small molecute-macromotecule binding in complex system. Analyt. Biochem. 63, 99-109. Klotz I. M. (1982) Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 217, 1247-1249. M6hl W., Motschi H. and Schweiger A. (1988) EPR, ENDOR and ESEEM investigations of adsorbed Cu(II) on the surface of the bacterium Klebsiella pneumoniae. In preparation. Sigg L. (1979) Die Wechselwirkung yon Anionen und schwachen S/iuren mit ~-FeOOH (Goethit) in w/issriger L6sung. Diss. ETH No. 6417. Sigg L. (1985) Metal transfer mechanisms in lakes; the role of settling particles. In Chemical Processes in Lakes (Edited by Stumm W.), pp. 283-307. Wiley-lnterscience, New York.

Sigg L. (1987) Surface chemical aspects of the distribution and fate of m¢~l ions in lakes. In Aquatic Surface Chemistry. Chemical Processes at the Particle-Water Interface (Edited by Stumm W.)i Wiley-lnterscience, New York. Sposito G. (1983) On the surface complexation model of the oxide--aqueous solution interface. J. Colloid Interface Sci. 91, 329-340. Staub R. (1961) Ernfihrungsphysiologisch-aut6kologische Untersuchungen an der planktischen Blaualge Oscillatoria rubescens D.C. Schweiz. Z. Hydrol. XXHI, 82-192. Stroes-Gascoyne S., Kramer J. R. and Snodgrass W. J. (1986) A new model describing the adsorption of copper on MnO2. Envir. Sci. Technol. 20, 1047-1050. Stumm W. and Morgan J, J. (1981) Aquatic Chemistry. Wiley-Interscience, New York. WestaU J. and Hohl H. (1980) A comparison of electrostatic models for the oxide solution interface. Adv. Colloid Interface Sci. 12, 265-294. Whitfield M. and Turner D. R. (1979) Critical assessment of the relationship between biological thermodynamic and electrochemical availability. In Chemical Modeling in Aquatic Systems (Edited by Jenne E. A.), pp. 657~580. ACS Symposium Series 93. American Chemical Society, Washington, D.C. Whitfield M. and Turner D. R. (1987) The role of particles in regulating the composition of natural waters. In Aquatic Surface Chemistry. Chemical Processes at the Particle-Water Interface (Edited by Stumm W.). WileyInterscience, New York. Williams R. J. P. (1981) Physico-chemical aspects of inorganic element transfer through membranes. Phil. Trans. R. Soc. Land. B 294, 57-74. Wilson D. E. and Kinney P. (1977) Effects of polymeric charge variations on the proton-metal ion equilibria of humic materials. Limnol. Oceanogr. 22, 281-289.