APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p.

1274-1282

Vol. 59, No. 5

0099-2240/93/051274-09$02.00/0 Copyright 1993, American Society for Microbiology

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain Al

KAY L. SHUFTLEWORTHt AND RICHARD F. UNZ* Laboratory of Environmental Microbiology, Intercollegiate Program in Ecology and Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
Received 23 October 1992/Accepted 10 February 1993

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain Al to sorb heavy metals from solution. Cells of Thiothrix strain Al were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl2. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain Al grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.
Filamentous sulfur bacteria, including Thiothrix spp., occur in natural aquatic environments (14) and in biological wastewater treatment systems, particularly those which are oxygen deficient or septic (31, 36). When conditions are favorable for their growth, Thiothrix spp. may become dominant in the microbial flocs of the activated-sludge process of wastewater treatment and contribute to the phenomenon of filamentous sludge bulking, which is characterized by poor settling and limited compaction of the sludge biomass. Heavy metals occur both in domestic and industrial wastewaters (24), and the U.S. Environmental Protection Agency, through the 1987 Water Quality Act, seeks to stringently limit the discharge of bioavailable heavy metals to the environment (10). It is particularly important, therefore, to understand the extent to which microorganisms indigenous to biological treatment systems may mobilize or immobilize metals present in wastewaters. One important factor in metal sorption is the available surface area. According to the hypothesis of Pipes (25), a filament possesses a greater surface area-to-volume ratio than a clump of cells does; consequently, flocs in bulking sludges may have a larger surface area than those in nonbulking sludges and may therefore exhibit a greater potential for metal uptake. Lawson et al. (15) were unable to establish definitive proof of enhanced metal uptake by bulking sludges, however, and studies of metal interactions with axenic cultures of wastewater filamentous bacteria have been limited to Sphaerotilus spp. (3, 17). Shuttleworth and Unz (26) reported that several heavy metals had toxic effects on Thiothric strain Al, which was an

isolate from bulking activated sludge (36). It was envisioned that this organism may differ from unicellular bacteria in its uptake of heavy metals as a result of its filamentous growth habit and the utilization of reduced sulfur for energy (37). In this report, the performance of Thiothrix strain Al in the removal of heavy metals from solution is described and the implications of metal sorption on metal toxicity are discussed.

Corresponding author.

Toxicological Research, Jefferson, AR 72079.

t Present address: Division of Microbiology, National Center for

1274

MATERUILS AND METHODS Bacteria. Thiothrix strain Al is a member of our culture collection. The bacterium was maintained and grown on lactate-thiosulfate-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (LTH) medium, described elsewhere (26). Sorption experiments. LTH medium was inoculated with growing broth culture (log phase) of Thiothrix strain Al to produce an optical density at 400 nm (OD400) of 0.0075. Incubation was carried out at 25C on an orbital shaker (110 rpm) for 2.5 to 3 days unless otherwise stated. Biomass was harvested by centrifugation (7,000 x g at 25C for 15 min), washed twice, and resuspended in 20 mM HEPES buffer (pH 7.20). Concentrated cell suspensions were kept aerobic by bubbling with air until needed. The cell density was approximated by measuring the OD400. Subsamples of cells were frozen at -20C for subsequent protein analysis. The standard reactor conditions for sorption experiments, unless otherwise noted, consisted of 20 ml of cells (OD400 of 0.3 for Cu and 1.6 for Zn and Ni) suspended in 20 mM HEPES buffer (pH 7.20) in 125-ml high-density polyethylene bottles. Preliminary experiments revealed that nonspecific metal sorption was lower with high-density polyethylene than polypropylene bottles. All metals were added as chloride salts at appropriate concentrations. The cell-metal mixtures were incubated for 6 h at 25C and 110 rpm. Bacteria

VOL. 59, 1993

were

SORPTION OF HEAVY METALS TO THIOTHRIX STRAIN Al

1275

always the last component to be added to the reaction mixtures, and all experiments were performed in duplicate. At intervals, cells were recovered by filtering 15 ml of sample through 0.2-,um-pore-size filters (diameter, 25 mm). Polysulfone filters (Gelman Sciences, Ann Arbor, Mich.) were used for experiments involving nickel or zinc. Polycarbonate filters were required for the copper study to minimize nonbiological metal sorption. For all metals, the use of polysulfone filter support units (Gelman Sciences) was necessary to prevent sorption of metals to the filter apparatus. Cells were rinsed twice in situ with 2.5 ml of ultrapure water and then quantitatively removed from the filter with approximately 7 ml of ultrapure water. Then 1 ml of concentrated HNO3 was added to each sample, and the volume was reduced to 5 ml by gentle heating. Cells were digested by addition of 6 ml of concentrated HNO3 and evaporation to dryness at just below the boiling point. Digested samples were resuspended in 15 ml of 1% HNO3 in preparation for analysis by flame atomic absorption spectrophotometry (AAS) or in 0.2% HNO3 for analysis to be completed by graphite furnace AAS. In preliminary experiments, filtrates were also kept for metal analysis. Metal recovery (sum of cells and filtrates) was based on the measured total metal added and was between 92 and 103%, with an average of 98.8%. The difference in results between duplicates was typically less than 4%. For one study done in triplicate, the range for the standard deviation was 0.3 to 2.4% of the mean and the average standard deviation was 1.4% of the mean. Sorption under anoxic conditions. Cells containing an abundance of sulfur granules (20 h old) and cells virtually devoid of sulfur granules (2.5 days old) were harvested. The cells were washed and resuspended, and oxic and anoxic conditions were created by bubbling humidified air or nitrogen, respectively, through the solutions for 30 min. Then metal chloride was added to give a final concentration of 50 FLM. The cells were bubbled for another 3 h and then separated from the bulk solution by filtration. Metal analyses were performed only on the filtrates because metal sulfide precipitates might not be quantitatively recovered from the filter along with the cell fraction. Metal uptake during growth. LTH medium was supplemented with NiCl2 and 63NiC12 (specific activity, 52.7 MBq/ ,mol; Amersham, Arlington Heights, Ill.) to produce the desired nickel concentration and an activity of 200 Bq/ml. A
sufficient inoculum of washed Thiothrix strain Al cells was added to produce an initial OD4. of 0.01, and the cultures were incubated at 25C on an orbital shaker (110 rpm). At intervals, aliquots were removed from the cultures for measurement of OD4., pH, and cell-bound nickel. The OD of sonicated samples was used to quantify cell growth (26). Cell-bound nickel was determined by filtering samples through a 0.2-,um filter, rinsing twice with 5 ml of ultrapure water, and air drying. The cells were partially digested by being incubated with 2 ml of concentrated HCI for 5 h. Each 2-ml HCl sample was mixed with 15 ml of Scintiverse II cocktail (Fisher Scientific, Pittsburgh, Pa.), and cell-bound nickel was determined from radioactive counts obtained with a liquid scintillation counter (model 1217 Rackbeta; LKB Instruments, Gaithersburg, Md.). The external standards ratio method was used for quench corrections. Metal uptake in the presence of ligands. The effect of various ligands on metal speciation was predicted by using the computer model MINEQL (35) as described previously (26). Equilibrium constants which were substituted for values in the MINEQL data base and constants which were not part of the original data base are given in Table 1. All

TABLE 1. Additions to and changes in the thermodynamic constants used in the MINEQL program'
Compound (ID no.)b
Log K Component stoichiometry

Type speciesd
II
II II

Ni acetate (6910) Zn acetate (6140) H Tris (13590) Ni Tris (7570) Ni Tris (7580) Zn Tris

1.435
1.58 8.28' 3.03e 5.12' 2.67T -8.68 35.3 3.83 2.90

Cu(OH)2 (20500) Zn3(PO4)2 (20650) H glycolate Cu glycolate Ni glycolate Zn glycolate H lactate Ca lactate Mg lactate Cu lactate Cu lactate Ni lactate Ni lactate Zn lactate Zn lactate

Ni (1) acetate (1) Zn (1) acetate (1) H (1) Tris (1) Ni (1) Tris (1) Ni (1) Tris (2) Zn (1) Tris (1) Cu (1) H (-2)

2.26 2.38 3.86 1.42 1.37 3.02 4.84 2.22 3.62 2.22 3.75

Zn (3) P04 (2) H (1) glycolate (1) Cu (1) glycolate (1) Ni (1) glycolate (1) Zn (1) glycolate (1) H (1) lactate (1) Ca (1) lactate (1) Mg (1) lactate (1) Cu (1) lactate (1) Cu (1) lactate (2) Ni (1) lactate (1) Ni (1) lactate (2) Zn (1) lactate (1) Zn (1) lactate (2)

II II II V V II II II II II II II II II II II II II

a All other thermodynamic constants used were those contained in the data base (therm.dat) supplied with the program. The MINEQL ionc.dat data base was modified by addition of the ion charge values (-1) for glycolate and lactate. The ionc.dat changes were necessary for the program to properly calculate the changes in lactate and glycolate K values as a function of ionic strength. b ID no. is the number in the therm.dat data base used to identify the compound. Only the numbers that were included in the original data base are given here. c The log K values are the thermodynamic constants and are given in the convention used in the MINEQL program. d The type species are listed according to the MINEQL format. ' The literature values for these compounds were concentration equilibrium constants (KI). The extended Debye-Huickel equation was used to determine the activity coefficients necessary to calculate the Kvalues from the K values (32).

substituted values, except those for zinc phosphate, were obtained from tables of critical stability constants (18, 19, 28-30); the zinc phosphate constant was obtained from

Lindsay (16). Desorption of metals. Batches of cells were incubated for 8 h at 25C and 110 rpm in 21.5 ,uM Cu, 63.3 ,uM Ni, or 57.0 ,M Zn. Aliquots (20 ml) of cells were removed, filtered, and rinsed twice with 2.5 ml of ultrapure water. The cells were quantitatively removed from the filter by being washed off with exactly 20 ml of the desired desorption solution. The desorption solutions were 6 mM KCl, 5 mM CaCl2, and 5 mM EDTA; each compound was dissolved either in ultrapure water (pH unadjusted) or in 20 mM HEPES (pH 7.20). The cells, thus resuspended in the various desorption solutions, were again incubated for 8 h. Aliquots of cells (15 ml) were then processed for metal analysis as stated above. Isotherm models. The sorption data were fitted to Freundlich and Langmuir isotherm models (34). The linearized form of the Freundlich equation is as follows: log qe = (log Kf) + [(1/n) (log C)], where C is the concentration in solution at equilibrium, qe is the moles of solute sorbed per unit weight at concentration C, and Kf and n are constants. The Langmuir equation was linearized by the method of Dowd and Riggs (4) and is as follows: qe = Q - [(1Ib)(q,/ C)], where qe and C are the same as for the Freundlich equation, Q is the moles of solute sorbed per unit weight of

1276

SHUTTLEWORTH AND UNZ

APPL. ENVIRON. MICROBIOL. TABLE 2. Constants for Freundlich isothermsa,b

c .5_ 1000

Metal

logKf
2.22

1/n

r2

a
m

a)

100

Cu
10Zn

E
0 cr

,/
=

Copper Nickel C < 4.8 C > 4.8 Zinc C c 4.9 C > 4.9

0.650 0.885 0.250 0.598 0.236

0.999
0.992 0.996 0.988 0.954

1.34 1.79
1.53 1.80

.Ni
1

5qe
0.1 1

KfC /

10 100 1000 C (,omol/L) FIG. 1. Freundlich sorption isotherms for Thiothrix strain Al. C is the concentration of metal in solution at equilibrium, and Kf and n are constants. The ranges of total metal concentrations tested were 2.5 to 100 ,uM Cu, 0.5 to 500 ,uM Ni, and 2.5 to 1000 ,uM Zn. The concentrations of cell protein were 39.5 ,ug/ml (Cu), 168 pg/ml
(Ni), and 182 ,ug/ml (Zn).

a The log Kf is the sorption capacity for C = 1, and the slope indicates sorption intensity. b Cell protein concentrations for copper, nickel, and zinc were 39.5, 168, and 182 ,ug/ml, respectively.

adsorbent to form a complete monolayer, and b is a constant. AAS. AAS analyses were completed with a Perkin-Elmer model 3030B atomic absorption spectrophotometer equipped with a model HGA-600 furnace. All zinc and high-concentration copper samples were analyzed by airacetylene flame AAS under the conditions recommended by the instrument manufacturer. Low-concentration copper samples and all nickel samples were analyzed by the graphite furnace technique. No matrix interferences were found. Other procedures. Cell protein was determined by the Lowry method with the digestion procedure modified to prevent potential interference from sulfur granules (37). Ultrapure water was obtained from a Milli-Q four-bowl system (Millipore Corp., Bedford, Mass.). Glassware and plasticware were washed in Linbro 7x detergent, rinsed, soaked for a minimum of 4 h in 20% nitric acid, and rinsed four times with ultrapure water. Chemicals were reagent grade and commercially available.
RESULTS

The capacity of Thiothrix strain Al cells to sorb metals

was a function of the metal type and metal concentration (Fig. 1). Within the equilibrium concentration (C) range of 0.178 to 33 ,uM Cu (2.5 to 100 ,uM total copper), copper sorption data fit the Freundlich isotherm. The sorption of nickel and zinc between C ranges of 0.08 to 450 ,uM and 0.2 to 930 ,uM, respectively, did not fit either the Freundlich isotherm or the Langmuir isotherm. However, both nickel and zinc sorption could be described by biphasic Freundlich isotherms. The equation was applied once for C c 4.8 ,uM and a second time for C 2 4.8 ,uM (Fig. 1). At the lower metal concentrations the cells had a greater affinity for zinc than for nickel, but at C 2 4.8 ,uM there were no differences in the sorption of nickel and zinc (Fig. 1). The K and n values for the Freundlich isotherms are given in Table 2. The sorption of Ni and Zn was very fast. Most of the metal ions which were taken up from solution were bound within 10 min, and almost no increase in the level of bound metal occurred after 1 h (Fig. 2). Metal uptake was also a function of cell age and was not linear with cell protein concentration (Fig. 3). There was virtually no difference in metal uptake

between cells which were 2, 3, 4, or 5 days old, but cells which were 1 day old sorbed substantially less metal than older cells did. One-day-old cells were in the early to mid-log phase and were packed with sulfur granules (Fig. 4A). By day 2, cells were in the late log to early stationary phase and were virtually devoid of sulfur granules (Fig. 4B). The sorption of zinc was inhibited by the presence of other divalent cations, and calcium was a somewhat more effective inhibitor than was magnesium (Fig. 5). Cells in zinc solution with no additional cations sorbed 57% of the metal; however, in the presence of calcium or magnesium at 10 times the zinc concentration, 23 and 30%, respectively, of the zinc was sorbed. At each concentration, there was a statistically significant difference (P < 0.02; Student's t test) between the effects of calcium and of magnesium on zinc uptake. The presence of up to 2.0 mM KCI had virtually no effect on zinc sorption (Fig. 5). Zinc and nickel in combination also appeared to mutually compete for sorption sites (Fig. 6). The amount of zinc sorbed was decreased by the addition of nickel (Fig. 6B), and, conversely, the amount of nickel sorbed was lowered by the presence of zinc (Fig. 6A). For example, 154 ,umol of zinc per g protein was sorbed from a 50 ,uM solution but only 112 ,umol of zinc per g of protein was sorbed from a 100 ,uM solution which was 50% zinc and 50% nickel. However, 219 ,umol of zinc per g of protein was sorbed from a 100 ,uM zinc solution. Similarly, 128 ,umol of nickel per g of protein was sorbed from 50 ,uM nickel; 91.6 ,umol of nickel per g of protein was sorbed from a 100 ,uM solution of 50% nickel and 50% zinc, but 166 ,umol of nickel per g of protein was sorbed from 100 ,uM nickel. The

c .5

0- 1250.
00- 100.0
a)

Ni

7550-

:3l

252 4 6

10

Time (hrs)
FIG. 2. Kinetics of nickel and zinc binding to Thiothrix strain Al. Metal concentrations were 53.2 F.M nickel and 45.0 ,uM zinc. The protein concentration was 197 ,ug/ml for both nickel and zinc.

VOL. 59, 1993

SORPTION OF HEAVY METALS TO THIOTHRIX STRAIN Al

1277

co

2
6040-

20
20

1 00

200

300

400

Protein concentration (mg/L)

100-

N.U
40~~~~~~~~
0
~

80

buffered 5 mM EDTA solution removing 80 to 83% of the initial metal sorbed. Calcium chloride was the second best metal desorption agent, and it removed nickel and zinc more effectively than it removed copper (Fig. 8). More metal was removed by CaC12 in unbuffered solution (pH approximately 5.6 to 6.0) than in HEPES-buffered solution (pH 7.2), even though the presence of 20 mM HEPES substantially increased the ionic strength of the solution. The presence of KCl had virtually no impact on the desorption of copper, but KCI had a pronounced effect on the retention of nickel and zinc by cells in unbuffered ultrapure water. There was no desorption of zinc by ultrapure water, although 21% of the sorbed copper was removed by water alone. In previous studies, observations of hydrogen sulfide odor and blackish grey discoloration in some unaerated Thiothrix cultures were taken as evidence of possible sulfur reduction. It was believed that under anoxic conditions the bacterium might cause metals to precipitate as sulfides. However, there was no evidence of metal sulfide precipitation when cells, either with or without sulfur granules, were subjected to a

2000

00

40

nitrogen atmosphere. Growing cultures of Thiothrix strain Al were capable of taking up nickel from the growth medium (Fig. 9); however,
the amount of cell-associated nickel was much smaller than would be predicted from the information obtained on uptake under nongrowth conditions. Only 17% of 1.19 ,uM nickel was removed during growth in nickel-containing medium, whereas 79% of 1 ,uM nickel was sorbed to cells resuspended at a comparable cell density in HEPES buffer. Cell growth, cell-associated nickel level, and pH all increased concomitantly. Attempts to prevent pH changes during growth were unsuccessful because cell growth was inhibited by higher concentrations of HEPES buffer. The cell-associated nickel was not readily desorbed. No metal was removed from 3-day-old cells (grown in 1.19 p,M Ni) by incubating harvested cells for 4 h in 10 mM HEPES buffer (pH 5, 6, 7, or 8) or in HEPES-buffered 0.5 mM CaCl2 (pH 7.5). Furthermore only 6% of the cell-associated nickel was removed by 0.1 mM EDTA in 10 mM HEPES buffer (pH 7.5).
DISCUSSION The Freundlich and Langmuir isotherm models are commonly used to describe the sorption of metals onto microbial surfaces and are usually in agreement over moderate ranges of the equilibrium sorbate concentration (34). In the present study, sorption data best fit the Freundlich isotherm, although the curves were biphasic for nickel and zinc. Because the curves are biphasic, there are probably at least two types of binding sites for nickel and zinc (6). The chemistry of cell surfaces is complex, and the potential therefore exists for metals to sorb at a variety of sites (1, 20). Additionally, metals may not only be sorbed to the surface but may also be taken up into the interior of the cells. Metal sorption can vary according to pH and ionic strength. In this study, the pH was maintained at 7.20 because (i) Thiothrix strain Al is pH sensitive and does not grow well below pH 7 (personal observation) and (ii) organic wastewaters typically have a circumneutral pH. Maintaining this pH necessitated using an effective buffer (HEPES), which undoubtedly affected the results of metal uptake experiments. Although the metal-binding capacity of HEPES has been reported to be "negligible" (9), metal complexation by the buffer may nevertheless occur (12). Metal binding by bacteria has been shown to be profoundly influenced by pH, buffer type, ionic strength, incu-

0
100

200

300

400

Protein concentration (mg/L)

FIG. 3. Effect of cell age and cell concentration on the sorption of nickel (A) and zinc (B) to Thiothrix strain Al. The nickel and zinc concentrations were 52.4 and 48.9 ,uM, respectively. Symbols: 0, 1 day old; A, 2 days old; *, 3 days old; E, 4 days old; V, 5 days old.

comparative ability of nickel and zinc to compete for sorption sites was analyzed by linear regression. If nickel and zinc competed equally well for the same sites, a plot of the ratios of nickel sorbed/total metal sorbed versus nickel added/total metal added should produce a slope of 1. If nickel was not bound as effectively as zinc, the slope should be <1, and, conversely, if nickel bound more readily than zinc, the slope should be >1. The slope was 0.91 and was significantly different from 1.00 at P < 0.005, indicating that there was a slight selectivity for zinc over nickel. The binding of nickel and zinc was also affected by the presence of various organic ligands (Fig. 7). It was assumed that only free metal ions would be available for binding to the cells, and, as expected, the chelated metal species (NiEDTA, Ni-nitrilotriacetic acid [NTA], Zn-EDTA, and ZnNTA) did not bind to the cells. Similarly, the presence of acetate, glycolate, Tris, or citrate inhibited the sorption of nickel and zinc to biomass. However, acetate, glycolate, and Tris are relatively weak ligands for nickel and zinc; therefore, high concentrations of these ligands were needed to achieve a 30 to 50% reduction in the predicted metal species available for sorption. The ionic strength of the solutions was greatly increased in the trials containing such high concentrations of ligand, and controls with 30 mM KCI showed that high ionic strength alone substantially reduced the sorption of nickel and zinc (Fig. 7). It was noteworthy that lactate did not cause a decrease in the sorption of nickel. The desorption of metals by water, HEPES, KCI, CaCl2, and EDTA is shown in Fig. 8. The chelating agent EDTA was the most efficient desorption agent tested, with HEPES-

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SHUTTLEWORTH AND UNZ

APPL. ENVIRON. MICROBIOL.

VOL. 59, 1993

60i
-o

SORPTION OF HEAVY METALS TO THIOTHRIX STRAIN Al

I
0.40

1279

a)

A KCI

X
.0
0

Ni

o
U)

40*

N
4-

c 0
0

\ 0@~~~ \
0 O 0

0-

IL)
a-

20

O - --

= -

gCIa OCaCI2
E

0.0

0.0

0.5 1.0 1.5 2.0 Cation concentration (mM)

a.)

fo alcae10

20

KCI

het 10ct

Glyc TRIS Citr

EDTA

NTA

FIG. 5. Effect of cations on the sorption of zinc to Thiothrix strain Al. The zinc concentration was 50 ,uM, and the protein concentration was 173 ,ug/ml.
0

0.40

Zn

FIG

o.2

Efec oflgnsoEh opio fnce n ict

0.30-

bation time, metal concentration, and other parameters (20). In addition, under identical conditions metal uptake has been shown to be dependent on the test organism (21). It is therefore not surprising that reports on the extent of metal accumulation in bacterial cells give widely different results; the highest reported metal concentration in cells is 44% of cell dry weight (8). In the current study, nickel or zinc contributed 0.7 to 0.8% of Thiothrix strain Al cell dry weight when the bacterium was exposed to a metal concentration of 100 ,uM. Under identical conditions, copper amounted to 6.3% of cell dry weight. These concentrations are not

0~~~~~~~~~~~~~~~~

i1.050

0.0

cocetato wa 16 gm.Abeiton:1,1 u ea 10 20 KCI MCet Lact Glyc TRIS citr EDTA NTA actae LatE0m oimlcat;Gy,1 Msdu Omiu FIG. 7. Effect of ligands the sorption of nickel and zinc to Thiothrix strain Al. The total metal concentration 0.40 pmol (20 10 controls. Complexed metal cpM) for all cases except thei.M
on
was

species were considered

concentration
was

to be unavailable for

sorption.
10,
10

The

protein

168

p.g/ml.

Abbreviations:

.c

control; 20, 20 p.M metal control; KCI, 30 mM KCI; sodium acetate; Lact, 10 mM sodium lactate; Glyc, 10 mM sodium glycolate; TRIS, 20 mM Tris; Citr, 20 p.M sodium citrate; EDTA, 10 p.M sodium EDTA; and NTA, 10 p.M sodium NTA.

p.M metal Acet, 30 mM

oL
0.

unusual among bacteria

(8);

hence the filamentous

morphol-

~0

m
0

L._

ogy of the Thiothrix strain did not appear to enhance the capacity for metal adsorption.
The

affinity

of the cells for metals tested

was

Cu

>

Zn

1000

Ni concentration (pM)
c
._

-o

200C.
at
-

M. 0AM Ni M, 5s MNi M, 1 0 sM Ni

0, 20 FM Ni
S,

50OAMNi

'a

150100

.0

c,,L
400

00

8
C

o6

50

Cu
20
60; 70; 100 50 Zn concentration (pM)

Ni

Zn

n
W

FIG. 6. Competition between nickel and zinc for binding sites on Thiothrix strain Al. (A) Effect of zinc on the sorption of nickel; (B) effect of nickel on the sorption of zinc. The protein concentration was 173 p.g/ml.

FIG. 8. Desorption of metals from metal-laden Thiothrixc strain Al cells. The initial amount of metals sorbed was 431 pmol of Cu per g of cell protein, 160 pLmol of Ni per g of cell protein, or 174 ,umol of Zn per g of cell protein. The desorption medium consisted of 6 mM KCI, 5 mM CaC12, or 5 mM EDTA made either in ultrapure water or in 20 mM HEPES buffer at pH 7.20. Symbols: _, water; =, HEPES buffer; ED1, KCI; K3 KCI in HEPES buffer; GMZ, CaCl2; 1, CaC12 in HEPES buffer; M, EDTA in HEPES buffer. *, no

desorption.

1280
25
0

SHUTTLEWORTH AND UNZ

0

APPL. ENVIRON. MICROBIOL.

20
0
x
-o

15
10-

E c
0

(n

L-0

5
z

researchers working with a dissimilar type of bacterium, Zoogloea ramigera, also found that metal uptake was culture age dependent (23). The presence of organic ligands is usually assumed to reduce the sorption of metals to bacteria because only free metal ions are bioavailable. This is true provided that (i) ligands are not degraded by the bacteria and (ii) stability constants for the organic ligands are greater than constants
for the sites on the bacterial surface. In studies on ligand effects, most researchers have used very strong ligands which are generally not degraded. In our work we not only examined the ability of weak ligands to influence bacterial metal sorption but also used a computer model of the theoretical metal speciation to assist in the interpretation of the results. The observed effects of the strong chelating agents EDTA and NTA on nickel and zinc sorption to Thiothrix strain Al were accurately predicted from these theoretical considerations. The EDTA and NTA species were exactly 50% of the 0.4 pumol of total metal; therefore, 0.2 ,umol was predicted to be available for biosorption. The amounts of nickel and zinc which were sorbed from the EDTA and NTA solutions were nearly identical to the amounts sorbed from the controls, which contained 0.2 ,umol of total metal. The effects of acetate, lactate, glycolate, and Tris on metal sorption were more difficult to interpret. Because the stability constants of these compounds are relatively low, high concentrations were required to significantly alter metal speciation. The high concentration of ligands elevated the ionic strength of the reaction medium, and in these cases, it was not possible to separate the ionic strength effects from the ligand effects, even though a control for ionic strength was included in the experiments. Of the ligands used, only acetate and lactate are degradable by Thiothrix strain Al (37). Degradation of these ligands was expected to cause an increase in the amount of metal sorbed over the amount predicted to be available. There was no evidence for increased sorption with acetate, possibly because the lactategrown cells did not degrade enough acetate to affect metal speciation. In contrast, nickel sorption in the presence of lactate was not only higher than predicted in the absence of lactate degradation but also higher than that of the control (no lactate present). This result was reproducible. The trials with citrate were the most difficult to interpret, and it is possible that the predicted speciation was not accurate. It is well known that thermodynamic constants may be inaccurate because of analytical limitations (32), and in many instances the equilibrium constants in the MINEQL computer program were substantially different from constants found in the literature (18, 19, 28-30). However, it was not possible to replace the citrate thermodynamic data in the MINEQL program, because some of the needed critical stability constants were not available. The effect(s) of ligands on the sorption of metals has also been noted in other studies. For instance, Singh and Yadava (27) demonstrated that citrate stimulated cadmium uptake in the cyanobacterium Anacystis nidulans whereas EDTA inhibited cadmium uptake. These workers attributed the citrate-enhanced uptake to the availability of citrate as a carbon source for the organism. Similarly, Jardim and Pearson (13) found that citrate degradation during growth of Plectonema boryanum could cause an increase in the amount of free ionic copper in the growth medium. Predicting and interpreting metal uptake by growing cultures of bacteria is complex because media contain many

-a
.2

0-

Time (days) Uptake of nickel by Thiothrix strain The initial nickel concentration was 1.19 ,uM.
FIG. 9.

Al

during growth.

Ni. In experiments with mixtures of metals, there appeared to be little selectivity between nickel and zinc. The regression of the ratios method used to determine selectivity of nickel and zinc was unique, and it is suggested that this method be used in the future to provide a quantitative assessment of the competition of cations for binding sites. Although others have shown competition between metals for binding sites and metal selectivity (5, 7, 21), the potential for preferential uptake of metals at specific sites on microbial surfaces merits further study because of its importance to

biosorption technology. The sorption of metal ions to resting cells of Thiothrix strain Al appeared to involve primarily an ion-exchange type of reaction. This contention is supported by the data obtained in the kinetics, cation competition, and desorption experiments. It is well substantiated that metabolically independent uptake of metals is usually very rapid (8), and metal uptake by Thiothric strain Al was very rapid. In addition, metal desorption from Thiothrix strain Al was enhanced by increasing the ionic strength and by adding competing cations and chelating agents; therefore, most of the metal was rather loosely associated with the cells. However, approximately 20% of the bound metal ions were not removed from Thiothrix strain Al by the strong chelating agent EDTA; this
nonextractable metal could have diffused into the interior of (38) reported that Cu uptake in the alga Chlamydomonas rheinhardii occurred in two steps and presented evidence that the second step was a result of the diffusion of metals into the cells. Alternative explanations of the observed phenomenon in Thiothrix strain Al include (i) complexation of metals at sites with higher stability constants than EDTA and (ii) the presence of metal-binding sites which are inaccessible to EDTA. It seems unlikely, however, that there were surface-binding sites with higher affinities for metals than EDTA, because there was no evidence of any bacterial metal uptake from preformed metal-EDTA
the cells. Xue et al.

complexes.
Thiothrix strain Al has an unusual mode of metabolism in
that it appears to be an obligate mixotroph and requires a reduced inorganic sulfur source for growth (37). In a growth medium of acetate and thiosulfate, most of the thiosulfate is utilized in the early log phase of growth and sulfur granules are formed during this period of thiosulfate utilization (33). As the culture ages, stored sulfur disappears. Therefore, cells in early log phase differ metabolically from those in late

log phase,

and metabolic differences may have contributed

to the observed effect of cell age on nickel and zinc uptake. However, it is also possible that other time-dependent

changes accounted for the observed variations, because

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components which can influence metal toxicity and/or metal speciation. In this study the medium contained substantial amounts of calcium and magnesium, both of which were shown to inhibit the sorption of zinc in short-term experiments. The lactate in the medium must have formed complexes with the metals, but these complexed metal ions were probably made available by microbial degradation of the lactate. Furthermore, the pH of the LTH medium rose significantly during growth of Thiothrix strain Al, and the solution pH can affect the extent of metal binding. The removal of nickel from the growth medium did not, however, appear to be a result of pH-related sorption, since the cell-associated nickel was not desorbed by lowering the pH. In fact, the limited removal of cell-associated nickel by EDTA suggests that the metal uptake during growth was not an ion-exchange phenomenon; therefore, the mechanism for metal uptake in growing cells of Thiothrix strain Al may differ from the mechanism for uptake in resting cells. The availability of an energy source(s) may cause actively growing cultures to respond differently from resting cells, because energy-dependent metal efflux and energy-dependent metal uptake systems have both been reported (2, 22). Furthermore, microorganisms have been shown to alter their growth medium in ways which can inhibit metal uptake (11, 12). Shuttleworth and Unz (26) determined that copper was more toxic than either nickel or zinc to Thiothrix strain Al, and results from the present study revealed that Thiothrix strain Al had the highest sorption capacity for copper, although the highest adsorption intensity occurred at low concentrations of nickel. Calcium and magnesium were found to reduce the toxicity of heavy metals (26) and to reduce the sorption of zinc to the bacterium. Similarly, nickel toxicity was diminished by the presence of zinc, and the ability of zinc and nickel to compete for sorption sites was confirmed. The chelating agent EDTA effectively prevented nickel and zinc sorption, as well as decreasing the toxicity of these metals to the bacterium. Thus, all of the toxic effects of metals on Thiothrix strain Al, except the synergistic toxicity of copper-nickel and copper-zinc mixtures, can be accounted for by metal sorption.
ACKNOWLEDGMENTS This study was funded, in part, by NSF grant ECE 8417663 and by the Environmental Resources Research Institute, College of Engineering, and Department of Civil Engineering, all of The Pennsylvania State University. We thank Chris Cox for his instruction on the MINEQL program and William Tarutis for his help with the statistical analyses.
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