microbial cells as biosorbents forheavy metals: pseudomonas

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1981, p. 237-2450099-2240/81/010237-09$02.00/0

Vol. 41, No. 1

Microbial Cells as Biosorbents for Heavy Metals:Accumulation of Uranium by Saccharomyces cerevisiae and

Pseudomonas aeruginosaGERALD W. STRANDBERG,* STARLING E. SHUMATE II, AND JOHN R. PARROTT, JR.Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Uranium accumulated extracellularly on the surfaces of Saccharomyces cere-

visiae celLs. The rate and extent of accumulation were subject to environmentalparameters, such as pH, temperature, and interference by certain anions andcations. Uranium accumulation by Pseudomonas aeruginosa occurred intracel-lularly and was extremely rapid (<10 s), and no response to environmentalparameters could be detected. Metabolism was not required for metal uptake byeither organism. Cell-bound uranium reached a concentration of 10 to 15% of thedry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P.aeruginosa cells within a given population possessed visible uranium depositswhen examined by electron microscopy. Rates ofuranium uptake by S. cerevisiaewere increased by chemical pretreatment of the cells. Uranium could be removedchemically from S. cerevisiae cells, and the cells could then be reused as abiosorbent.

The use of microbial cells as biosorbents forheavy metals offers a potential alternative toexisting methods for decontamination or recov-ery or both of heavy metals from a variety ofindustrial aqueous process streams (3, 14, 20, 21,24). Biosorptive extraction of uranium from nat-ural waters has been proposed as well (18; E.Heide, M. Paschke, K. Wagener, and M. Wald,German patent 2,345,430, 1975). Metal accumu-lation by biosorption is generally considered tobe a rapid physical/chemical phenomenonwhich uses preexisting biomass. Two principlesites of uranium complexation in biological sys-tems have been proposed. Rothstein and Meier(17) suggested that on the surfaces of yeast cellsthere are reactive groups which are chemicallysimilar to high-molecular-weight polyphos-phates and are responsible for uranium com-plexation. Dounce and Flagg (7) provided evi-dence that the carboxyl groups of proteins effec-tively complex uranium. Recently, Beveridgeand Murray (4) and Matthews and Doyle (T. H.Matthews and R. J. Doyle, Abstr. Annu. Meet.Am. Soc. Microbiol. 1979, K86, p. 159) reportedthat the carboxyl groups of the peptidoglycansin the cell walls of Bacillus subtilis are theprimary sites of divalent metal complexation.Our investigations have been directed toward

the use of microbial biosorption of metals forthe removal of radionucides (e.g., uranium)from waste streams generated by nuclear fuel-processing plants (20, 21). Although our empha-sis has been on process development, we alsowant to understand the mechanism of uranium

uptake to determine whether it can be enhancedthrough environmental or genetic manipulationof microbial cells. Two organisms, Saccharo-myces cerevisiae and Pseudomonas aeruginosa,were selected from an earlier survey (20) for adetailed characterization of uranium uptake.Both had been used in previous metal uptakestudies (11, 14, 17; H. R. Meyer, Ph.D. thesis,Colorado State University, Fort Collins, 1977),and many aspects of their physiology, structure,etc., were documented in the literature. Theselection of these two organisms was fortuitousin that the evidence which we report belowsuggests two separate biosorption mechanisms.

MATERIALS AND METHODSUranium uptake experiments. The organisms

used in this study were S. cerevisiae NRRL Y-2574(Agricultural Research Service Culture Collection,Peoria, Ill.) and P. aeruginosa CSU (obtained from H.R. Meyer and S. Johnson, Colorado State University).Details of the methods for culture maintenance, cellproduction, and uranium assay were described previ-ously by Shumate et al. (21). Both organisms wereroutinely cultured in a rich medium containing thefollowing (in grams per liter): glucose, 10.0; yeast ex-tract, 3.0; malt extract, 3.0; and peptone, 5.0 (22). Theuranium uptake ability of S. cerevisiae cultured in thesucrose mineral salts vitamin medium described byWinge and Roberts (23) was also examined. Briefly, todetermine uranium uptake, a suspension of washedcells from a 24-h culture was added to a uranyl nitratehexahydrate solution (-100 g of uranium per m3)equilibrated to the desired temperature (40°C unlessnoted otherwise). This cell suspension was preparedto provide about 0.4 mg (dry weight) of cells per ml in

237

238 STRANDBERG, SHUMATE, AND PARROTT

a uranyl nitrate cell mixture. Dry cell weights wereobtained by air-drying (100°C) known volumes of cellsuspensions before they were exposed to uranium.Additions or adjustments to the uranyl nitrate solutionwere made before cells were added. In those instanceswhere the cells were treated chemically, the washedcells were exposed to the chemical agent at roomtemperature for a given time and then rewashed threetimes with deionized, distilled water before contact (orin some cases, recontact) with uranyl nitrate. Theuranyl nitrate cell mixture was shaken at 100 rpm(2.54-cm stroke) on a rotary shaker. At the desiredtime, cells were removed from samples of the primarysuspension by centrifugation at -2,500 x g for 3 to 5min. The remaining soluble uranium was assayed spec-trophotometrically by using Arsenazo III reagent (16,19, 21). Cell-free controls were run concurrently in allexperiments.We tried methods other than centrifugation to ob-

tain a more rapid separation of P. aeruginosa cellsfrom the uranium solution. Filtration under positiveor negative pressure through 0.22-, 0.45-, and 1.2-,ummembrane filters (Millipore Corp., Bedford, Mass.)and a variety ofsintered glass filters was unsatisfactorybecause the cells rapidly plugged the filters and thefilters themselves adsorbed some uranium. Some suc-cess was obtained in preliminary experiments in whichthe cells were passed rapidly (under air pressure of -1to 2 lb/in2) through a small glass column (inside di-ameter, -1 cm) containing a 2-cm bed of Bio-Rad AG50W-X12 ion-exchange resin (200 to 400 mesh; hydro-gen form; Bio-Rad Laboratories, Richmond, Calif.) toadsorb the remaining soluble uranium. The resin bedwas then washed extensively with water to remove theremaining cells. Uranium was eluted from the resinbed with 1.0 M nitric acid and analyzed by the stan-dard Arsenazo III assay after pH adjustment (to pH4.0 with NaOH) of the eluate.

Electron microscopy. Transmission electron pho-tomicrographs and energy-dispersive X-ray data wereobtained by L. K. West (Department of Biochemistry,University of Tennessee, Knoxville) and J. Bently(Metals and Ceramics Division, Oak Ridge NationalLaboratory, Oak Ridge, Tenn.). The general procedureinvolved fixation of uranium-exposed cells with 1%osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2)for 3.5 h, dehydration in a graded series of acetonesolutions, and embedment in plastic (L. K. West, Ph.D.thesis, University of Tennessee, Knoxville, 1977). En-ergy-dispersive X-ray analyses with JEM-100CX andPhillips EM 400T/FEG analytical electron micro-scopes (10- to 100-nm variable beam size) of thin (100-nm) and thick (250-nm) sections were used to verifythe location of uranium.Calcium and potassium effects. To determine

the effect of calcium on uranium uptake, the uranylnitrate solution (100 g of uranium per mi3) was supple-mented with 8 x 10-2 Ci of 45CaC12 (New EnglandNuclear Corp., Boston, Mass.) per m3 and 100 g ofunlabeled CaCl2 per m3. After contact with the cells,soluble uranium was measured by using the standardArsenazo III assay, except that 8 x 10-4 M ethylene-diaminetetraacetic acid was included in the assay mix-ture to complex the calcium (16). Soluble calcium andcell-bound calcium (sulfuric acid digest) were meas-

ured by standard liquid scintillation counting tech-niques.

In the case of potassium, 100 g of K+ (as KCl) perm3 was added to the uranyl nitrate solution beforecells were added. Soluble uranium was measured bythe standard Arsenazo III assay.Complexation of uranium by amino acids.

Amino acids (0.1%, wt/vol) were added to the uranylnitrate solution, and the pH was adjusted to 4.0 withNaOH. Uranium uptake was followed as describedpreviously.Phosphomannan. Phosphomannans from Han-

senula holstii NRRL Y-2449 and Hansenula capsu-lata NRRL Y-1842 (kindly supplied by M. E. Slodki,Northem Regional Research Center, Peoria, Ill.) weredissolved in deionized, distilled water and then mixedindividually with uranyl nitrate hexahydrate solutionsto give a final uranium concentration of 478 or 1,044g/m3 and a final phosphomannan concentration of0.2% (wt/vol). After the solutions were stirred for 1 hat room temperature, the phosphomannans were pre-cipitated by adding 1.5 volumes of 95% ethanol-0.1%(wt/vol) KCl. The precipitates were removed by cen-trifugation at 7,000 x g for 20 min, and soluble uraniumin the supematant was measured by the Arsenazo IIIassay. A uranyl nitrate hexahydrate solution wastreated in the same manner, as a control.

RESULTSThe microbial cells used in these experiments

were cultured in the absence of uranium. Inaddition, they were washed free of extraneousnutrients before exposure to uranium. There-fore, the measured uranium uptake was consid-ered to be an inherent property of the cells andnot associated with cell growth. This was furtheremphasized by the rapid rates of metal uptakeby S. cerevisiae and P. aeruginosa (Fig. 1). Therate of uptake by P. aeruginosa was extremelyrapid. Although attractive for process applica-tion, this rapid rate has hampered studies of theeffects of environmental conditions on uraniumuptake by this organism. Although used rou-tinely in our experiments, centrifugation to sep-arate the cells from the metal solution signifi-cantly extends the time of exposure. Throughoutthis study, we examined alternative separationtechniques. Filtration proved to be impracticalsince the cells rapidly plugged membrane andglass filters and the filter materials themselvesadsorbed some uranium. Using an ion-exchangeseparation technique (see above), we recentlyobtained preliminary evidence that uranium isassociated with P. aeruginosa cells within 5 to10 s after the cells contact uranium. Thus, as aconsequence of this rapid uptake rate, the ap-parent lack of any effect of the conditions im-posed on uranium uptake by P. aeruginosa inthe experiments described below may have beendue to our inability to detect it within the timeframe of the measurements. However, uranium

APPL. ENVIRON. MICROBIOL.

MICROBIAL ACCUMULATION OF URANIUM 239

100

on

80

z0

-i0

60

z0

(, 40z

z4 4

0 2

0 1 2 3 4 5 6 22

TIME (h)

FIG. 1. Removal of uranium from aqueous solu-tions by S. cerevisiae and P. aeruginosa.

accumulation by S. cerevisiae was sufficientlyslow so that the time required for centrifugation(3 to 5 min) was a less significant fraction of thetotal time before equilibrium was reached be-tween soluble uranium and cell-bound uranium.

Despite differences in the rates of uraniumuptake, the total capacity for metal accumula-tion (10 to 15% of the original dry cell weight),was the same for both species. Although gener-ally the uranium concentration on the cells wasinferred by the measured loss of soluble ura-

nium, our results were confirmed by an inde-pendent analysis of the exposed cells by theAnalytical Chemistry Division, Oak Ridge Na-tional Laboratory.

Electron microscopic examinations and en-

ergy-dispersive X-ray analyses showed that ura-

nium accumulated as needle-like fibrils in a layerapproximately 0.2 ,tm thick on the surfaces of S.cerevisiae cells (Fig. 2). Little or no uranium wasfound inside these cells or inside cells withoutvisible uranium deposits. In contrast, uraniumformed dense intracellular deposits in P. aerugi-nosa (Fig. 3). This is surprising in view of therapid rate of metal uptake. The cells shown inFig. 3 were exposed to uranium for 2 h. Becausethe rate of uranium uptake was so rapid, we didnot attempt an electron microscopic examina-tion of these cells after shorter exposure times.Although we lack visual evidence of immediateintracellular deposition of uranium, we do knowthat uranium is fimly associated with the cells

within a few seconds (see above).It is evident in Fig. 2 and 3 that not all of the

cells possessed visible uranium deposits. Thosecells which had uranium deposits (32 and 44% ofall S. cerevisiae and P. aeruginosa cells, respec-tively [averages from several fields of view hav-ing 20 to 30 cells of S. cerevisiae and -100 cellsof P. aeruginosa]) showed no apparent struc-tural differences from those cells which did nothave such deposits; both budding and nonbud-ding S. cerevisiae cells had deposits. R. Mc-Kracken (University of Vermont) developed atechnique whereby uranium-exposed cells ofboth species could be separated into two bandsby light centrifugation (2,000 to 3,000 x g, 5 to10 min) after layering on 40% (wt/vol) CsCl. Asindicated by its reaction with the Arsenazo IIIreagent, the heavier cell band contained the bulkof the sorbed uranium. We were able to culturea few viable cells from the separated bands onstreak plates, but there was no difference inuranium uptake by isolates of either speciescompared with the parent cultures.The conditions used to culture cells can affect

uranium uptake. S. cerevisiae cultures grown ona synthetic medium (23) had a faster rate ofuptake by a factor of 2.5 than cultures grown ona rich organic medium (22). Although not quan-titated, the growth rate and cell yield were re-duced in the synthetic medium. On the otherhand, there was no difference in metal uptakebetween aerobically and anaerobically grownyeast cells.

Published reports of metal biosorption andour own experience with S. cerevisiae indicatedthat uranium uptake did not require cellularmetabolism. However, the rapid intracellularuptake of uranium by P. aeruginosa suggestedthe possible involvement of metabolically me-diated active transport. Cells of both specieswere exposed separately to uranium in the pres-ence of the metabolic inhibitor 2,4-dinitrophenol(5 x 10-3 M) or sodium azide (1 x 10-4 M), orthey were pretreated for 5 to 10 min with HgC12(1.0% solution) or formaldehyde (10% solution)before exposure to uranium. These latter twocompounds were lethal for both species (no cellscould be cultured from the treated cell prepa-rations). Uranium uptake by both species wasnot affected by either metabolic inhibitor. Al-though formaldehyde and HgCl2 treatments hadno apparent effect on P. aeruginosa, the rate ofuranium uptake by S. cerevisiae (Fig. 4) wasincreased by these treatments. The cellular Hgconcentrations before and after exposure to ura-nium were not determined.Temperature and the initial solution pH had

a dramatic effect on metal uptake by S. cerevi-

VOL. 41, 1981


U

FIG. 2. Electron micrograph of S. cerevisiae showing surface accumulation of uranium. x35,000.

FIG. 3. Electron micrograph of P. aeruginosa showing intracellular accumulation of uranium. x27,000.


MICROBIAL ACCUMULATION OF URANIUM

siae. As Fig. 5 shows, the rate of uptake in-creased with temperature between 20 and 500C.Although the initial rate of uptake increased as

the pH was raised from pH 2.5 to 5.5, maximalequilibrium distributions were obtained betweenpH 3.0 and 4.0 (Fig. 6). There was no discernible

1001I

* UNTREATED

o FORMALDEHYDE TREATED

E A UNTREATEDso6 a H9CI2 TREATED

z

0

60

Z

z

0

Z 40w

z

0

'q20

FIG.treatme

100

E

z

0en 60

z

Z 4C0

z

tr2C

FIG.take by

100 I

pH 2.5

Ego

FIG. \.Effect of initialpH 3.0

60

S0

z

40 pH

20 pH 435

TIME (hI

FIG. 6. Effect of initial pH on uranium uptake byS. cerevisiae.

._A § $t+------response ofP. aeruginosa to temperature or pH,_______________________' although it must be remembered that we were

0 2 3 4 5 6 22 unable to observe the transition between the

TIME (h) initial and equilibrium stages. Since the normal

4. Effects of HgCl2 and formaldehyde pre- pH of the uranyl nitrate solution was about 4.0,ants on uranium uptake by S. cerevisiae. no routine pH adjustment was necessary.

Water washing was ineffective in removing> < uranium from the cells. Several agents that sol-

ubilize or complex with uranium were used totreat cells of S. cerevisiae that had been exposedto uranium; 16-h suspensions in 0.1 M nitric

o * acid, 0.1M disodium ethylenediaminetetraacetic0 \20- acid, and 0.1 M ammonium carbonate removed

only 59.3, 72.3, and 83.5%, respectively, of thebound uranium. To determine whether surfacebinding sites were altered by these treatments,

the treated cells were washed and reexposed touranium. As Fig. 7 shows, all three treatments

\30C enhanced the initial rate of uranium uptake, butnitric acid and sodium ethylenediaminetetraa-cetic acid greatly reduced the concentration ofuranium on the cells at equilibrium. Two other

40C \ agents (data not shown), sodium citrate (0.1 M)and potassium oxalate (1.0 M), removed 57 and

50so c 14%, respectively, of the bound uranium and

i1_ iI~ Iiiir:nup-- mresulted in an increased rate of metal uptakesimilar to the increased rate observed after am-

I monium carbonate treatment. Whereas ammo-)) I1 2 3 4 5 6 22 nium carbonate treatment before or after theTIME (h) initial exposure to uranium increased the rate of

5. Influence of temperature on uranium up- metal uptake, sodium citrate and potassium ox-S. cerevisiae. alate were effective only after prior exposure of

VOL. 41, 1981 241


100

W;-E

.Tz0

D-i0V)

z0

.4ll.

zuiuz0u

2

z-4mD

s0

60

40

20

0 1 2 3 4 5 6 22TIME (hI

FIG. 7. Uranium uptake by S. cerevisiae (preex-posed to uranium) after treatment with nitric acid,di,sodium ethylenediaminetetraacetic acid (EDTA),or ammonium carbonate.

the cells to uranium. Sodium ethylenediamine-tetraacetic acid and nitric acid were not testedin this way.

It is known (7) that insoluble complexes ofproteins, such as casein, and uranium occur.

However, we found that soluble Casamino Acids(Difco Laboratories, Detroit, Mich.) also ex-

hibited this phenomenon. Amino acid analysesof a Casamino Acid solution before and afterexposure to uranium tentatively implicated cys-teine and glutamic acid as the amino acids in-volved in the complexation and precipitation ofuranium. To determine whether these and otheramino acids were strong enough complexingagents to compete with cells for uranium, theywere incorporated individually (0.1%, wt/vol) inthe uranyl nitrate solution (pH 4.0) before cells

were added. The presence of several amino acidshad no effect on uranium uptake by P. aerugi-nosa. Whereas glutamic acid and aspartic acid(dicarboxylic amino acids) strongly inhibiteduranium uptake by S. cerevisiae (Fig. 8), none

of the monocarboxylic amino acids, includingthe sulfur-containing amino acids cysteine andmethionine, had any effect. Substitutions on theamino group (e.g., N-methylglycine, N,N-dime-thylglycine, and glycylglycine) of a monocarbox-ylic amino acid did not result in interference,whereas the corresponding organic acids (e.g.,acetic acid) did interfere with metal uptake.

Rothstein and Meier (17) observed that cer-

tain divalent cations (Ba2", Ca2") interfered withuranium uptake by S. cerevisiae, whereas mon-ovalent cations had no effect. We wanted toverify this with our yeast strain and to determinewhether there was a similar effect on internaluranium accumulation in P. aeruginosa. Also,potentially interfering metals would be of con-cern in any biological treatment process for ura-nium removal. Potassium (100 g of K+ per m3,as KCl) had no effect on uranium uptake byeither organism, nor did Ca2" (100 g/m3, asCaCl2) interfere with uptake by P. aeruginosa.45Ca was taken up concomitantly with uraniumin this organism. Both the initial rate of uraniumuptake and the ultimate equilibrium distributionin S. cerevisiae were altered by Ca2" (Fig. 9).Calcium was bound at a slightly slower rate thanuranium during the first 2 h, but uranium be-came displaced from the cells as calcium uptakecontinued. The presence of uranium enhancedcalcium uptake.Phosphate groups have been implicated as

sites of uranium complexation by S. cerevisiae(17). Phosphorylated polysaccharides (phospho-mannans) comprise a portion of the cell wall inseveral yeast species, including S. cerevisiae (9).We were able to obtain samples of purified andwell-characterized (particularly with regard tothe phosphate/mannose ratios) yeast phospho-mannans from two species of Hansenula. AsTable 1 shows, uranium complexation by these

100 , ,

E go CELLS PLUSGLUTAMICACID

2

CELLS PLUSO0 ASPARTIC60 -ACID

z0

4 CELLSONLY

wZ 40-z0

z~200

0~~~~~~~~~~0 1 2 3 4 5 6 22

TIME (h)

FIG. 8. Inhibition of S. cerevisiae uranium uptakeby glutamic acid and aspartic acid.

UNTREATED

AMMONIUM CARBONATE -

0& TREATED

0

l l



-J0.10

U (U ONLY)

6a 0.06 - //-

gr ~ o (U+Co)

IL / CaCaOLY

0.06

EA. 0.04J

ICa (Ca ONLY)

0.02

0

0 1 2 3 4 5 6 24

TIME (h)

FIG. 9. Effect of Ca2 on uranium uptake by S.cerevisiae.

TABLE 1. Complexation of uranium by yeastphosphomannans

Solution ura- Phospho-Mannose/ nium concnPhosphomantemanna

nan from: phspateio/m' uraniumInitial Final concn (%)

H. holstii -5 478 224 12.7NRRL Y- 1,044 754 14.52448

H. capsulata -2.5 478 48 21.5NRRL Y- 1,044 432 30.61842a Final concentration, 0.2% (wt/vol).b Mannose/phosphate ratios were supplied by M. E.

Slodki, Northern Regional Research Center, Peoria,Ill.'Percentages were calculated as follows: [(grams of

uranium)/(grams of phosphomannan)] x 100.

phosphomannans was related to their phosphatecontents.

DISCUSSIONUranium uptake by S. cerevisiae and uranium

uptake by P. aeruginosa differ in many respects.The surface-associated accumulation ofuraniumexhibited by S. cerevisiae is consistent with theview that metal biosorption occurs by the com-plexation of positively charged metal ions withnegatively charged reactive sites (e.g., R-COO-,P042-) on the cell surface (2-4, 17) or in extra-cellular polymers (8). As expected, metal up-take was affected by environmental parameters,such as pH, temperature, and competing cations(i.e., Ca2). Not only can environmental changes

affect reactive metal-binding sites, but alsothe solution chemistry of uranium is quite com-plex. In the pH range of the optimal uraniumuptake (pH 3.0 to 4.5), soluble uranium existsas UO22" (1) and other hydrolysis products[(UO2)2(OH)22+, U02(OH)+, (UO2)3(OH)5+].Since carbon dioxide was not excluded in ourexperiments, carbonate complexation reactionswith the uranyl ion could also take place. At-tempts to determine the chemical state of ura-nium as it exists on cell surfaces have beenunsuccessful. We have observed that as uraniumis taken up by cells, the pH increases from -4.0to 5.5 to 6.0, indicating a release of free hydroxylions. This suggests that UO22+ could be the formof the bound metal, and in fact, UO22+ complexesreadily with a variety of anions (7).The relatively large amounts of uranium ac-

cumulated (10 to 15% of the dry cell weight)were of concern. These values are similar to thevalue obtained for a mixed culture of denitrifyingbacteria (Shumate et al., Biotechnol. Bioeng., inpress) and Rhizopus arrhizus (B. Volesky, per-sonal communication). However, it was difficultto imagine that there were sufficient bindingsites to account for this much metal. Actually,since only 32% of the cells possessed measurableamounts of uranium, the metal concentration onthat fraction of the cells approached 50% on adry weight basis. Beveridge (2) also observed anon-stoichiometric accumulation of metals onisolated cell walls of B. subtilis. He suggestedthat metal molecules complex with existing re-active sites and that additional metal "crystal-lizes" on these bound molecules. It would helpto know the chemical nature of the bound metal.Although no physiological or genetic basis forthe distribution of metal uptake in cell popula-tions has been found to date, this phenomenonis not only of basic scientific interest, but also ofgreat practical importance. If an entire popula-tion could be induced to accumulate metalthrough environmental or genetic manipulation,the potential of the process would be enhanced.

Rothstein and Meier (17) provided evidencethat polyphosphate groups of S. cerevisiae com-plex with uranium. Our findings that uraniumcomplexed with yeast phosphomannans andthat the uranium complexation capacity of thepolymers appeared to be related to their phos-phate contents support the hypothesis thatphosphate groups play a role in metal binding.Carboxyl groups are also active in metal com-plexation (4, 7; Matthews and Doyle, Abstr.Annu. Meet. Am. Soc. Microbiol. 1979). The factthat yeast cells grown on a synthetic mediumshowed enhanced uranium uptake rates mightbe due to an increase in the phosphate andprotein contents of the cell walls, which is known

VOL. 41, 1981


to occur in yeast cultured at reduced growthrates (13). It would be worthwhile to examinethe effects of growth conditions and nutrientlimitations in both batch and continuous cul-tures on the cell wall compositions of the metal-binding and -nonbinding yeast cells separable byCsCl centrifugation. Unfortunately, we have yetto find a method for removing bound uraniumwithout an apparent effect on the cell surface(as evidenced by changes in subsequent metaluptake).Dounce and Flagg (7) carried out extensive

studies on the complexation of uranium withorganic acids, proteins, and a few individualamino acids, using a titration technique. Organicacids and proteins were very effective in com-plexing with uranium. Although these authorsconcluded that the free carboxyl groups of pro-teins were the active sites of uranium complex-ation, they were unable to explain the very lim-ited interaction between uranium and the mon-ocarboxylic amino acids serine and glycine.Rothstein and Meier (17) considered these stud-ies in interpreting the results of their own equi-librium dialysis experiments, which measuredthe affmities of various complexing agents, in-cluding proteins, for uranium. They observedthat the maximum amount of uranium boundby a protein accounts for only 20% of the avail-able "free" carboxyl groups. Their suggestionthat "as in the case of copper, only those car-

boxyl groups which have adjacent accessorygroups such as carboxyl and hydroxyl groupscan form stable complexes with uranyl ion" wasone of their reasons for discounting yeast cellsurface proteins as important for uranium com-

plexation.Our experiments on the competition between

yeast cells and soluble amino acids for uraniumshowed that dicarboxylic amino acids are effec-tive complexing agents for uranium. We ob-served that monocarboxylic and N-substitutedamino acids were ineffective in the competitionexperiments. Molecular models indicated nobasis for a steric hinderance of uranium com-plexation by these compounds. We have con-cluded that the positive charge on the aminogroup, which exists in the pH range of our ex-periments, is sufficient to prevent uranium fromcomplexing with the proximal carboxyl group.The distant carboxyl group of the dicarboxylicamino acid is free to complex, as are the carboxylgroups of the corresponding organic acids. Thisinterpretation would explain the lack of com-

plexation by serine and glycine observed byDounce and Flagg (7). It would also provide analternative explanation for the less-than-stoichi-ometric complexation of uranium by the availa-ble free carboxyl groups in a protein, as reported

by Rothstein and Meier (17); i.e., there are rel-atively few carboxyls (based on their proximityto an amino group) that are free to complex withuranium. Two other related points should bementioned in regard to this general question.The first is that reducing the positive charge oncell wall components by chemical treatment(e.g., formaldehyde) can enhance metal uptake(5). The second is that there is recent evidencethat the most effective site for metal complexa-tion on the cell walls ofB. subtilis is the carboxylgroup of glutamic acid in the peptidoglycan (4).With time, the competitive effect of glutamic

and aspartic acids diminish, and uranium be-comes associated with the cells (Fig. 8). Weconsidered the possibility that the cells mightmetabolize these amino acids, thus relieving thecompetitive effect, but we did not attempt tomeasure changes in amino acid concentrations.However, initially there was a considerableamount of amino acid present (0.1%, wt/vol),and uranium is to some extent toxic to S. cere-visiae (17; unpublished data). Also, as men-tioned above, uranium uptake is normally ac-companied by an increase in the pH of thesolution. No substantial change in pH occurredin the competition experiments. These factorsindicate that relief of the competitive effect isnot due to amino acid depletion. The fact thaturanium becomes preferentially associated withthe cells cannot be fully explained at this time.The chemistry of uranium is quite complex, andwe do not know the chemical nature of thebound uranium. The theory of Beveridge con-cerning nucleization and crystallization of ura-nium on the cell surface deserves further atten-tion. Rothstein and Meier (17) stated that theyeast-uranium complex was the most stablecomplex which they observed.

Since the presence of active metal-bindingsites on the surfaces of S. cerevisiae cells has notbeen established, we have no explanation for theincrease or decrease in uranium uptake ratesresulting from chemical pretreatment (e.g., am-monium carbonate, HgCl2, formaldehyde). Asnoted above, formaldehyde could enhance metaluptake by reducing the overall positive chargeof cationic sites on cell walls. Again, both thetheoretical considerations and the practical ben-efit of increasing microbial metal uptake are ofimportance.The responses of uranium uptake by S. cere-

visiae to pH, temperature, and interfering metalions (e.g., Ca2+) can be understood if the uptakeprocess is similar to an ion-exchange type ofmechanism. However, how do we explain therapid intracellular uptake of uranium by P.aeruginosa? Within the limits of our ability tomeasure uranium uptake, the process in this



species appears to be insensitive to environmen-tal conditions, and we do not know how uraniumenters the cell so rapidly (metabolism has beendiscounted). Once inside the cell, uranium ap-pears to be localized. Perhaps the heavy metal-binding protein metallothionein, whose presencehas now been verified in the procaryote Syne-chococcus sp. (15), is involved. Interestingly, inanother bacterial system which we have exam-ined (Shumate et al., in press), a mixed cultureof denitrifying bacteria behaves in a mannersimilar to P. aeruginosa (i.e., there is a veryrapid uranium uptake and an apparent insensi-tivity to environmental parameters). This mixedculture is believed to be composed predomi-nantly of pseudomonad-like organisms (10). Therates of uranium uptake in this culture wererapid enough to prevent an assessment of masstransfer phenomena.

Since only 44% of the cells in the electronmicrographs of P. aeruginosa contained visibledeposits of uranium, there is again the theoreti-cal and practical question of whether genetic orenvironmental factors govern metal uptake.We originally hoped to be able to extrapolate

the results of our studies with the two speciesused to enhance our knowledge of metal bio-sorption in a variety of biomass sources. Thefact that there are at least two different biosorp-tion systems, which are subject to different pa-rameters, precludes any generalizations. En-hancement by any means is dependent on thetype of biosorption system involved. Futurestudies will be directed toward elucidating thetwo mechanisms observed. We are particularlyinterested in why only a portion of the cells in apopulation take up uranium and how this pro-portion can be increased. We also intend toinclude other radionucides and heavy metals inour metal uptake studies, using the same generalapproach described in this paper.

ACKNOWLEDGMENTSThis research was sponsored by the Office of Nuclear Waste

Management, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corp.

LITERATURE CITED

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2. Beveridge, T. J. 1978. The response of cell walls ofBacillus subtilis to metals and to electron-microscopicstains. Can. J. Microbiol. 24:89-104.

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VOL. 41, 1981

microbial cells as biosorbents forheavy metals: pseudomonas

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