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638 Biofechnol.Prog. 1995, 11 , 638-642

Adsorption of Copper and Chromium by Aspergillus carbonariusSameer Al-Ashehand Zdravko Duvnjak"Chemical Engineering Department, University of Ottawa, 161 Louis Pasteur, Ottawa, Ontario, Canada K1N 6N5

Aspergil lus carbonarius NRC 401121 adsorbs some copper and chromium from theirsolutions. The amount of the adsorbed metal per unit of biomass increased with adecrease in the biomass concentration. The increases in th e initial concentrations ofmetals and pH of the solutions resulted in an increase in copper and chromium uptake.The optimum temperature for copper uptake was 25 "C.Heating of the biomass priorto utilizing it in the adsorption tests decreased its metal adsorption capacity.Preincubation of the biomass with glucose enhanced the metal adsorption. Theoptimum glucose concentration in this process was 0.1%.

IntroductionThe utilization of microbial biomass for the removalof metals from industrial waste waters and pollutedwaters has been already recognized. The biomass has

to satisfy the following requirements to be considered asan adsorbent (Volesky, 1987): (1) he efficient and rapiduptake and release of metals, (2) he low production costof the biosorbent and possibility of its reutilization, (3)the efficient, rapid, and cheap separation of the biosor-bent from the solution, and (4) a high selectivity of metaladsorption and desorption. If they are fulfilled, theutilization of the biomass would make the biosorptionprocess more attractive compared to processes which useother types of adsorptions.The mechanism of metal uptake by microorganisms isnot well-known, but some attempts had been reportedin order to explain it. Harris and Ramelow (1990) statedthat the accumulation of metals by microorganism maytake place by any or a combination of the followingprocesses: (1)entrapment by cellular components, (2)active transport across the cell membrane, (3) cationexchange or complexation, and (4) adsorption. Kuyucakand Volesky (1988a) reported that cell walls of microbialbiomass, mainly composed of polysaccharides, proteins,and lipids offer particularly abundant metal-bindingfunctional groups such as carboxylate, hydroxyl, sul-phate, phosphate, and amino groups. Muraleedharanand Venkobachar (1990) attributed the metal-bindingcapacity of microbes to their proteineous component.Some microorganisms have been studied for the ad-sorption of copper(I1) and chromium(II1). Gandermalucidum (Muraleedharanand Venkobachar, 1990), Rhizo-pus arrhizus, Cladosporium resinae, and Pencilliumitalicum (Rome and Gadd, 19871,Arthrobacter giacomell(Grappelli et al., 1992), Escherichia coli (Baldry andDean, 19801,Aspergillus niger and Mucor rouxii (Mullenet al., 1992),Saccharomyces cerevisiae (Avery and Tobin,19931, and Bacillus megaterium (Mohapatra et al., 1993)are microorganisms that have been used fo r copper(I1)uptake. There are also microorganisms which were usedfor chromium(II1)uptake, such asA. giacomell (Grappelliet al., 19921,R . arrhizus (Tobin et al., 1984),Streptomycesnoursei (Mattuschka and Straube, 1993), Halimedaopuntia, Ascophyllum nodosum, and S. cerevisiae (Kuyu-cak and Volesky, 1988a).In this work, Aspergillus carbonarius is used to testits ability fo r copper(I1) and chromium(II1) uptake. Themicroorganism has been previously used for the produc-tion of phytase and reduction of phytic acid content in

canola meal during a solid-state fermentation process (Al-Asheh and Duvnjak, 1994). The objective of this workis to study the effects of the biomass and metal concen-trations, pH, temperature, and addition of glucose to themetal-containing solutions on the adsorption of themetals.Material and Methods

Microorganism, Media, and Culture Conditions.A. carbonarius NRC 401124 was used to study theadsorption of Cu2+and C?+ from their solutions. Thesolid and liquid media for growth of this microorganismwere prepared as previously reported (Al-Asheh andDuvnjak, 1994). The microorganism grew in the liquidmedium in the form of small pellets ranging from 0.5 t o5 mm in diameter. The pellets were separated bycentrifugation (1OOOOg x 15 min) and washed three timeswith saline solution (2%NaC1). After that the cells werefiltrated and kept at 4 "C before their utilization in theadsorption experiments.Adsorption Experiment. A required amount of thecells was weighed and transferred into a 250 mL Erlen-meyer flask containing 49 mL of 5mM 2-(N-morpholino)-ethanesulphonic acid (MES) buffer, adjusted to pH 6(unless otherwise specified) with 1N NaOH. This bufferhas negligible metal-complexing properties (Good et al.,1966). Dry weight of biomass was obtained by drying at105 "C for at least 48 h.One milliliter of either 5000 ppm Cu2+ in the form ofCuS04.5Hz0)or 5000 ppm C13+ (in the form of [Cr(H20)4-Cl21Cl2.H~O)was added to the biomass suspension in thebuffer to make the final metal concentration 100 ppm(unless otherwise stated). The mixture was agitated ona shaker and samples were taken for analysis. Thebiomass was separated by vacuum filtration using 0.45pm filter paper, and the filtrate was analyzed for themetal, the adsorption of which was studied, using atomicabsorption spectrophotometer (Varian AA-1475 series).Each experiment was carried out in duplicate, and theaverage results are presented in this work.

Results and DiscussionThe effect of the biomass concentration on the copperuptake was studied using the biomass in the range of1.15-9.20 mg per milliliter of a 100 ppm CuZfsolution.The results indicate (Figure 1) hat the uptake of Cu2+per unit of biomass increased with a decrease in thebiomass concentration. Depending on the biomass con-

8756-7938/95/3011-0638$09.00/0 995 American Chemical Society and American Institute of Chemical Engineers

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Ill

0 10 20 30 40 50 60 70Time (min)

Figure 1. Effect of biomass concentration on copper up tak e atp H 6 of a 100 ppm copper solution. Symbols are experimenta land solid lines are data predicted us ing eq 1.Biomass concen-t ra t ion (mg/mL); 0 , 1.15; 0, 2.3; A, 4.6; 0, 9.2.Table 1. Constants a and c of Eq 1 for Various BiomassConcentrations

biomass (mg/mL) a (pg/(mL-min')) C1.152.34.69.240.17 0.043218.8 0.06123.941 0.24571.391 0.2569

centration, 31-55% of the initial Cu2+ oncentration wasadsorbed. The higher metal loading of biomass with adecrease of its concentration has also been noticed byGadd and White (1989) fo r the uptake of thorium by S.cerevisiae. The biosorption kinetics are also shown inFigure 1. The kinetics s tudy is essential to establish therates of metal uptake and release. A rapid uptake wouldallow a short solution-biosorbent contact time and wouldresult in the use of much shallower contact beds ofsorbent materials in their column applications (Kuyuacakand Volesky, 1988a). The results of Figure 1,as mostothers in this work, show that the largest amount of theadsorbed metal ions was attached to the biomass withinthe firs t 10 min of the adsorption process and, after 30min, further metal uptake was almost negligible. Theresults from Figure 1 can be very well represented bythe following empirical correlation (Figure 1):

q = atc (1)where q is the metal uptake, t is the contact time, and aand c are empirical constants which were evaluated byfitting eq 1 o the experimental data using the programSCIENTIST (Micromath Scientist Software). These con-stants for different biomass concentrations are presentedin Table 1.The Cu2+ solutions which contained from 25 to 400ppm of this ion and 9.20 mg of biomass per milliliter ofsolution were used to study the effect of the initial Cu2+concentration on its uptake by the biomass. The resultsshow (Figure 2) that the increase in the Cup+uptake isassociated with an increase in the initial metal concen-tration. This can be explained by the progressive in-crease in electrostatic interactions, relative to covalentinteractions, with sites of progressively lower affinity forCuf2 when Cuf2 initial concentrations increased (VanCutsem et al., 1984). Another experiment was performedto test the biomass saturation by this metal; the adsorp-tion was carried out fo r 15 min, and the initial Cu2+

0 IO 20 30 40 50 BO 70Time (min)

Figure 2. Effect of copper concentration on i t s up ta ke by A.carbonarius at pH 6 of a 9.2 mg/mL biomass su spensio n. Copperconcentration (ppm): 0, 25; +, 50; 100; A, 200; A, 400.concentrations were extended to 3500 ppm. The resultsof the equilibrium concentrations, i.e. the Cu2+concen-tration in the suspension after adsorption, and Cu2+uptake indicate (Figure 3) that the biomass of A. car-bonarius was rather saturated with Cu2+when its initialconcentration was 3500 ppm. This isotherm curve wastested with the following Langmuir model which fits theexperimental data reasonably well (Figure 3):

where q e s the amount of the adsorbed Cua+at the finalequilibrium concentration, C,, and K and b are theLangmuir constants. In fitting eq 2 t o the experimentaldata (Figure 31, using the SCIENTIST software, theconstantsK and b were found to be 0.480 68 mLJmg and0.002 36 mLJpg, respectively. Similar isotherm curvesfor Cu2+uptake were also obtained by other authorsusing various microorganisms such as R. arrhizus (Tobinet al., 1984),S. noursei (Mattuschka and Straube, 1993),and C. resinae and P. italicum (Rome and Gadd, 1987).Adsorption of metals by microorganisms is highly pHsensitive. Aksu et al. (1991) noticed an increase in Cu2+uptake by activated sludge bacteria with an increase inpH up to 5; a very low uptake was obtained at pH 2.Almost no Cu2+ ptake by Chlorella vulgaris was noticedat pH 3, while the maximum uptake was obtained at pH5 (Harris and Ramelow, 1990). A further increase in pHup to 7 resulted in a slight decrease in Cu2+ dsorption.In this work, the effect of pH on the adsorption of Cu2+by A. carbonarius biomass was studied in deionizedwater, the pH of which was adjusted using 1N NaOH or1N HCl. The biomass concentration in a 100 ppm coppersolution from which the metal adsorption was carried outwas 1.94 mg/mL. A t pH values of 1.5,3.5, and 7.0,3.22,6.32, and 19.47 pg of Cu2+were adsorbed per milligramof biomass, respectively. The increase in biosorption byraising the pH may be, in general, ascribed t o theincrease of negatively charged groups at the surface ofthe microbial cells as it was reported by Luef et al. (1991).Temperature is another parameter that alters adsorp-tion process. Suspensionsof 1.565 mg/mL biomass a t pH5 were prepared and transferred t o water baths set atvarious temperatures. After 10 min, 100 ppm Cu2+wasadded, and adsorption was followed fo r 15 min. Theoptimum temperature for the uptake is 25 "C (Table 2).

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640 Biotechnol. Prog., 1995, Vol. 11, No. 6200 ,160- 4012 20vH 1004

op 60 -' " 7 , , , , ,0

0 500 1000 1500 2000 2500 3000Equilibrium concentration (ppm)

Figure 3. Relationship between equilibrium copper concentra-t ion and i ts uptake at p H 6 of a 3.365 mg/mL biomasssuspension. Symbols ar e experim ental and solid line representsda ta predicted us ing eq 2.Table 2. Effect of Temperature on Copper Uptake by A.carbonarius

~ ~~~~

temp Cu2+uptake temp Cu2+uptake("C) W m d ("C) b d m d4 8.3 40 1.271 0 10.5 50 0.9625 11.6

Similar trends were obtained for Zn2+uptake by Candidautilis (Failla et al., 1976); he optimum temperature was30 "C, and the uptake was depressed with furtherincrease in temperature. The increase in the uptake withtemperature could be due to the increase in the energyof the system tha t facilitates the attachment of Cu2+ othe surface of the cells, while the decrease with fur therincrease in temperature could be a result of the distortionof some sites of the cell surface available for metaladsorption o r due to the desorption process that mightoccur at high temperatures.To examine the ability of the preheated cells t o adsorbCu2+, hree pH 5 suspensions of cells, each containing6.2 mg/mL dry biomass, were preheated a t 50 "C; the firstand the second suspension were exposed to this temper-ature for 1and 2 h, respectively, and then allowed to coolt o the room temperature. The third served as the control.Each system was supplemented with Cu2+ n a concen-tration of 100 ppm, and the metal adsorption wasmeasured after 15 min. The Cu2+uptakes of 2.94 and0.806 pg/mg were noticed in the systems preincubatedfor 1 and 2 h, respectively. The adsorption of 3.46 pgCu2+ per milligram of biomass was observed in thecontrol. Such an effect of exposure of the cells to a highertemperature was expected because of the protein andlipid constituents of microbialcell surfaces, that are foundt o be a t least partially responsible for metal adsorption,and their sensitivity t o a heat treatment.The effect of glucose on the metal adsorption bymicrobial cells has been reported. Norris and Kelly(1977) observed higher Co2+ and Cd2+ uptakes by S.cerevisiae in the presence of 10 mM glucose than withoutit. Fuhramann and Rothestein (1968) studied the trans-port of Zn2+,Co2+,and Ni2+ nto bakers' yeast cells. Theyreported that the uptake was enhanced 5-20-fold in thepresence of glucose and phosphate compared to thestarved cells. In our study it was also noticed tha tglucose affected the amount and the rate of Cu2+uptakeby the cells of A. carbonarius (Table 3, Figure 4). A

54. 5

4h3 3.5i;o5 3f 1.5P8 1.5

10.5

0

E '

0 5 IO 15 20 25 30 35Time (min)

Figure 4. Effect of glucose concentration on copper upta ke byA. carbonarius at p H 5 of 100 ppm copper and a 3.16 mg/mLbiomass suspension. Glucose concentration (%I: A, 0.0;+, 0.05;Table 3. Effect of Glucose on Copper Uptake by A.carbonarius at pH 6 f 100 ppm Cu2+and 3.23 mg/mLBiomass Suspension

.,.1; A,0.3;0, .5; 0 , 1.0.

system Cu2+uptake eglmg)no glucose0.1% glucose"0.1% glucose*0.1% glucosee

6.917.158.519.43" Cu2+added after shakin g the suspension w ith glucose for 15min. * Cu2+ added af ter sh akin g the suspension with glucose for1h. e Cu2+added after s hak ing the suspension with glucose for 2h .

longer pre-exposure of the cells to glucose resulted in ahigher Cu2+uptake (Table 3). It was also noticed thatlower concentrations of glucose resulted in higher ratesand amounts of Cu2+ dsorption, while its concentrationabove 0.1% (wh) reduced substantially the ability of thecells to adsorb this metal. The increase in the adsorptioncan be the result of an increase of cells' activities,including the metal adsorption activity, caused by theincrease in the energy available to the cells in thepresence of glucose. At higher glucose concentrations,it may happen that this compound interferes with thecells' metal active sites, thus preventing their interactionswith metal ions. Therefore, it is necessary to find theoptimum glucose concentration for the adsorption ofmetals by microbial biomass. In this study, the highestCu2+adsorption was noticed when the glucose concentra-tion was 0.1% (w/v) (Figure 5).Adsorption of Cr3+byA. carbonarius was also studiedin this work. The effect of biomass concentration on CP+uptake (Table 4) was similar to that of the Cu2+uptake(Figure 1). Comparing these two sets of results, it canbe noted that more copper than chromium was adsorbed

per unit of biomass when the two systems contained thesame concentrations of biomass. In this case a 30-35%of the initial C P + concentration was reduced in all of thesystems. Grappelli et al. (1992) obtained 75.5% adsorp-tion of C P +byA .giacomell at pH 4.7. Tobin et al. (1984)investigated the adsorption of 17 different metals by R.arrhizus, and the highest uptakes were observed withU023+and C P + , 0.82 and 0.59 mM/g, respectively.Mattuschka and Straube (1993) obtained 46% removalof Crf3by S.noursei at pH 4.9 and 2.3 g/dm3dry biomass.The effect of the initial C$+ concentration on its uptakeby the cells of A . carbonarius is also shown in Table 5.From these data, the equilibrium chromium concentra-tions, C,, are calculated and plotted against the uptakeof this metal, qe (Figure 5). This equilibrium relation-

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lZO t /10014 80$9 60'a40

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00 50 100 150 200 250

Equilibriumconcentration@pm)Figure 5. Relationship between equilibrium chromium con-centration and its uptake at pH 6 of a 1.44 mg/mL biomasssuspension. Symbols are experimental,and solid line representsdata predicted using eq 3.Table 4. Effect of Biomass and C P+ Concentrations onC P + UDtake by A. carbonarius at DH 6

contact biomassa (mg/mL) C P+ (ppm)*time(min) 0.6 0.72 1.44 2.88 25 50 10 0 200 400C$+Uptake Gglmg)2 69.8 22.9 18.2 6.2 11.8 16.9 18.2 34.7 54.210 90.3 34.0 26.0 9.1 12.7 20.5 26.0 38.4 74.760 102 43.9 34.8 13.0 13.7 24.3 34.8 74.3 114.3

a Using 10 0 ppm initial Cr3+ concentration. Using 1.44 mglmL biomass suspension.Table 5. Constants Kl and K1 of Eq 8 for Various pHs

4 9.1695 5.2256 1.8317 1.4540.01950.02530.10100.1155

ship can be represented by the following Freundlichisotherm model:4 e = K&,n (3)

where Kf nd n are Freundlich's constants. The valuesof these constants are 4.26 pug0 396/(mg.mL-o.604)nd 0.604,respectively; they were evaluated by fitting eq 3 to thedata in Figure 5 using the SCIENTIST software package.The effect of pH on Cr3+ uptake was also studied.These results (Figure 6) confirmed the previous resultsobtained for Cu2+uptake, where increasing pH resultedin an increase of the metal uptake. Kuyucak and Volesky(1988b) studied the adsorption of CS+byH . opuntia andA. nodosum. With H. opuntia, the uptake was increasingwith increasing pH from 2 to 4, while there was nosignificant change in the CS+ uptake with a furtherincrease in pH up to 11. In the case ofA. nodosum, theauthors obtained a steady increase in the metal uptakewith the increase in pH from 2 to 8. However, with afurther increase in pH up to 11, a slight decrease in theC13+ uptake was noticed.To model the results from Figure 6, it was assumedthat the metal uptake occurred a t the surface only andthe metal ions moved from the bulk solution and attachedto the cell surface. Therefore, the rate of metal transferis assumed to be proportional to the biomass concentra-tion and t o the difference of chromium concentration in

O K I0 10 20 30 40 50 60 70

Time (min)Figure 6. Effect of pH on chromium uptake by A. carbonariusat 100 pp m chromium and a 1.44 mg/mL biomass suspension.Symbols are experimental and solid lines are data predictedusing eq 8. pH: A, ; e, 5 ;.,; 0, 7.the bulk solution and tha t a t the biomass surface. Thiscan be written as:

- _m - Fo(m- m,)d t (4)where m is the metal concentration in the bulk solution,m , is the metal concentration at the cell surface, Xo isthe biomass concentration, and K I is the mass transfercoefficient. At the cell surface, the metal concentration,m,, is in equilibrium with the amount of metal beingadsorbed, q. For the purpose of this model the followinglinear equilibrium relationship is assumed between m ,and q :

4 =4% ( 5 )The mass balance for the metal is

mo= m + X o q (6)where mo is the initial metal concentration in thesolution. Combining eqs 4, 5, and 6 gives the followingexpression:

(7 )Equation 8 relates the metal uptake with the contacttime at a given biomass and initial metal concentrationsand is obtained by integration of eq 7:

The parameters K I and Kl were determined for each pH(Table 5) by fitting eq 8 to the data of Figure 6 usingSCIENTIST. Although Figure 6 indicates that thismodel can be used t o fit the experimental data for pH 4and 5, it is evident that it should be improved to give abetter representation for the pH values of 6 and 7 .

ConclusionsA. carbonarius is able to adsorb copper and chromiumfrom their solutions. In both cases more metal uptakeper unit of biomass was noticed with lower biomassconcentrations than with higher ones. A Langmuir-typemodel fits the equilibrium data of copper adsorption,while a Freundlich isotherm model fits better the equi-librium data of chromium adsorption. The uptake of themetals increases by increasing the initial copper and

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642 Biotechnol. Prog., 1995, Vol. 11 , No . 6Gadd, G. M.; White, C. Removal of Thorium from Simula tedAcid Process St rea m s by Fung al Biomass. Biotechnol. Bioeng.1989,3 3, 592-597.Good, N. E. ;Winget , G. D.; Winter, W.; Connolly, T. N.; Izawa ,S.; Singh, R. M. M. Hydrogen Ion Buffers for BiologicalResearch. Biochem. J . 1966, 2, 467-477.Grappelli , A.; Campan ella, L.; Cardare l l i , E.; Mazzei, F.; Cor-datore, M.; Pietrosanti, W . Metals Removal and Recovery byArthrobacter sp. Biomass. W at. Sci. Technol. 1992,26,2149-2152.Ha rr i s , P. 0.; Ramelow, G. J. Binding of Metal Ions byPart icu la te Biomass derived from C hlore lla vulgaris andScenedesmus quad ricauda . Environ. Sc i . Technol. 1990, 2,220-228.Kuyucak, N.; Volesky, B. biosorbents for Recovery of Metalsfrom In dus tria l Solutions. Biotechnol. L ett. 1988a, 2, 137-142.Kuyucak, N.; Volesky, B. A method of Metal Removal. WaterPollut. Res. J . 1988b,23, 424-433.Luef, E.; Prey, T.; Kubicek, C. P. Biosorption of Zinc by Funga lMycelial Wa stes. Appl. M icrobiol. Biotechnol. 1991,34,688-692.Mattu schka, B.; Straub e, G. Biosorption of Metals by a WasteBiomass. J . Chem. Technol. Biotechnol. 1993, 58, 57-63.Moha pa t ra , S. P.; Siebel, M. A.; Alaerts , G. J. Effect ofBacillusmegaterium on Removal of Copper from Aqueous Solutionsby Activated Carbon. J . Environ. Sci. Health 1993,28, 615-629.Mullen, M. D.; Wolf, D. C.; Beveridge, T. J.; Bailey, G. W.Sorpt ion of Heavy M etals by Soil Fun gi Aspergillus niger an dMucor rouxii. Soil Biol. Biochem. 1992,24 , 129-135.Mu raleedharan , T. R.; Venkobachar, C. M echanism of Biosorp-tion of Copper(I1) by G anode rma lucidu m. Biotechnol. Bioeng.1990,35, 320-325.Norris, P. R.; Kelly, D. P. Accumulation of Cadm ium an d Cobaltby Saccharomyces cerevisiae. J . Gen. Microbiol. 1977, 99,Rome, L.; Gadd, G. M. Copper Adsorption by Rhizopus arrhiz us,Cladosporium resinae and Penicillium italicum. Appl. Mi-crobiol. Biotechnol. 1987,26, 84-90.Tobin, J. M.; Cooper, D. G.; Neufeld, R. J. Up tak e of Metals byRhizopus ar rhiz us B iomass. Appl. Environ. M icrobiol. 1984,47, 821-824.Van Cutsem, P.; Metdagh, M. M.; Rouxhet, P. G.; Gillet, C.Pre l iminary ESR Study and Rela t ion with Ion ExchangeThermodynamics of Copper Adsorbed on a Biological IonEx chan ger- the Nitella fzexilis Cell Wall. React. Polym. 1984,2, 31-35.Volesky, B. Biosorbents for M etal Recovery. Tibtechnology 1987,5. 96-101.

317-324.

chromium concentrations. pH has a significant effect onthe copper and chromium uptakes. The optimum tem-perature for copper uptake is 25 "C.Preheating of thebiomass suspension depressed copper adsorption com-pared to that of the control. Addition of glucose enhancescopper adsorption; the optimum glucose concentration forthis adsorption is 0.1%.Notation

n44 et

equilibrium metal concentration, &mL (ppm)Langmuir constant, mUmgFreundlich constant, y,g0.396/(mg.mL-0,604)linear equilibrium constant, mumgbiomass concentration, mg/mLempirical constant, yg/(mL.minc)Langmuir constant, muygempirical constant, dimensionlessmass transfer coefficient, mU(mg.min)metal concentration at the bulk solution, yg/mLinitial metal concentration, yg/mL (ppm)metal concentration at the cells' surface, yg/mLFreundlich constant, dimensionlessmetal uptake, yglmgmetal uptake at equilibrium, yglmgcontact time, min

( P P d

(PPm)

Literature CitedAksu, Z.; Kutsal, T.; Gu n, S.;Haciosmanoglu, N. ; GholaminejadM. Inv estig atio n of Biosorption of Cu(II ), Ni(I1) an d Cr(V I)Ions to Activated Sludge Bacteria. Environ . Tech. 1991, 12,915-921.Al-Asheh, S.; Duvnjak D. Effect of Glucose Concentration onthe B iomass and Phy tase Product ions an d the Reduct ion ofthe Phyt ic Acid Content in Canola Meal by Aspergi l luscarbonarius D uring Sol id -s ta te Fermenta t io n Process. Bio-technol. Prog. 1994, 10, 353-359.Avery, S.V.;Tobin, J. M. Mechanism of Adsorption of Ha rd a ndSoft Metal Ions to Saccharom yces cerevisiae a nd Influence ofH a r d a n d Soft Anions. Appl. E nviron. Microbiol. 1993, 9,2851-2856.Baldry, M. G. C.; Dean, A. C. R. Copper Accumulation byEscherichia coli s train FE 12/5. 1 Uptake During BatchCulture. Microb. Lett. 1980, 15, 83-87.Failla, M. L.; Benedict, C. D.; Weinberg, E. D. Accumulationand Storage of Zn2+ by C and ida utilis . J. Gen. Microbiol.1976,94 , 23-36.Fuh ram ann , G.; Roths t ien , A. The Transport of Zn2+,Co2+,a n dNi2+ nto Ye ast Cells. Biochem. Biophys. Acta 1968,163,325-330.

Accepted August 4, 1995.@BP950053Y

@ Abstract published in Advance ACS Abstracts, October 1,1995.