638

Biofechnol.Prog. 1995, 11, 638-642

Adsorption of Copper and Chromium by Aspergillus carbonarius

Sameer Al-Ashehand Zdravko Duvnjak"
Chemical Engineering Department, University of Ottawa, 161 Louis Pasteur, Ottawa, Ontario, Canada K1N 6N5

Aspergillus carbonarius NRC 401121 adsorbs some copper and chromium from their solutions. The amount of the adsorbed metal per unit of biomass increased with a decrease in the biomass concentration. The increases in the initial concentrations of metals 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 prior to utilizing it in the adsorption tests decreased its metal adsorption capacity. Preincubation of the biomass with glucose enhanced the metal adsorption. The optimum glucose concentration in this process was 0.1%.

Introduction
The utilization of microbial biomass for the removal of metals from industrial waste waters and polluted waters has been already recognized. The biomass has to satisfy the following requirements to be considered as the an adsorbent (Volesky, 1987): (1) efficient and rapid uptake and release of metals, (2) the low production cost of the biosorbent and possibility of its reutilization, (3) the efficient, rapid, and cheap separation of the biosorbent from the solution, and (4) a high selectivity of metal adsorption and desorption. If they are fulfilled, the utilization of the biomass would make the biosorption process more attractive compared to processes which use other types of adsorptions. The mechanism of metal uptake by microorganisms is not well-known, but some attempts had been reported in order to explain it. Harris and Ramelow (1990) stated that the accumulation of metals by microorganism may take place by any or a combination of the following processes: (1)entrapment by cellular components, (2) active transport across the cell membrane, (3) cation exchange or complexation, and (4) adsorption. Kuyucak and Volesky (1988a) reported that cell walls of microbial biomass, mainly composed of polysaccharides, proteins, and lipids offer particularly abundant metal-binding functional groups such as carboxylate, hydroxyl, sulphate, phosphate, and amino groups. Muraleedharan and Venkobachar (1990) attributed the metal-binding capacity of microbes to their proteineous component. Some microorganisms have been studied for the adsorption of copper(I1) and chromium(II1). Ganderma lucidum (Muraleedharanand Venkobachar, 1990), Rhizopus arrhizus, Cladosporium resinae, and Pencillium italicum (Rome and Gadd, 19871,Arthrobacter giacomell (Grappelli et al., 1992), Escherichia coli (Baldry and Dean, 19801,Aspergillus niger and Mucor rouxii (Mullen et al., 1992),Saccharomyces cerevisiae (Avery and Tobin, 19931, and Bacillus megaterium (Mohapatra et al., 1993) are microorganisms that have been used for copper(I1) uptake. There are also microorganisms which were used for chromium(II1)uptake, such as A. giacomell (Grappelli et al., 19921,R. arrhizus (Tobin et al., 1984),Streptomyces noursei (Mattuschka and Straube, 1993), Halimeda opuntia, Ascophyllum nodosum, and S. cerevisiae (Kuyucak and Volesky, 1988a). In this work, Aspergillus carbonarius is used to test its ability for copper(I1)and chromium(II1) uptake. The microorganism has been previously used for the production of phytase and reduction of phytic acid content in

canola meal during a solid-state fermentation process (AlAsheh and Duvnjak, 1994). The objective of this work is to study the effects of the biomass and metal concentrations, pH, temperature, and addition of glucose to the metal-containing solutions on the adsorption of the metals.

Material and Methods Microorganism, Media, and Culture Conditions. A. carbonarius NRC 401124 was used to study the adsorption of Cu2+and C?+ from their solutions. The solid and liquid media for growth of this microorganism
were prepared as previously reported (Al-Asheh and Duvnjak, 1994). The microorganism grew in the liquid medium in the form of small pellets ranging from 0.5 to 5 mm in diameter. The pellets were separated by centrifugation (1OOOOg x 15 min) and washed three times with saline solution (2%NaC1). After that the cells were filtrated and kept at 4 "C before their utilization in the adsorption experiments. Adsorption Experiment. A required amount of the cells was weighed and transferred into a 250 mL Erlenmeyer flask containing 49 mL of 5 mM 2-(N-morpholino)ethanesulphonic acid (MES) buffer, adjusted to pH 6 (unless otherwise specified)with 1N NaOH. This buffer has negligible metal-complexing properties (Good et al., 1966). Dry weight of biomass was obtained by drying at 105 "C for at least 48 h. One milliliter of either 5000 ppm Cu2+(in the form of CuS04.5Hz0)or 5000 ppm C13+(in the form of [Cr(H20)4Cl21Cl2.H~O) added to the biomass suspension in the was buffer to make the final metal concentration 100 ppm (unless otherwise stated). The mixture was agitated on a shaker and samples were taken for analysis. The biomass was separated by vacuum filtration using 0.45 pm filter paper, and the filtrate was analyzed for the metal, the adsorption of which was studied, using atomic absorption spectrophotometer (Varian AA-1475 series). Each experiment was carried out in duplicate, and the average results are presented in this work.

Results and Discussion

The effect of the biomass concentration on the copper uptake was studied using the biomass in the range of 1.15-9.20 mg per milliliter of a 100 ppm CuZfsolution. The results indicate (Figure 1)that the uptake of Cu2+ per unit of biomass increased with a decrease in the biomass concentration. Depending on the biomass con-

0 8756-7938/95/3011-0638$09.00/0 1995 American Chemical Society and American Institute of Chemical Engineers

Biotechnol. Prog., 1995, Vol. 11, No. 6

639

Ill

10

20

30

40

50

60

70

IO

20

30

40

50

BO

70

Time (min)

Time (min)

Figure 1. Effect of biomass concentration on copper uptake at pH 6 of a 100 ppm copper solution. Symbols are experimental and solid lines are data predicted using eq 1. Biomass concentration (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 Biomass Concentrations
biomass (mg/mL) 1.15 2.3 4.6 9.2
a (pg/(mL-min'))
C

Figure 2. Effect of copper concentration on its uptake by A. carbonarius at pH 6 of a 9.2 mg/mL biomass suspension. Copper concentration (ppm): 0, 25; +, 50; 100; A, 200; A , 400.

40.17 18.8 3.941 1.391

0.0432 0.0612 0.2457 0.2569

concentrations were extended to 3500 ppm. The results of the equilibrium concentrations, i.e. the Cu2+concentration in the suspension after adsorption, and Cu2+ uptake indicate (Figure 3) that the biomass of A. carbonarius was rather saturated with Cu2+when its initial concentration was 3500 ppm. This isotherm curve was tested with the following Langmuir model which fits the experimental data reasonably well (Figure 3):

centration, 31-55% of the initial Cu2+ concentration was adsorbed. The higher metal loading of biomass with a decrease of its concentration has also been noticed by Gadd and White (1989) for the uptake of thorium by S. cerevisiae. The biosorption kinetics are also shown in Figure 1. The kinetics study is essential to establish the rates of metal uptake and release. A rapid uptake would allow a short solution-biosorbent contact time and would result in the use of much shallower contact beds of sorbent materials in their column applications (Kuyuacak and Volesky, 1988a). The results of Figure 1, as most others in this work, show that the largest amount of the adsorbed metal ions was attached to the biomass within the first 10 min of the adsorption process and, after 30 min, further metal uptake was almost negligible. The results from Figure 1 can be very well represented by the following empirical correlation (Figure 1):
q = atc

(1)

where q is the metal uptake, t is the contact time, and a and c are empirical constants which were evaluated by fitting eq 1to the experimental data using the program SCIENTIST (Micromath Scientist Software). These constants for different biomass concentrations are presented in Table 1. The Cu2+solutions which contained from 25 to 400 ppm of this ion and 9.20 mg of biomass per milliliter of solution were used to study the effect of the initial Cu2+ concentration on its uptake by the biomass. The results show (Figure 2) that the increase in the Cup+uptake is associated with an increase in the initial metal concentration. This can be explained by the progressive increase in electrostatic interactions, relative to covalent interactions, with sites of progressively lower affinity for Cuf2 when Cuf2 initial concentrations increased (Van Cutsem et al., 1984). Another experiment was performed to test the biomass saturation by this metal; the adsorption was carried out for 15 min, and the initial Cu2+

where qeis the amount of the adsorbed Cua+at the final equilibrium concentration, C,, and K and b are the Langmuir constants. In fitting eq 2 to the experimental data (Figure 31, using the SCIENTIST software, the constants K and b were found to be 0.480 68 mLJmg and 0.002 36 mLJpg, respectively. Similar isotherm curves for Cu2+uptake were also obtained by other authors using various microorganisms such as R. arrhizus (Tobin et 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 pH sensitive. Aksu et al. (1991) noticed an increase in Cu2+ uptake by activated sludge bacteria with an increase in pH up to 5; a very low uptake was obtained at pH 2. Almost no Cu2+ uptake by Chlorella vulgaris was noticed at pH 3, while the maximum uptake was obtained at pH 5 (Harris and Ramelow, 1990). A further increase in pH up to 7 resulted in a slight decrease in Cu2+adsorption. In this work, the effect of pH on the adsorption of Cu2+ by A. carbonarius biomass was studied in deionized water, the pH of which was adjusted using 1N NaOH or 1N HCl. The biomass concentration in a 100 ppm copper solution from which the metal adsorption was carried out was 1.94 mg/mL. At pH values of 1.5,3.5, and 7.0,3.22, 6.32, and 19.47 pg of Cu2+were adsorbed per milligram of biomass, respectively. The increase in biosorption by raising the pH may be, in general, ascribed to the increase of negatively charged groups at the surface of the microbial cells as it was reported by Luef et al. (1991). Temperature is another parameter that alters adsorption process. Suspensionsof 1.565mg/mL biomass at pH 5 were prepared and transferred t o water baths set at various temperatures. After 10 min, 100 ppm Cu2+was added, and adsorption was followed for 15 min. The optimum temperature for the uptake is 25 "C (Table 2).

640
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Biotechnol. Prog., 1995, Vol. 11, No. 6

,
h

5
4.5

1
v

160
140

3 3.5
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2 120

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op

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0 0
, , , ,

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15

20

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30

35

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500

1000

1500

2000

2500

3000

Equilibrium concentration (ppm)

Figure 3. Relationship between equilibrium copper concentration and its uptake at pH 6 of a 3.365 mg/mL biomass suspension. Symbols are experimental and solid line represents data predicted using eq 2. Table 2. Effect of Temperature on Copper Uptake by A. carbonarius
~ ~~~~

Figure 4. Effect of glucose concentration on copper uptake by A. carbonarius at pH 5 of 100 ppm copper and a 3.16 mg/mL biomass suspension. Glucose concentration (%I: A, 0.0; 0.05; . A, 0.3;0, 0 , 1.0. , 0.1; 0.5;

+,

Table 3. Effect of Glucose on Copper Uptake by A. carbonarius at pH 6 of 100 ppm Cu2+and 3.23 mg/mL Biomass Suspension
system no glucose 0.1% glucose" 0.1% glucose* 0.1% glucosee Cu2+uptake eglmg) 6.91 7.15 8.51 9.43

temp ("C) 4 10 25

Cu2+uptake W m d 8.3 10.5 11.6

temp ("C) 40 50

Cu2+uptake bdmd 1.27 0.96

Similar trends were obtained for Zn2+uptake by Candida utilis (Failla et al., 1976);the optimum temperature was 30 "C, and the uptake was depressed with further increase in temperature. The increase in the uptake with temperature could be due to the increase in the energy of the system that facilitates the attachment of Cu2+to the surface of the cells, while the decrease with further increase in temperature could be a result of the distortion of some sites of the cell surface available for metal adsorption or due to the desorption process that might occur at high temperatures. To examine the ability of the preheated cells to adsorb Cu2+,three pH 5 suspensions of cells, each containing 6.2 mg/mL dry biomass, were preheated at 50 "C; the first and the second suspension were exposed to this temperature for 1and 2 h, respectively, and then allowed to cool to the room temperature. The third served as the control. Each system was supplemented with Cu2+in a concentration of 100 ppm, and the metal adsorption was measured after 15 min. The Cu2+uptakes of 2.94 and 0.806 pg/mg were noticed in the systems preincubated for 1 and 2 h, respectively. The adsorption of 3.46 pg Cu2+ per milligram of biomass was observed in the control. Such an effect of exposure of the cells to a higher temperature was expected because of the protein and lipid constituents of microbial cell surfaces, that are found t o be at least partially responsible for metal adsorption, and their sensitivity to a heat treatment. The effect of glucose on the metal adsorption by microbial 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 without it. Fuhramann and Rothestein (1968) studied the transport of Zn2+, Co2+,and Ni2+into bakers' yeast cells. They reported that the uptake was enhanced 5-20-fold in the presence of glucose and phosphate compared to the starved cells. In our study it was also noticed that glucose affected the amount and the rate of Cu2+uptake by the cells of A. carbonarius (Table 3, Figure 4). A

" Cu2+added after shaking the suspension with glucose for 15 min. * Cu2+ added after shaking the suspension with glucose for 1h. e Cu2+added after shaking the suspension with glucose for 2 h.
longer pre-exposure of the cells to glucose resulted in a higher Cu2+uptake (Table 3). It was also noticed that lower concentrations of glucose resulted in higher rates and amounts of Cu2+adsorption, while its concentration above 0.1% (wh) reduced substantially the ability of the cells to adsorb this metal. The increase in the adsorption can be the result of an increase of cells' activities, including the metal adsorption activity, caused by the increase in the energy available to the cells in the presence of glucose. At higher glucose concentrations, it may happen that this compound interferes with the cells' metal active sites, thus preventing their interactions with metal ions. Therefore, it is necessary to find the optimum glucose concentration for the adsorption of metals by microbial biomass. In this study, the highest Cu2+adsorption was noticed when the glucose concentration was 0.1% (w/v) (Figure 5). Adsorption of Cr3+by A. carbonarius was also studied in 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 can be noted that more copper than chromium was adsorbed per unit of biomass when the two systems contained the same concentrations of biomass. In this case a 30-35% of the initial C P + concentration was reduced in all of the systems. Grappelli et al. (1992) obtained 75.5% adsorption of CP+ by A. 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 with U023+ and CP+, 0.82 and 0.59 mM/g, respectively. Mattuschka and Straube (1993) obtained 46% removal of Crf3by S. noursei at pH 4.9 and 2.3 g/dm3dry biomass. The effect of the initial C$+ concentration on its uptake by the cells of A. carbonarius is also shown in Table 5. From these data, the equilibrium chromium concentrations, C,, are calculated and plotted against the uptake of this metal, qe (Figure 5). This equilibrium relation-

Biofechnol. Prog., 1995,Vol. 11, No. 6

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$
'a

80
60

40

O K
20
0

1 0

20

30 40 Time (min)

50

60

70

50

100

150

200

250

Equilibrium concentration @pm)

Figure 5. Relationship between equilibrium chromium concentration and its uptake at pH 6 of a 1.44 mg/mL biomass suspension. Symbols are experimental,and solid line represents data predicted using eq 3.
Table 4. Effect of Biomass and C P + Concentrations on C P + UDtake by A. carbonarius at DH 6

Figure 6. Effect of pH on chromium uptake by A. carbonarius at 100 ppm chromium and a 1.44 mg/mL biomass suspension. Symbols are experimental and solid lines are data predicted using eq 8. pH: A, 4; e, 5 ; . 0, 7. , 6; the bulk solution and that a t the biomass surface. This can be written as:

- _ - kFo(m- m,) dm
dt

(4)

contact

biomassa (mg/mL)

CP+ (ppm)*
50
100 200

time(min) 0.6 0.72 1.44 2.88 25

2 10 60

400

C$+Uptake Gglmg) 69.8 22.9 18.2 6.2 11.8 16.9 18.2 34.7 54.2 90.3 34.0 26.0 9.1 12.7 20.5 26.0 38.4 74.7 102 43.9 34.8 13.0 13.7 24.3 34.8 74.3 114.3

a Using 100 ppm initial Cr3+ concentration. Using 1.44 mgl mL biomass suspension.

where m is the metal concentration in the bulk solution, m, is the metal concentration at the cell surface, Xo is the biomass concentration, and K I is the mass transfer coefficient. At the cell surface, the metal concentration, m,, is in equilibrium with the amount of metal being adsorbed, q. For the purpose of this model the following linear equilibrium relationship is assumed between m, and q: 4 = 4% The mass balance for the metal is (5)

Table 5. Constants Kl and K1 of Eq 8 for Various pHs

4 5 6 7 9.169 5.225 1.831 1.454 0.0195 0.0253 0.1010 0.1155

mo = m +Xoq

(6)

ship can be represented by the following Freundlich isotherm model:

4 e = K&,n

where mo is the initial metal concentration in the solution. Combining eqs 4, 5, and 6 gives the following expression:

(3)

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

where Kfand n are Freundlich's constants. The values u0 and of these constants are 4.26 p g 396/(mg.mL-o.604) 0.604, respectively; they were evaluated by fitting eq 3 to the data 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 results obtained for Cu2+uptake, where increasing pH resulted in an increase of the metal uptake. Kuyucak and Volesky (1988b) studied the adsorption of CS+ by H. opuntia and A. nodosum. With H. opuntia, the uptake was increasing with increasing pH from 2 to 4, while there was no significant change in the CS+ uptake with a further increase in pH up to 11. In the case ofA. nodosum, the authors obtained a steady increase in the metal uptake with the increase in pH from 2 to 8. However, with a further increase in pH up to 11, a slight decrease in the C13+ uptake was noticed. To model the results from Figure 6, it was assumed that the metal uptake occurred at the surface only and the metal ions moved from the bulk solution and attached to the cell surface. Therefore, the rate of metal transfer is assumed to be proportional to the biomass concentration and to the difference of chromium concentration in

The parameters K I and K were determined for each pH l (Table 5) by fitting eq 8 to the data of Figure 6 using SCIENTIST. Although Figure 6 indicates that this model can be used to fit the experimental data for pH 4 and 5, it is evident that it should be improved to give a better representation for the pH values of 6 and 7.

Conclusions A. carbonarius is able to adsorb copper and chromium from their solutions. In both cases more metal uptake per unit of biomass was noticed with lower biomass concentrations than with higher ones. A Langmuir-type model fits the equilibrium data of copper adsorption, while a Freundlich isotherm model fits better the equilibrium data of chromium adsorption. The uptake of the metals increases by increasing the initial copper and

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Biotechnol. Prog., 1995, Vol. 11, No. 6

Gadd, G. M.; White, C. Removal of Thorium from Simulated Acid Process Streams by Fungal Biomass. Biotechnol. Bioeng. 1989,33, 592-597. Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. M. Hydrogen Ion Buffers for Biological Research. Biochem. J . 1966, 2, 467-477. Grappelli, A.; Campanella, L.; Cardarelli, E.; Mazzei, F.; Cordatore, M.; Pietrosanti, W. Metals Removal and Recovery by Arthrobacter sp. Biomass. Wat. Sci. Technol. 1992,26,21492152. Harris, P. 0.; Ramelow, G. J. Binding of Metal Ions by Particulate Biomass derived from Chlorella vulgaris and Scenedesmus quadricauda. Environ. Sci. Technol. 1990, 2, 220-228. Kuyucak, N.; Volesky, B. biosorbents for Recovery of Metals from Industrial Solutions. Biotechnol. Lett. 1988a, 2, 137142. Kuyucak, N.; Volesky, B. A method of Metal Removal. Water Pollut. Res. J . 1988b,23, 424-433. Luef, E.; Prey, T.; Kubicek, C. P. Biosorption of Zinc by Fungal Mycelial Wastes. Appl. Microbiol. Biotechnol. 1991,34,688692. Mattuschka, B.; Straube, G. Biosorption of Metals by a Waste Biomass. J . Chem. Technol. Biotechnol. 1993, 58, 57-63. Mohapatra, S. P.; Siebel, M. A.; Alaerts, G. J. Effect ofBacillus megaterium on Removal of Copper from Aqueous Solutions by Activated Carbon. J . Environ. Sci. Health 1993,28, 615629. Mullen, M. D.; Wolf, D. C.; Beveridge, T. J.; Bailey, G. W. Sorption of Heavy Metals by Soil Fungi Aspergillus niger and Mucor rouxii. Soil Biol. Biochem. 1992,24, 129-135. Muraleedharan, T. R.; Venkobachar, C. Mechanism of Biosorption of Copper(I1) by Ganoderma lucidum. Biotechnol. Bioeng. 1990,35, 320-325. Norris, P. R.; Kelly, D. P. Accumulation of Cadmium and Cobalt by Saccharomyces cerevisiae. J . Gen. Microbiol. 1977, 99, 317-324. Rome, L.; Gadd, G. M. Copper Adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum. Appl. Microbiol. Biotechnol. 1987,26, 84-90. Tobin, J. M.; Cooper, D. G.; Neufeld, R. J. Uptake of Metals by Rhizopus arrhizus Biomass. Appl. Environ. Microbiol. 1984, 47, 821-824. Van Cutsem, P.; Metdagh, M. M.; Rouxhet, P. G.; Gillet, C. Preliminary ESR Study and Relation with Ion Exchange Thermodynamics of Copper Adsorbed on a Biological Ion Exchanger-the Nitella fzexilis Cell Wall. React. Polym. 1984, 2, 31-35. Volesky, B. Biosorbents for Metal Recovery. Tibtechnology 1987, 5. 96-101. Accepted August 4, 1995.@

chromium concentrations. pH has a significant effect on the copper and chromium uptakes. The optimum temperature for copper uptake is 25 "C. Preheating of the biomass suspension depressed copper adsorption compared to that of the control. Addition of glucose enhances copper adsorption; the optimum glucose concentration for this adsorption is 0.1%.

Notation
equilibrium metal concentration, &mL (ppm) Langmuir constant, mUmg Freundlich constant, y,g0.396/(mg.mL-0,604) linear equilibrium constant, mumg biomass concentration, mg/mL empirical constant, yg/(mL.minc) Langmuir constant, muyg empirical constant, dimensionless mass transfer coefficient, mU(mg.min) metal concentration at the bulk solution,yg/mL
(PPd

n
4 4e

initial metal concentration, yg/mL (ppm) metal concentration at the cells' surface, yg/mL (PPm) Freundlich constant, dimensionless metal uptake, yglmg metal uptake at equilibrium, yglmg contact time, min

Literature Cited
Aksu, Z.; Kutsal, T.; Gun, S.; Haciosmanoglu, N.; Gholaminejad M. Investigation of Biosorption of Cu(II), Ni(I1) and Cr(VI) Ions to Activated Sludge Bacteria. Environ. Tech. 1991, 12, 915-921. Al-Asheh, S.; Duvnjak D. Effect of Glucose Concentration on the Biomass and Phytase Productions and the Reduction of the Phytic Acid Content in Canola Meal by Aspergillus carbonarius During Solid-state Fermentation Process. Biotechnol. Prog. 1994, 10, 353-359. Avery, S. V.; Tobin, J. M. Mechanism of Adsorption of Hard and Soft Metal Ions to Saccharomyces cerevisiae and Influence of Hard and Soft Anions. Appl. Environ. Microbiol. 1993, 9, 2851-2856. Baldry, M. G. C.; Dean, A. C. R. Copper Accumulation by Escherichia coli strain FE 12/5. 1 Uptake During Batch Culture. Microb. Lett. 1980, 15, 83-87. Failla, M. L.; Benedict, C. D.; Weinberg, E. D. Accumulation and Storage of Zn2+ by Candida utilis. J. Gen. Microbiol. 1976,94, 23-36. Fuhramann, G.; Rothstien, A. The Transport of Zn2+,Co2+,and Ni2+into Yeast Cells. Biochem. Biophys. Acta 1968,163,325330.

BP950053Y
Abstract published in Advance ACS Abstracts, October 1, 1995.
@