Environ. Sci. Technol. 1904, 18, 106-109
Gamble, D. S.; Schnitzer, M. "Trace Metals and MetalOrganic Interactions in Natural Waters"; Singer, P. C., Ed.; Ann Arbor Science: Ann Arbor, MI, 1974; Chapter 9. Martell, A. E. Pure Appl. Chem. 1976, 44, 81-113. Perdue, E. M. Geochim. Cosmochim. Acta 1978, 42, 1351-1358. Davis, J. A.; Leckie, J. 0. Environ. Sci. Technol. 1978,12, 1309-1315.
(53) Rubio, J.; Matijevic, E. J . Colloid Interface Sci. 1979,68, 408-42 1. Received for review March 15,1983. Revised manuscript received July 1,1983. Accepted August 8,1983. This research was funded by a grant from the Department of Energy under Contract DE- AC02-80EV10467.
Bioaccumulation of Nickel by Algae Hong-Kang Wang" and John M. Wood Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392
Six strains of algae and one Euglena sp. were tested for their ability to bioaccumulate nickel. Radioactive @Niwas used together with a microplate technique to determine the conditions for nickel removal by axenic cultures of cyanobacteria, green algae, and one euglenoid. The cyanobacteria tested were found to be more sensitive to nickel toxicity than the green algae or the Euglena sp. The concentration factor (CF) for nickel was determined under a variety of conditions and found to be in the range from 0 to 3.0 X lo3. The effect of environmental variables on nickel uptake was examined, and a striking pH effect for bioaccumulation was observed, with most of the algal strains accumulating nickel optimally at approximately pH 8.0. Competition experiments for binding sites between nickel and other cations, as well as with other complexing anions, showed that 63Niuptake was affected only by cobalt and by humic acids. Introduction Microorganisms adopt a number of strategies to maintain low intracellular concentrations of heavy metals. These include (I) biomethylation and transport through cell membranes of the resulting metal alkyl by diffusioncontrolled processes, (11) the biosynthesis of intracellular polymers which serve as traps for the removal of metal ions from solution, (111) the binding of metal ions to cell surfaces, and (IV) the precipitation of insoluble metal complexes (e.g., metal sulfides) at cell surfaces (1). Biomethylation appears to be a strategy employed by certain microorganisms for the effective decrease of total concentrations of nonessential inorganic ions, (e.g., Hg*, PbN, and Sn'I) in ecosystems such as sediments and soils. The biosynthesis of organometallic compounds from inorganic precursors is quite well understood at the biochemical level; however, there is a need for fundamental studies of strategies 11, 111, and IV listed above. These are especially pertinent to our understanding of the transport of essential metal ions such as CuII, Ni", and Cr'" and the prevention of such metal ions from reaching toxic levels inside cells. Nickel and chromium transport are of special interest since certain complexes of these metals are known to be carcinogenic. Recently we have investigated several strains of algae, both prokaryotes and eukaryotes, which provide examples of strategies 11, 111, and IV listed above. Also we have begun a basic study of Ni", CuII, and Cr"I transport and resistance in these strains of metal-tolerant algae. Most of our preliminary work has dealt with nickel transport and resistance by using radioactive a3Ni11. Heavy metals such as Ni" still cannot be removed entirely from industrial wastewater by traditional treatment methods. Since algae are known to bioaccumulate these metals, a basic study was undertaken to determine some 106 Environ.
Sci. Technol., Vol.
18, No. 2, 1984
of the parameters necessary for their effective removal from the aqueous phase. Early in 1963, Timofeyeva-Resovskaya et al. (2)reported on the usefulness of concentration factors and the ability of microorganisms and plants to bioaccumulate elements. Concentration factor (CF) is simply defined as the ratio of the concentration of an element taken up by a specific organism over that concentration of the element in the aqueous phase (3). Hassett et al. developed a microplate technique for the determination of concentration factors for mercury, cadmium, and lead by algae (4);however, their study did not examine essential heavy metals such as nickel, copper, and chromium. A considerable literature exists on the toxicity of nickel to algae (5-8) and on the bioconcentration of nickel by and by cyanobacteria and green algae brown algae (9,10) (11-13). However, to date there is no real understanding of the environmental factors which affect nickel uptake by microorganisms. For this reason we have examined the uptake of nickel by six different strains of algae and by one Euglena sp., some of which were already known to be resistant to nickel. Using 63Ni11,and a microplate technique (141,we have developed an extremely accurate and sensitive method for determination of concentration factors (CF's) for this element under a variety of conditions. The effects of pH, culture age, exposure time, light, nickel concentrations in solution, and competing cations and anions are considered. We believe that most of the nickel coordinates to functional groups at the cell surface, because the CF's which we measured are sufficient to cause toxic effects intracellularly. Recently we have shown that our nickel-resistant mutant Synechococcus Nic7 will tolerate an internal CF of 219 by comparison with a combined internal and external CF of 3 X lo3. Materials and Methods Organisms. Synechococcus sp. ATCC 27146 isolated by R. Y. Stanier, a nickel-resistantmutant of that organism Synechococcus sp. nic', and Oscillatoria sp. UTEX 1270 were chosen as representative cyanobacteria for this study. Scenedesmus sp. ATCC 11460 and Scenedesmus sp. B4 (a nickel-tolerant strain isolated near a copper-nickel mine, Sudbury, Ontario, was provided by Dr. P. Stokes, University of Toronto) and two organisms from the University of texas culture collection (Chlamydomonas sp. UTEX 89 and Euglena sp. UTEX 753) were selected as representative cultures. Chlamydomonas and Euglena were grown in 60-mL cultures of Cramer-Myers medium (15). The other four strains of algae were grown in 60-mL cultures of BG-11 medium (16). Chlamydomonas and Euglena were grown in stationary cultures at 20 O C with constant irradiation. The others were grown at 30 "C in a shaker. Growth was monitored at 546 nm. The incubation time
0013-936X/84/0918-0106$01.50/0
0 1984 Amerlcan Chemical Society
Table I. Uptake of 63Niab y Scenedesmus ATCC 11460 a t pH 8.0 and the Determination of Parameters RE, AC, and C F algab control
n
XabC
5
2000
un-' d
cv
n
xcontrolc
on-1 d
cv
83.4
4.1%
5
5134
146.7
2.9%
RE 61.0%
AC 61
CF 3.1 x 103
X'&a and xcontrol are cpm of 'j3Nin remaining Dry weight of alga = 0.2 mg/mL. a [63Ni11]= 0.02 fig/mL (0.34 pM). in the supernatant after 6 h a t 20 "C exposure by comparison with a distilled water control. d Standard deviation. Table 11. CF for 63Ni11a by Different Strains of Algae a t Different pH Conditions CF at pH algae
4
Scenedesmus ATCC 11460 NSb Scenedesmus B-4 NS NS Synechococcus ATCC 17146 NS Synechococcus Nic' NS Oscillatoria UTEX 1270 Chlamydomonas UTEX 8 9 2.7 X 10' NS Euglena UTEX 753 a ['j3NiI1] = 0.02 pg/mL (0.34 pM),incubated for confidence level.
5
NS 9.0 X 10' NS NS 2.9 x 101 3.8 X 10' NS
6 7.9 x 1 0 2 2.8 X l o 2 4.2 x 1 0 2 2.2 x 1 0 2 9.5 x 1 0 2 5.4 X 10' NS
6 h a t 20 "C in the light.
for green algae and Euglena was 10-15 days and for the cyanobacteria 5-10 days. Growth was also monitored by measuring the dry weight which was between 0.2 and 0.4 mg/mL for organisms in the logarithmic growth phase. Isotope. e3Ni11(Sp Act = 50 mCi/mL) was added to cultures as its chloride salt. This isotope was obtained from New England Nuclear Corp. Unlabeled NiC12 was added where indicated. Preparation of Algae for the Bioaccumulation Assay. From each culture, four aliquots of 12.0 mL were pipetted into graduated centrifuge tubes. The filamentous Oscillatoria cultures were homogenized with glass beads before centrifugation. After centrifugation the supernatant was discarded, and the cells were washed twice with distilled water before being suspended in a 3.0-mL final volume. One algal suspension was used for exposure to 63Ni11by the microplate technique, while the other three suspensions were used to determine the dry weight (mg/mL) by drying at 103 "C. Microplate Technique. Microculture plates were obtained from Titertek Corp. and consisted of 96 wells with disposable harvesting frames, a transfer fork, and a press from Flow Laboratories, Inc. Each well was filled to a final volume of 100 pL and included, in order of addition, 25 pL of buffer (0.1 M) (through the pH range 4-9 (In),25 pL of Cramer-Myers medium, or B G l l medium, diluted 1:lO with distilled water, 25 pL of s3NiC12,and either algal suspension or distilled water control (25 pL). Therefore, the algal population of each well was diluted to that of each original culture. Microplates were incubated in the light at 20 "C for the times of each experiment. After each preincubation time period, a harvesting plate was attached to each microplate, and a plastic holder containing 48 cellulose-acetate absorption cartridges, each tipped with a filter paper disk, was placed over the plate. A press was then used to force the cartridges simultaneously into the wells. These cartridges absorbed the supernatant, leaving the algal cells and the filter disk behind. The cartridges were then pushed with a plastic fork into scintillation vials containing 2.0 mL of distilled water. The scintillation vials were shaken and allowed to stand for 1 h to ensure solubilization of the filtrate and then counted in 10.0 mL of PCS scintillation fluid, after standing for 20 h. This procedure was found to be necessary to ensure precision for all experiments. Aquasol-2 scintillation
7 1.8 x 103
6.6 X 10' 3.0 x 103 4.4 x l o 2 1.1x 103 NS 1.7 X 10'
8 2.2 x 103 1.0 X l o 3 3.3 x 103 5.5 x l o 2 1.1x 101 NS 6.9 X 10'
9 2.0
x 103
4.0 X 10' 3.1 x 103 NS NS NS NS
NS = no significant uptake a t the 95%
cocktails can be used in place of PCS, and in this case there was no necessity to add distilled water. The data obtained from the above procedure (i.e., cpm of 63Niremaining in the supernatant) was used together with algal dry weight to determine the following parameters where X = cpm: removal efficiency (RE) =
Xcontrol- Xalga
x 100
*control
accumulation coefficient (AC) =
pg of Ni removed g of alga (dry weight)
concentration factor (CF) =
AC pg of 63Ni/mL of culture medium Table I presents representative data for Scenedesmus ATCC 11460 at pH 8.0 where each of the above parameters are determined. Five replicates for each experiment were performed (i.e., n). The coefficient of variance (CV) for all experiments ranged from 0.24% to 7.8%. All experiments with 'j3Ni were performed either 2 or 3 times. Therefore, this method gives reproducible results with a small experimental error. Results and Discussion The published work on nickel toxicity to algae is rather confused, because not only does nickel toxicity vary from species to species as expected but also results on individual species are inconsistent due to variables in culture conditions. Therefore, the reported minimum lethal doses for nickel poisoning, even within individual species, often varies by an order of magnitude (5-8,18,19). However, some general conclusions can be drawn. For example, the growth of cyanobacteria we studied is generally more sensitive to nickel toxicity than for the green algae. Our results show that cyanobacteria will tolerate 0.02 Mg/mL (0.34 pM NiC12)at pH 7.0 but at concentrations above 0.78 pg/mL (13.3 pM) cell lysis occurs. Even a nickel-tolerant mutant of Synechococcus was inhibited by 13.3 pM NiC12. Resistance to nickel toxicity is extremely pH dependent. For example, Scenedesmus ATCC 11460 will resist nickel poisoning at concentrations as high as 33 pM at pH 7.0, but at pH 4.0 these cells cannot tolerate 0.34 pM nickel. These initial experiments prompted a more detailed study of the effect of pH on nickel uptake by three strains Environ. Sci. Technol., Vol. 18, No. 2, 1984
107
Table 111. Uptake o f "NiI' by Scenedesmus ATCC 11460 by Cells Harvested a t Different Times culture age, days
optical density
dry weight, bioaccumulation mg/mL ability
10
0.170
0.100
17
0.493
0.225
24
1.07
0.558
RE AC CF RE AC CF RE AC CF
PH
4
5
6
4.5% 9 4.5 x i o 2 0.76% 0.68 3.3 x i o 1 23% 0.24 4.1 X 10'
5.6% 11 5.6 x 1 0 2 8.2% 7.2 3.6 x 1 0 2 6.8% 1.7 8.6 X 10'
12.8% 25 1.3 x 103 21.7% 19 9.6 x 1 0 2 60.1% 14 7.1 X 10'
of cyanobacteria and four strains of green algae. Table I1 shows that the accumulation of nickel by algae was a function of pH. Concentration factors vary from 0 to lo3. Similar results have been reported for metal uptake by several different species of algae (20). Most algal species showed maximum CF values between pH 7 and pH 9, with a maximum about pH 8. This would be consistent with surface binding of the nickel to functional groups on extracellular mucopolysaccharides (20). At pH 8.0 the nickel in aqueous solution largely exists as Ni2+with NiOH+ and Ni(OH), becoming more significant at pHs of 9.0 and above (19). (Ni(OH), precipitates out at [Ni2+]N 2 mM and pH 8. Basic conditions would facilitate the ionization of more oxygen-donor ligands and amino groups at the cell surface to provide a large number of Ni'I-bonding sites. However, Chlamydomonas shows a higher CF at pH -6,O. This probably reflects the unusual glycoprotein structure of the cell wall of this alga. At this point we selected Scenedesmus ATCC 11460 for a more detailed study of nickel uptake, because not only does this organism have the highest CF for this element but also it shows the higher resistance to nickel among the algae studied. Table I11 shows that the age of culture is extremely important, i.e., the older the culture the greater the RE, but the lower the AC. However, the CF decreases with age. The cultures tested in Table I11 were all in the exponential growth phase, but the older cultures had greater cell densities (Le., dry weight) than the younger cultures. This difference in CF between old cultures and young cultures probably reflects differences in structure at the cell surface which occurs with age, and this could be due to a decreasing nutrient supply etc. Other parameters which we found to be important to nickel uptake were (i) the exposure time and (ii) light vs. dark conditions. Figure 1shows that the CF increased rapidly for 24 h until the Ni" binding sites became saturated when cells were exposed to light. However, in the dark, the CF increases more slowly without saturation after 3 days. Apparently the kinetics for nickel uptake is affected by the metabolic activity caused by photosynthesis. Next, in order to study the surface interactions of Ni", we examined the effect of [Ni"] on the adsorption isotherm, This was accomplished by using the Freundlich equation
where y = the mass of substance absorbed ( x ) per unit mass of absorbent ( m ) . c = the equilibrium concentration of the solute being absorbed ( k and n are empirical parameters). By taking logarithms of both sides of the above equation we obtain 1 log y = log k - log c (2) n
+
108 Envlron. Sci. Technol., Vol. 18, No. 2 , 1984
P
g X
8
7
9
21.6% 3 0.5% 43 61 2.2 x 103 3.1 x 103 37.0% 64.9% 33 44 1.6 x 103 2.2 x 103 68.3% 78.6% 24 28 1.2 X l o 3 1.4 X l o 3
23.0% 46 2.3 x 103 49.3% 43.8 2.19 x 103 65.0% 23 1.2 X l o 3
I I
1
2
3
EXPOSURE TIME (days) Flgure 1. Effect of light vs. dark on the uptake of e3Ni1'by Scenedesmus ATCC 11460, at pH 8 ((0)light; (A)dark).
-3.0
-1.0
-2.0
0
LOG C Flgure 2. Adsorption isotherm for 63N11i by Scenedesmus ATCC 11460. From these data we find that x l m = 2.09 and n = 0.943, and therefore, y = 20.9(x/c)(1/1.06)at 20 OC in the light, at pH 8.
Figure 2 shows that a plot of log y vs. log c is linear over the concentration range 0.1-1.0 pg/mL. This range of [Nin]was chosen because it is more representative of nickel concentrations in mine water, although the alga does have the capacity to bioaccumulate much more nickel at the cell surfaces. In the next series of experiments we compared the ability of Scenedesmus ATCC 11460 to bioaccumulate nickel in the presence of competing cations and competing anions. Table IV shows that the CF for nickel is only significantly affected by Co" (by t-test, a = 0.05). Therefore, the binding of nickel to functional groups at the cell surface probably is influenced by the ionic radius of the metal ion in addition to charge and chemical re-
Table IV. C F for 63Ni11by Scenedesmus ATCC 1 1 4 6 0 in the Presence of a Variety of Cations Nizt a other cationb CF Ni”, 1pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL NiZ+,1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL
Hg”, 1 pg/mL Ca2+,1 pg/mL Pbz+,1 pg/mL Cu2+,1 pg/mL Zn2+,1 pg/mL Fe3+,1 pg/mL Coz+,1 ,ug/mL Cr”, 1 pg/mL MgZt, 100 pg/mL Ca2+,100 pg/mL
2.1 x 103 2.0 x 103 1.9 x 103 1.9 x 103 1.8 x 103 2.0 x 103 1.8 x 103 1 . 5 x 103 2.0 x 103 1.8 X l o 3 1.7 X l o 3
a 63Niztwith 4 / 5 “cold” carrier. Inorganic salts as their nitrates or acetates, the Ni”, and other cation were added simultaneously with the inoculum medium. 63Ni2+ exposure was for 24 h at 20 “ C in the light, at pH 8.
Table V. CF for 63Ni11by Scenedesmus ATCC 1 1 4 6 0 in t h e Presence of Competing Anions N ~ Za+ other anionsb CF 2.7 x 103 Cl-, 250 pg/mL 2.6 x 103 SO,’-, 250 pg/mL 2 . 5 x 103 CO,’-, 25 pg/mL 2.7 x 103 P o d 3 - ,1 pg/mL 2.4 x 103 CN-, 5 pg/mL 2.4 x 103 humic acid, 5 pg/mL 2.3 X l o 3 All as their sodium a 63Ni2+ with 4 / 5 “cold” carrier. or potassium salts. Ni” and other anion were added simultaneously with the inoculum medium. 63Ni2+ exposure was for 24 h at 20 “C in the light, at pH 8. Ni”, 1 pg/mL Ni”, 1 pg/mL Ni2+,1 Mg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL Ni”, 1 pg/mL NiZ+,1 pg/mL
activity (i.e., ionic radius for Co” = 0.72 A, and for Ni” it is 0.70 A). Among the other cations Fe”’ and Cr”I have smaller Pauling ionic radii and are more charged than Nin or CoII, but at pH 8.0 these metals are hydrated more rapidly so that their coordination sites are filled with OHligands before competition at the cell surface becomes a factor. Table V shows the effect of competing anions on nickel bioaccumulation. Concentrations of anions were selected which more closely represent those found in fresh-water systems with the exception of CN- and PO4*. CN- was examined because of the utilization of this anion in the extraction of precious metals by the mining industry, where this ion is a common contaminant of raffinate streams. Only humic acid affected significantly the CF for nickel. Despite the formation constant for Ni(CN)42-(i.e., log Kf = 31.31, the polydentate ligands at the algal surface effectively compete in complexing most of the nickel. However, the polydentate ligands on humic acids [Na+ salt] (Aldrich) do compete to some extent in preventing nickel uptake (by t-test, CY = 0.05). We have shown that Scenedesmus ATCC 11460 is tolerant to relatively high concentrations of nickel, and this organism is extremely effective at removing nickel from solution within the pH range 7-9. Using 63Niand a microplate technique, we have developed an extremely accurate method for measuring nickel uptake by algae under a variety of environmental conditions. Research on the ability of microorganisms to bioaccumulate metals is of appreciable practical importance. The chemical processing
industries are faced with the problem of removal technology for low concentrations of toxic metals in industrial effluents. This problem may be alleviated by the selection of special strains of algae which specifically remove the metal ion under consideration. Algae can be used to monitor the presence of toxic metals in wastewaters and therefore serve as biological indicators. This study points out how selective algae can be used in the bioconcentration of specific metal ions even in the presence of competing cations and anions. This selectivity appears to be considerably better than current ion-exchange resin technology. Since algae are inexpensive to grow in continuous culture, they offer an alternative approach to industrial metal recovery processes which are presently employed. Acknowledgments We thank Florence Gleason for her advice on growing algae. H.-K. Wang is part of a cultural exchange program from Beijing Agricultural University, Beijing, China. We especially thank Steve Michurski for his technical assistance in the early phases of this research. Registry No. Nickel, 7440-02-0; mercury, 7439-97-6; calcium, 7440-70-2; lead, 7439-92-1; copper, 7440-50-8; zinc, 7440-66-6; iron, 7439-89-6; cobalt, 7440-48-4; chromium, 7440-47-3;magnesium, 7439-95-4; calcium, 7440-70-2; chloride, 16887-00-6; sulfate, 14808-79-8; carbonate, 3812-32-6; phosphate, 14265-44-2; cyanide, 57-12-5.
Literature Cited Wood, J. M. Proc. Nobel Conf. Inorg. Biochem. Chem. Scr. 1983,21, 155-160. Timofeyeva-Resovskaya, YeA. Tr. Inst. Biol., Akad. Nauk. SSSR, Ural. Fil. 1963, 30, 1 (JPRS 21, 816). Polikarpov, G. G. ”Radioecology of Aquatic Organisms”; translated from the Russian by Vincent Schultz et al.; Reinhold Book Division: New York, 1966. Haasett, J. M.; Jennett, J. H.; Smith, J. E. Appl. Environ. Microbiol. 1981, 41, 1097. Fezy, J. S. Environ. Pollut. 1979, 20, 131. Stratton, G. W.; Corke, C. T. Chemosphere 1979,8, 731. Spencer, D. F.; Green, R. W. Environ. Pollut. 1981,25,241. Wong, P. T. S.; et al. J. Fish. Res. Board Can. 1981,38,479. Foster, P. Enuiron. Pollut. 1976, 10, 45. Fuge, R. Mar. Chem. 1973,1, 281. Sivalingam, P. M.; Rodziah, I.; Sorui, Microbiol. Immunol. 1981, 29, 171. Kurata, A.; Yoshida, Y.; Taguchi, F. J . Biochem. (Tokyo) 1980, 18, 1, Mer. Ballester, A,; Castelvi, J. Invest. Pesq. 1980, 44, 1. Hischberg, H.; Skane, H.; Thorsby, E. Plant Cell Physiol. 1977, 16, 1167. Cramer, M.; Myers, J. Arch. Mikrobiol. 1952, 17, 384. Stanier, R. Y.; Kunisawa, R.; Mandel, G.; Cohen-Bazire, F. Bacteriol. Rev. 1971, 35, 171. Robert, C. W. “Handbook of Chemistry and Physics”;CRC Press: Boca Raton, FL, 1974; p 113. Parasad, P. V. D.; et al. Water, Air, Soil Pollut. 1982,17, 263. Nriagu, 0. J. “Nickel in the Environment”; Wiley-Interscience: New York, 1980. Trollope, D. R.; Evans, B. Environ. Pollut. 1976, 11, 109.
Received for review March 28,1983. Revised manuscript received July 25, 1983. Accepted August 10,1983. This work was supported by a grant from Atlantic Richfield Co.
Envlron. Sci. Technol., Vol. 18, No. 2 , 1984
109
