Europ. J. Protisto!. 36, 443-450 (2000) December 29, 2000 http://www.urbanfischer.de/journals/ejp
European Journal of
PROTISTOLOGY
Influence of pH, Temperature, Culture Age and Cations on Adsorption and Uptake of Ni by
Chlorella vulgaris
S. K. Mehta, B. N. Tripathi and J. P. Gaur" Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi 221 005, India; Fax: 91-542-368174; e-mail:
[email protected]
Summary Nickel accumulation by C. vulgaris was studied distinguishing adsorption and intracellular accumulation. The surface adsorption contributed maximally «80%) to total Ni accumulation by the test alga. It was maximal and of equal magnitude at pH 3.5 and 5.5 accordingly suggesting the participation of strong and weak acidic functional groups of C. vulgaris with relatively low and high affinity for Ni adsorption. Nickel uptake was greatly reduced at acidic pH. The cultures in the decline phase of growth showed highest adsorption of Ni but the rate of Ni uptake was greatly reduced when cultures were in the decline or stationary phase of growth. A better exposure of Ni binding sites, or generation of new sites was perhaps responsible for greater Ni adsorption by old cultures of C. vulgaris. Sodium and K caused mixed inhibition whereas Ca and Mg caused noncompetitive inhibition of adsorption and uptake of Ni. Chromium was not able to competitively inhibit adsorption and uptake of Ni by the test alga. The competitive inhibition of Ni adsorption by Cu and Zn seems to be related to their similar ionic properties. Cu stimulated Ni uptake due possibly to increased permeability of the plasma membrane. The present study disagrees with the general conception that cations are competitive inhibitors of metal adsorption and uptake by algae. Key words: Adsorption; Uptake; Heavy metal; Chlorella
vulgaris; Nickel.
Introduction Heavy metal pollution deleteriously affects algae, the principal primary producers of aquatic ecosystems. Many algae inhabiting metal enriched water bodies tend to accumulate high concentrations of these toxic elements [6]. However, there is very limited understanding "corresponding author © 2000 by Urban & FischerVerlag
of the basic processes involved in metal accumulation by algae. A majority of earlier studies describe total metal accumulation by algae [6] despite the fact-that metal accumulated only inside the cell is responsible for toxicity. Furthermore, metal adsorption on the cell surface has been shown as one of the important mechanisms of metal tolerance in algae [6]. At this point it looks essential to distinguish metal accumulated in the intracellular compartments from that adsorbed on the cell surface. A few studies have been conducted to distinguish intracellular metal uptake from adsorption on the cell surface in lichens and bryophytes [3, 4, 19], and also in some algae [2, 11]. Nevertheless, there is a pressing need to understand these two major processes of metal accumulation in other important algal species also. Chlarella vulgaris, a virtually ubiquitous unicellular green alga, grows abundantly in wastewater and also in high rate oxidation ponds all over the world. This alga has a remarkable ability to accumulate high concentrations of various heavy metals from external environment [5, 9, 12]. Therefore, C. vulgaris could be employed for stripping of metals from wastewaters [14, 18]. Moreover, various species of Chlarella have often been used in toxicity bioassays [17]. These considerations necessitate understanding the mechanism(s) of metal accumulation by this organism. The present study deals with the accumulation of Ni by C. vulgaris distinguishing intracellular uptake from the surface adsorption, which has not been hitherto investigated. The effects of factors like pH, temperature, culture age and presence of cations and other metal ions on kinetics of Ni adsorption and uptake have also been investigated. Nickel was selected as the test metal because it is extremely toxic to organisms, and often occurs at high concentrations in industrial and domestic effluents [6, 13]. 0932-4739/00/36/04-443 $ 15.00/0
444
S. K. Mehta, B. N.Tripathi and J. P. Gaur
Material and Methods Microorganism: Cblorella vulgaris, isolated from a pond located within the campus of the Banaras Hindu University, Varanasi, India, was used as test organism. It was grown axenically in modified Chu-10 medium [10] prepared in MilliQ (Bangalore, India) deionized water. Stock cultures were grown in 250-ml Erlenmeyer flasks in a 14 h light (72 prnol m-2 S-1): 10 h dark cycle at 6.8 pH. The cultures were handshaken several times daily. All experiments, wherever not specified, were conducted with cultures in the logarithmic phase of growth. Three replicates were considered for all experiments. Kinetic Constants: The Michaelis-Menten equation, commonly used in enzyme kinetics, was employed to describe the uptake of Ni by the test organism, and the kinetic constants, i.e., apparent Krn (Michalis-Menten constant, the ion concentration required for half maximal uptake) and Vmax (maximal uptake rate) were calculated. These constants could not be calculated for Ni adsorption as it did not occur at a constant rate. Wellsand Brown [19] have advocated the use of alternate parameters for describing adsorption of metal ions by plant materials. Therefore, the parameters, apparent K, (dissociation constant, the ion concentration required for the half maximal adsorption) and U rnax (maximal possible adsorption during 30 min incubation) were determined using the procedure outlined by Wells and Brown [19]. Ni Analysis of Algal Cells: Methods as described by Bates et al. [2] were followed to distinguish the quantity of adsorbed versus intracellular metal content. After incubation in metal solution the cell pellets were washed with 2 mM EDTA (disodium salt) for 10 min. The fraction of Ni not extracted by EDTA was defined as the intracellular Ni, resulting due to the uptake process. Nickel desorbed with EDTA was defined as the adsorbed fraction. The amount of Ni adsorbed on the cell surface was determined by subtracting the intracellular Cu content from the total metal content of the test alga. Intracellular Ni content was determined by digesting the EDTA-washed cell pellets in a 10 ml digestion solution consisting of concentrated nitric acid, hydrogen peroxide (30% w/v) and Milli-Q water in a 1: 1: 3 ratio (v/v/v). Digestion was done on a hot plate till a clear solution of about 5 ml was left. This solution was cooled and then the final volume was adjusted to 5 ml by addition of 2% (v/v) nitric acid. Nickel content in the solution was determined by using a PerkinElmer atomic absorption spectrophotometer (model 2380). Time-Course of Adsorption and Uptake of Ni: Cells were harvested from stock cultures by centrifugation and pellets were washed twice with Milli-Q water. Pellets were then resuspended in 250 ml Ni-free unbuffered medium. The pH was adjusted to 5.5 ± 0.2 using 0.1 M HCI or NaOH at the beginning of the experiment. Ni was added to cell suspensions to give 10,25,50,80 and 100JlM Ni. At various time intervals 20 ml cell suspension was taken out from each flask. The suspension was divided into two portions for determining total and intracellular metal accumulation. The cells were centrifuged and pellets were analyzed for metal content. All flasks and tools used in metal accumulation studies and samples for analysis were repeatedly washed with 0.1 mM HN 0 3 and rinsed with Mill-Q water in order to minimize possible contamination. Adsorption and Uptake of Ni as a Function of Ni Concentration in the External Environment: Adsorption and uptake of Ni were measured from its various external concen-
trations ranging from 2.5 to 100 JlM. Cells were incubated in 100 ml medium containing different concentrations of Ni. The samples undergoing metal treatment were kept in a shaker at 30 rpm. The cellswere incubated in metal solution for 30 min (determined from the time-course study) as adsorption became saturated within this time period and the uptake occurred concomitantly at a constant rate. After incubation in Ni containing medium, the cells were harvested, pellets digested and analyzed for metal contents as described earlier. Effects of pH on Adsorption and Uptake of Ni: 50 ml medium having various concentration of Ni was taken into 150 ml Erlenmeyer flasks. The pH was adjusted to 3.5, 4.5, 5.5, 6.0, 6.5 or 7.5 before introducing cells into the medium. The cells were incubated in light in a shaker for 30 min at 30 rpm. Adsorption and uptake of Ni were determined as described earlier. Effects of Culture Age on Adsorption and Uptake of Ni: Cultures in the exponential phase of growth were incubated in fresh medium, and kept in conditions as described before. Cells were harvested 8, 16, and 32 days after incubation. The cell pellets were washed and resuspended in 50 ml medium containing various concentrations of Ni. The samples were incubated in conditions as detailed above. Effects of Cations on Ni Adsorption and Uptake: The effect of cations like Ca, Mg, K, Na, Cu, Zn and Cr on kinetics of adsorption and uptake of Ni by the test alga were studied. 100 JlM of each tested cation were added separately to 50 ml cell suspensions containing various concentrations of Ni. Salts of the cations used were CaCI 2.2H20 , MgCl 2 • 6H 20 , KCI, NaCl, CuCI 2.6H20 , ZnCl 2 and K2CrP7' Thereafter, the suspensions were incubated in light for 30 min in a shaker, and adsorbed and intracellular metal contents were determined.
Results The adsorption of Ni by C. vulgaris was very rapid and saturated within 10 min. After the first 10 min of incubation, the main fraction of Ni was found adsorbed on the cell surface (Fig. 1). The uptake of Ni increased at an almost constant rate up to 30 min of exposure of cells to Ni, and then slowed down. The rate of metal uptake remained linear only up to 30 min irrespective of metal concentration used. The ability of C. vulgaris to accumulate Ni was further tested at various concentrations of Ni. The adsorption and uptake of Ni increased depending on their external concentrations (Fig. 2). The U rnax (0.744 ± 0.058 fmol cell") was about five times greater than V max (0.160 ± 0.008 fmol cell" h'), Higher K; (21.41 ± 1.07 pM) than K, (12.83 ± 0.98 pM) indicated that adsorption reached saturation at a relatively lower concentration ofNi. The adsorption and uptake of Ni by C. vulgaris were highly dependent on pH of the external medium (Fig. 3). Adsorption was maximal and almost of equal magnitude at 3.5 and 5.5 pH. A relatively higher pH was favourable for the uptake of Ni by the test organism. Ni uptake was maximal in the pH range 5.5-6.5, but
Ni Adsorption and Uptake by C. vulgaris
.
0.6
0.6
"8
"8
0
0
.E
C
.E
0.4
C
e.
~ 0
0
III
III
Z
0.4
0
.Q
"0 11l
"0 11l
0.2
Z
0.2
0.0
0.0 0.10
0.16
-0-- 10 ~M
0 0
- 0 - 25~M
"
50~M
,
0.08
0
0.06
80 11M -0-- 10011M
"8
.E
445
~
v 0 0
0.12
~
0
.E
oj
a 0.04
~
pH 3.5 pH45 pH 5.5 pH60 pH 6.5 pH 7 5
0.08
oj
sc
a:>
11l
:>
Z
Z
0.02
0.04
0.00 -1--
0.00
o
60
45
30
15
0
Time, min
-,.-
20
-
-,--
---,r --
- .-
40
60
80
-
r'
100
Concentration of Ni, ~ M
Fig. 1. The time-course of Ni adsorption and uptake by C.
Fig. 3. Effect of pH on adsorption and uptake of Ni by C.
vulgaris when incubated in solutions containing 10, 25, 50, 80
vulgaris from varying concentration s of Ni in the medium
and 100 uM Cu. Vertical bars show S. D. of three replicates.
dur ing 30 min incubation . Vertical bars show standard deviation, n =3.
0.6
"8 0
.E
0.4
0 12
C
.,
0
~ 0
'"08
III
"0 11l
1;
Z 0.2
~
008
.
~ 0 04
Z
000 -1- -
o 0.0 -f--
o
-
-r---
20
-,-20
- , - ---,i 40
---,r --
--r-
40
60
-
60
,.------, I I 100 80
---r--
-
80
-
--.--
---'
100
Ni concentration, ~M
Fig.2. Adsorptio n and uptake of Ni by C. vulgaris as a function of Ni concentration in the medium during 30 min incubation. Error bars represent standard deviation, n = 3.
abruptly decreased with further rise in pH. In co ntrast to adsorption, the uptake of Ni by the test alga was ver y stro ngly inhibited at acidic pH. The kinetic constants for adsorption and uptake of Ni in relation to pH are plotted in Fig. 4. The U max for Ni adsorption showed two peaks of almost similar magnitude at 3.5 and 5.5 pH. The V max showed a relatively simpler pattern with change in pH of the suspension. It was invariab ly increased with a rise in pH from 3.5 to 5.5 and sta yed unchanged till 6.5 pH. The V max was considerabl y lowered when the suspension had alkaline pH. Besides V max' K, and K; were also affected by pH of the cell suspension. The K, was decreased with an increase in pH from 3.5 to 5.5 beyond which no further increase was evident. Contrary to it, K; was increased with an increase in pH from 3.5 to 6.5. K m always remained higher than K, at each tested pH. Adsorption and uptake of Ni were also affected by temperature. Nickel u ptake increased w ith a rise in temperature from 6 to 25 °C (Fig. 5). Fu rt her rise in
446
S. K. Mehta, B. N. Tripathi and J. P. Gaur 1.0
--
.,
-{]- Vmax' fmol cell
0.8
0.8 - r - - - - - - - - - - --
-,
U""",. fmol cell
-, -, h
~ 0.6 (5
.E
0.6
.~ 0.4
e-o
0.4
CIl
1J
co
Z 0.2
0.2 -
o
6 ·C
o "
15 · C 25 ·C 38 ·C
'L
0.08
7 may be ascribed to its precipitation as hydroxides. An increase in Ni up take with a rise in temperature from 6 to 25 °C suggested that the process is active. Non-competitive inhibition of Ni uptake by Cr can be att ributed to their very dissimil ar ionic properties. The co mpetitive inhibition of Ni adso rption by Cu and Zn suggests their binding onto common fun ctional groups on th e cell surface. Th e stimulation of Ni uptake by Cu may be related to th e great pot enti al of Cu in disrupting the membran e perm eability that perhaps increased permeation and intracellular accumulation of Ni into the cell [6, 13]. Zinc was howe ver inhibitory to Ni uptake by the test organism. Perhaps Zn does not have the ability to evoke stimulatory effects on Ni uptake by increasing membrane permeability. In fact, Ernst et al. [7] revealed that Zn doe s not damage isolat-
449
ed membranes, and the disruption of membrane permeability is generally not visible even at high concentrations ofZn. Calcium and Mg were non-competiti ve inhibitors of Ni uptake, and this is in disagreement with Mallick and Rai [12] and Wells and Brown [19]. It is well known th at high concentration s of Ca prevent the passage of heavy metals through the memb rane [6] due either to competition for binding sites, or precipitation or complexation of metals by carb onate, bicarbonate or hydr oxide of calcium [14]. A similar effect perhaps contributed to decreased Ni uptake by C. vulgaris in the presence of Ca. Moreover, since the mode of inhibition of Ni uptake by both Ca and Mg was similar, there is a possibility that both these cations exerted inhibitory effects in a similar manner. Potassium and Na caused mixed inhibition due perhaps to th e reason that they are monovalent cations and w ere th erefore not able to act dir ectly on th e Ni-uptake system so as to cause its co mpetitive inhibition. For Ni adso rption, K and Na decreased U rnax but K, was increased thus suggesting a mixed type inhibition. It seems th at binding of monovalent cations perhaps reduced th e space availabl e on th e cell surface for th e binding of Ni. Ca and Mg probably redu ced the surface charge density of anionic sites available on the surface for Ni binding [19] thereby resulting in reduced Ni adso rption. The present study shows that the widely held assumption th at cations inhibit metal accumulation by competing with them for intracellular transport as well as binding on the cell surface is not absolutely valid at least in C. v ulgaris. Acknowledgements: We th ank the Head, Department of Botany, and the Co-ordinator, Centre of Advanced Study in Botan y, Banaras Hindu University, for facilities.
References
2
3 4
5
Aksu Z. (1998): Biosorption of heavy metals by Microalgae in batch and cont inuous systems. In: Wong Y.-S. and Tam N .F.Y. (eds.): Wastewate r Treatment with Algae, pp. 38-53, Springer-Verlag, H eidelberg. Bates S.S., Tessier A., Campbell PG.c. and Buffle ]. (1982): Zinc adsorption and transport by Chlamydomonas variabilis and Scenedesmus subspicatus (Chloro phyceae) grow n in semicontin uous culture. ]. Phycol. 18, 521- 529. Beckett R.P. and Brown D.H. (1984): The cont rol of cadmium upt ake in the lichen genus Peltigera.]. Exp. Bot. 35, 1071-1082. Brow n D.H. and Beckett R.P. (1985): Int racellular and extracellular uptake of cadmium by the moss Rhytidiadelphus squarrosus. Ann. Bot. 55, 179-188. Ch o D.Y., Lee S.T., Park S.W. and Chung A.S. (1994): Studies on the biosorption of heavy metals into Chlorella vulgaris. J. Environ. Sci. H ealth Part A 29, 389-409.
450
S. K. Mehta, B. N. Tripathi and J. P. Gaur
6 DeFilippis L.F. and Pallaghy C.K. (1994): Heavy metals: Sources and Biological Effects. In: Rai L'C; Gaur J.P. and Soeder C]. (eds.): Algae and Water Pollution. pp. 31-37, E. Schweizerbart'sche Verlagsbuchhandlung (Nagele u. Obermiller), Stuttgart. 7 Ernst W.H.O., Verkleij J.A.C. and Schat H. (1992): Metal tolerance in plants. Acta Bot. Neerl. 41, 229-249. 8 Fourest E. and Volesky B. (1996): Contribution of sulfonate groups and alginate to heavy metal biosorption by the dry biomass of Sargassurn fluitans. Env. Sci. Technol. 30,277-282. 9 Garnham G.W., Codd G.A. and Gadd G.M. (1992): Kinetics of uptake and intracellular location of cobalt, manganese and zinc in the estuarine green alga Chlorella salina. Appl. Microbiol. Biotechnol. 37, 270-276. 10 Gerloff G.c., Fitzerald G.P. and Skoog F. (1950): The isolation, purification and culture of blue-green algae. Am. J. Bot. 27, 216-218. 11 Knauer K., Behra R. and Sigg L. (1997): Adsorption and uptake of copper by the green alga Scenedesrnus subspicatus (Chlorophyceae). J. Phycol. 33, 596--601. 12 Mallick N. and Rai LiC. (1993): Influence of culture density, pH, organic acids and divalent cations on the removal
of nutrients and metals by immobilized Anabaena doJ. Microbiol. Biotechnol. 9, 196-201. Mehta S.K. and Gaur J.P. (1999): Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris. New Phytol. 143,253-259. Rai L.c., Gaur J.P. and Kumar H.D. (1981): Phycology and heavy-metal pollution. BioI. Rev. 56, 99-151. Rai P.K., Mallick N. and Rai t.c, (1994): Effect of nickel on certain physiological and biochemical behaviors of an acid tolerant Chlorella vulgaris. Bio metals 7, 193-220. Sunda W.G. and Huntsman S.A. (1995): Regulation of copper concentration in the oceanic nutricline by phytoplankton uptake and regeneration cycles. Limnol. Oceanogr. 40,132-137. Shubert L.E. (editor) (1984): Algae as Ecological Indicators, Academic Press, London. Volesky B. and Holan Z.R. (1995): Biosorption of heavy metals. Biotechnol. Prog. 11,235-250. WellsJ.M. and Brown D.H. (1990): Ionic control of intracellular and extracellular cadmium uptake by the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. New Phytol. 116,541-553.
liolum and Chlorella vulgaris. World
13 14 15 16
17 18 19
