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: jpgaur@satyam.net.in
Summary
Nickel accumulation by C. vulgaris was studied distin-
guishing 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. vulgariswith 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 bind-
ing sites, or generation of new sites was perhaps responsi-
ble for greater Ni adsorption by old cultures of C. vulgaris.
Sodium and Kcaused mixed inhibition whereas Ca and Mg
caused noncompetitive inhibition of adsorption and up-
take of Ni. Chromium was not able to competitively inhib-
it adsorption and uptake of Ni by the test alga. The com-
petitive 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 ele-
ments [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 ac-
cumulated 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 essen-
tial to distinguish metal accumulated in the intracellular
compartments from that adsorbed on the cell surface. A
few studies have been conducted to distinguish intracel-
lular 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 under-
stand 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 concentra-
tions of various heavy metals from external environ-
ment [5, 9, 12]. Therefore, C. vulgaris could be em-
ployed for stripping of metals from wastewaters [14,
18]. Moreover, various species of Chlarella have often
been used in toxicity bioassays [17]. These considera-
tions 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 in-
vestigated. 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 be-
cause it is extremely toxic to organisms, and often oc-
curs 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 andJ. P. Gaur
Material andMethods
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 ax-
enically in modified Chu-10 medium [10] prepared in Milli-
Q (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 hand-
shaken several times daily. All experiments, wherever not
specified, were conducted with cultures in the logarithmic
phase of growth. Three replicates were considered for all ex-
periments.
Kinetic Constants: The Michaelis-Menten equation,
commonly used in enzyme kinetics, was employed to de-
scribe the uptake of Ni by the test organism, and the kinetic
constants, i.e., apparent K
rn
(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 adsorp-
tion during 30 min incubation) were determined using the
procedure outlined by Wellsand Brown [19].
Ni Analysis of Algal Cells: Methods as described by Bates
et al. [2] were followed to distinguish the quantity of ad-
sorbed 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 con-
sisting 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 Perkin-
Elmer atomic absorption spectrophotometer (model 2380).
Time-Course of Adsorption and Uptake of Ni: Cells
were harvested from stock cultures by centrifugation and pel-
lets 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 suspen-
sions to give 10,25,50,80 and 100JlMNi. At various time in-
tervals 20 ml cell suspension was taken out from each flask.
The suspension was divided into two portions for determin-
ing 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 sam-
ples for analysis were repeatedly washed with 0.1 mM HN0
3
and rinsed with Mill-Q water in order to minimize possible
contamination.
Adsorption and Uptake of Ni as a Function of Ni Con-
centration 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 shak-
er 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 oc-
curred concomitantly at a constant rate. After incubation in
Ni containing medium, the cells were harvested, pellets di-
gested 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 de-
scribed 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 kinet-
ics of adsorption and uptake of Ni by the test algawere stud-
ied. 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
2.2H2
0 , MgCl
2

6H
2
0 , KCI, NaCl, CuCI
2.6H2
0 , ZnCl
2
and K
2CrP7'
There-
after, 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 fur-
ther tested at various concentrations of Ni. The ad-
sorption 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 Vmax (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. vulgariswere
highly dependent on pH of the external medium (Fig.
3). Adsorption was maximal and almost of equal mag-
nitude 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 AdsorptionandUptake byC. vulgaris 445
0.00 -1-- -,.- - -,-- ---,r-- -.- - r'
100 80 60 40
Concentration of Ni, ~ M
20 o
0.0
0.16
0 pH 3.5
0 pH45
"
pH5.5
~
0.12
v pH60
0 pH6.5
~
0 pH 7 5
0
0.08
.E
oj
sc
11l
a
:>
0.04
Z
0.6
0.6
.
"8
"8
0
0
.E
0.4
.E
0.4
C
C
.Q
0
e.
~
0
0
III
III
"0
"0
0.2
11l 0.2
11l
Z
Z
0.0
0.10 -0-- 10~ M
-0- 2 5 ~ M
5 0 ~ M
0.08
80 11M
,
-0-- 10011M
"8
0
0.06
.E
oj
~
a0.04
:>
Z
0.02
0.00
0 15 30 45 60
Time, min
Fig. 1. The time-course of Ni adsorption and uptake by C.
vulgaris when incubated in solutions containing 10, 25, 50, 80
and 100uM Cu. Vertical bars show S. D. of three replicates.
Fig. 3. Effect of pH on adsorption and uptake of Ni by C.
vulgaris from varying concentrations of Ni in the medium
dur ing 30 min incubation. Vertical bars show standard devia-
tion, n =3.
0.0 -f-- - -r--- ---,r-- --r- - ---r-- - --.-- ---'
abruptly decreased with further rise in pH. In contrast
to adsorption, the uptake of Ni by the test alga was
ver y stro ngly inhibited at acidic pH. The kinetic con-
stants 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 Vmax showed a relatively simpler pat-
tern with change in pH of the suspension. It was invari-
ably increased with a rise in pH from 3.5 to 5.5 and
stayed unchanged till 6.5 pH. The Vmax was consider-
abl y lowered when the suspension had alkaline pH. Be-
sides Vmax' 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 upt ake increased with a rise in
temperature from 6 t o 25 C (Fig. 5). Furt her rise in
I
100
100
I
80
80
i
60 40
60
20
Ni concentration, ~ M
40
o
000 -1- - -,-- -,----,- - ,.------,
0 12
~
..
~ 0 04
Z
20
.,
'8
"0 008
1;
o
0.6
"8
0
.E
0.4
C
0
~
0
III
"0
11l
Z
0.2
Fig.2. Adsorption and uptake of Ni by C. vulgaris as a func-
tion of Ni concentration in the medium during 30 min incu-
bation. Error bars represent standard deviation, n = 3.
446 S. K. Mehta, B. N. Tripathi and J. P. Gaur
o 6 C
o 15 C
" 25 C
'<l 38 C
~ 0.6
(5
.E
. ~ 0.4
e-
o
CIl
1J
co
Z 0.2
0.0 -I-- --,- - r--- ,-- -r- - ,.--'
0.8 -r------------ -,
.,
-<>- U""",. fmol cell
-, -,
-{]- Vmax' fmol cell h
0.12
:c
~
(5
0.08
.E
oj
.x
co
i5..
::J
0.04
? Z
f-
2
~
0.00
5 6 7 8
0 20 40 60 80 100
1.0
0.8
0.6
0.4
0.2 -
0.0
28
24
20
16
-<>- ~ . ~
12 -<>- K m ~
3 4
pH of medium Ni concentration, ~ M
Fig. 4. Kinetic constants of Ni adsor ption (U
rnax
, K) and up-
take (Vmax> K
rn
) by C. vulgarisas functions of pH of the exter-
nal medium. Error bars represent standard deviation, n = 3.
Fig.5. Nickel adsorption and upt ake by C. vulgaris as at var-
ious temperature from various concent rations of Ni in the
medium. Error bars represent standard deviation, n = 3.
temperature to 38C, however, decreased the uptake.
Although adsorption was highest at 38 C, it was not
st ro ng ly influenced by small variations in temperature.
Effects of temperature on kinetic constants of adsorp-
tion and uptake are shown in Tabl e 1. Temperature had
no subst antial effect on U
rn

x
and K, The Vmax increased
with a rise in temperature from 6 to 25 C. But temper-
at ure did not display a significa nt eff ect on U rn, x.
The culture of C. vulgaris entered into the exponen-
tial phase 3 days after incubation into the fresh medium
(data not shown). The exponential phase continued for
7 days, foll owed by the station ar y phase for the next 10
days. The decline phase was induced 20 days after inoc-
ul ati on. The cult ures in the decl ine phase (32-day old)
showed maximum adsorption of N i wherea s the uptake
of Ni was maximal for the 8-day old (exponentially
Table 1. Effect of temperature on kinet ic constants for adsorption and upt ake of Ni in C. vulgaris.
Temperature Adsorption Uptake
(0C)
x, (pM) U
rnax
(fmol cell:') x, (pM) Vmax (fmol cell: ' h')
6.0 12.46' 1.5 0.664' 0.074 14.45' 1.30 0.034' 0.003
15.0 13.08' 1.07 0.682' 0.055 20.82
b
1.86 0.085
b
0.005
25.0 12.83' 0.71 0.742
b
0.025 21.45
b
1.37 0.163< 0.007
38.0 13.74' 1.02 0.819
b
0.025 22.04
b
1.66 0.142< 0.008
All data are means of three replicates; denotes S.D.
Figures in the same column followed by the same letter are not significantly different from each other according to Duncan's
Multiple Range Test (P < 0.05).
Ni Adsorptionand UptakebyC. vulgaris 447
100 80 60 40
Ni concentration, IJM
20 a
0.6
0.00 - p . . - - - r - - ~ - - . - - . - - - - ' - - '
L....
~
0
.E
0.4
C
.2
e-
o
'"
0 None
"0
til
0.2 0 Ca
Z
'"
Mg
v Na
0 K
0.0
0.12
:c
~
0
0.08
.E
ai
.>t:
til
a.
::J
0.04
Z
0 8 da yS
1.6 0 16 d8yS
'"
32 dayS
t,
~
1.2
0
.E
c
.g 08
a. .
0
'"
"0
til
Z 0.4
0.0
0.16
:c 0.12
~
0
.E 0.08
ai
.>t:
til
a.
::J
z
0.04
0.00 -
a 20 40 60 80 100
Ni concentration, IJM
Fig. 6. Effect of culture age on Ni adsorption and upt ake by
C. vulgarisfrom varyi ng concentrations of Ni in the medium.
Er ror bars repr esent standard deviation, n = 3.
Fig. 7. Nickel adsorption and upt ake by C. vulgaris from so-
lutions having various concent rations of Ni and 100 pM Ca,
Mg, K or Na during 30 min incubation. Err or bars represent
growing) culture (Fig. 6). In general, Ni adsorption and
uptake showed contrasting trends with increase in age
of the culture. Vmax was maximum when cultures were
in the exponential phase (Table 2). But U
max
was greater
for older cultures. Whil e K
m
remained unchanged, K,
decreased with increase in age of the cultu re.
Calcium, Mg, Na and K inhibited adsorption as well
as uptake of Ni by the test organism (Fig. 7). Monova-
lent cations were relatively stronger inhibitors of ad-
sorption than the bivalent cations. Potassium was the
strongest inhibitor of adsorption of Ni followed by Na,
Ca and Mg. The hierarchy of cations inhibiting Ni up-
take was Ca, K, Na and Mg in the decreasing order. The
kinetic constants for the inhibition of Ni adsorption
and uptake by cations are presented in Table 3. Unal-
tered K, and K
m
and reduced Umax and Vmax suggested
Table 2. Effect of culture age on kinetic constants for adsor ption and uptake of Ni by C. vulgaris.
Growth Phase
Exponential
Stationar y
Decline
Adsorpt ion Uptake
K
s
(pM) U
max
(fmol cell") x, (pM) Vmax (frnol cell:' h")
12.95
a
0.71 0.723" 0.025 21.54
a
0.57 0.158
a
0.008
22.76
b
1.09 1.325
b
0.045 19.36
a
0.72 0.102
b
0.004
30.08
c
1.64 2.071c 0.066 19.74
a
0.39 o.072' 0.004
All data are means of thr ee replicates; denotes S.D.
Figur es in the same column followed by the same letter are not significantly different according to Duncan's Multipl e Range
Test (P < 0.05).
448 S. K. Mehta, B. N. Tripathi and J. P. Gaur
Table3. Kineticconstants for the inhibitionof adsorption and uptake of Ni by cations and metalions in C. vulgaris.
Inhibiting
cation/
metal ion
Adsorption
K, (pM) U
max
(fmol cell:")
Uptake
Vmax (fmol cell:'h')
None
Ca
Mg
Na
K
Cu
Cr
Zn
12.78 1.08
13.25 0.78
12.12 0.81
1.01
1.53
01.74
13.08 1.05
23.38;:';:';:' 1.34
0.731 0.022
0.009
0.017
0.019
0.012
0.758 0.018
0.421 ;f;:-"-- 0.008
0.698 0.023
21.64 0.48
22.18 1.02
20.82 0.97
1.00
32.75;:-;:- 1.17
1.34
22.12 0.88
48.86;:;f;: 1.12
0.164 0.008
0.004
0.005
0.006
0.08r ;: 0.04
0.014
0.074;:' ;:' ;:- 0.002
0.149 0.007
All data are meansof three replicates; denotesS.D.
Data markedwith asterisks are significantly differentfromthe control ('f P < 0.05, ;:-;:-P < 0.01, ;:-;f;:- P <0.001).
0.00 -f-- -.-- - ,.-- -,-- - ,-- --,--l
0.0 -f---.--- ,.-- -,-- - ,-- --,--l
Fig. 8 shows the adsorption and uptake of Ni by the
test alga in presence or absence of 100 pM Cu, Zn or Cr.
All the metal ions inhibited the adsorption and uptake
of Ni. The only exception was Cu that accelerated Ni
uptake by C. vulgaris. Chromium did not change K,
and K
rn
but U
rnax
and Vmax were significantly decreased
thereby suggesting non-competitive inhibition of ad-
sorption and uptake of Ni by Cr. However, Zn and Cu
caused competitive inhibition of Ni adsorption.
Discussion
C. vulgaris accumulated high concentrations of Ni;
however, adsorption contributed < 80% to total Ni ac-
cumulation by the test alga. This agrees well with pre -
vious reports [2, 11] demonstrating a greater propor-
tion (90%) of metals on the cell surface in comparison
to the intracellular fraction. A large amount of Ni re-
maining adsorbed on the cell surface could be advanta-
geous in the sense that it could be recovered by using a
suitable desorbing agent [1, 18J, and the biomass of the
test alga could be repeatedly used for removing Ni
from wastewater. It was possible to distinguish adsorp-
tion and uptake of Ni in C. vulgaris, but whether Ni
adsorbed on the surface contributes to its uptake, or
both the processes are independent of each other need
to be elucidated. The time-course of Ni accumulation
showed two phases, an initial very rapid adsorption fol-
lowed by a slower uptake. Some earlier workers have
also made similar observations [2, 14, 15J. The present
work showed onl y one system for the uptake of Ni in
C. vulgaris, whereas high and low affinity uptake sys-
tems have been reported for Cu in some algal species
[11, 16].
The adsorption and uptake of Ni were greatly affect-
ed by the age of the culture. The cultures in the expo-
nential phase showed the maximal Ni uptake. The min-
100 80 60 40
Ni concentration, lJM
20 o
0.6

0
.s
0.4
c
.Q
e-
o
III
u
0.2
0 None
<U
Z
0 Cu
'"
c-
V Zn
0.16
B 0.12
o
.s
OJ " 0.08
.>L
<U
a.
:l
z 0.04
non-competitive inhibition of Ni adsorption and up-
take by Ca and Mg. Potassium and Na caused mixed in-
hibition of Ni adsorption and uptake by the test alga.
Fig.S. Nickel adsorption and uptake by C. vulgaris from so-
lutions havingvarious concentrations of Ni and 100 pM Cu,
Cr or Zn during 30 minincubation. Error bars representstan-
dard deviation, n = 3.
imal Ni uptake by cultures in the stationary or decline
phases was seemingly due to the fact that the cells were
not metabolically very active during these phases and
metal uptake is essentiall y a metabolism-dependent
process. Unlike uptake, the cultures in the stationary
and decline phases were more efficient than those in the
exponential phase in adsorbing Ni on the cell surface.
This may have resulted due to a better exposure of Ni
binding sites or creation of additional sites on the cell
surface.
Two peaks of U
rnax
at pH 3.5 and 5.5 suggest that C.
vulgaris has at least two types of functional groups dis-
sociating at strong and weak acidic pH which together
contributed to Ni adsorption. Almost similar U
rnax
but
different K, at pH 3.5 and 5.5 is suggestive of two types
of functional groups: (1) strongly acidic, having a low
affinity (higher K,) for Ni, and (2) weakl y acidic, with a
high affinity (lower K ) for Ni. Fourest and Volesky [8]
have also reported strong and weak functional groups
in the dried biomass of Sargassum fluitans. Although
adsorption of Ni at pH other than 3.5 and 5.5 was
greately reduced, it did not diminish completely. This
observation points at the invol vement of some other
fun ctional groups of relativel y minor importance in ad-
sorption of Ni by the test or ganism. However, inade-
quate understanding of the st ruc ture and nature of the
cell surface and the mechanism of met al adsorption on
it are major gaps in knowledge requiring thorough in-
vestigation.
A rise in pH from 3.5 to 6.5 led to an increase in in-
tra cellular level of Ni in the test alga. Likewise, Rai et
al. [15] have also shown a markedly increased Ni up-
take with an increase in pH from 3.5 to 6.8 in the wild
type Chlarella vulgaris. Ni uptake was reduced with a
decrease in pH due perhaps to increased competition
with protons for binding on the carrier molecules. It
may also be that the carrier mol ecul es were denatured
at acidic pH thereby reducing Ni uptake in the test alga.
The reduced uptake of Ni at pH >7 may be ascribed to
its precipitat ion as hydroxides. An increase in Ni up-
take with a rise in temperature from 6 to 25 C suggest-
ed 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 competitive inhibition of Ni adso rption by Cu and
Zn suggests their binding onto common fun ctional
groups on the cell surface. Th e stimulation of Ni uptake
by Cu may be related to the great pot enti al of Cu in
disrupting the membrane permeability that perhaps in-
creased permeation and intracellular accumulation of
Ni into the cell [6, 13]. Zinc was howe ver inhibitory to
Ni uptake by the test organi sm. Perhaps Zn does not
have the ability to evoke stimulatory effects on Ni up-
take by increasing membrane permeability. In fact,
Ernst et al. [7] revealed that Zn does not damage isolat-
Ni Adsorption andUptakebyC. vulgaris 449
ed membranes, and the disruption of membrane perme-
ability is generally not visible even at high concentra-
tions 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
that high concentrations of Ca prevent the passage of
heavy metals through the memb rane [6] due either to
competition for binding sites, or precipitation or com-
plexation of metals by carbonate, bicarbonate or hy-
dr oxide of calcium [14]. A similar effect perhaps con-
tributed 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 ef-
fects in a similar manner. Potassium and Na caused
mixed inhibition due perhaps to the reason that they
are monovalent cations and were therefore not able to
act dir ectly on the Ni-uptake syst em so as to cause its
competitive inhibition. For Ni adso rption, K and Na
decreased U
rnax
but K, was increased thus suggesting a
mixed type inhibition. It seems that binding of mono-
valent cations perhaps reduced the space availabl e on
the cell surface for the binding of Ni. Ca and Mg prob-
abl y reduced the surface charge density of anionic sites
available on the surface for Ni binding [19] thereby re-
sulting in reduced Ni adso rption. The present study
shows that the widely held assumption that cations in-
hibit metal accumulation by competing with them for
intracellular transport as well as binding on the cell sur-
face is not absolutely valid at least in C. v ulgaris.
Acknowledgements: We thank the Head, Department of
Botany, and the Co-ordinator, Centre of Advanced Study in
Botany, Banaras Hindu University, for facilities.
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