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Effect of copper and

cadmium on carbon
assimilation and uptake of
metals by algae
a a
P.A. Azeez & D.K. Banerjee
a
School of Environmental Sciences ,
Jawaharlal Nehru University , New Delhi, 110
067, India
Published online: 19 Sep 2008.

To cite this article: P.A. Azeez & D.K. Banerjee (1986) Effect of copper and
cadmium on carbon assimilation and uptake of metals by algae, Toxicological &
Environmental Chemistry, 12:1-2, 77-86, DOI: 10.1080/02772248609357151

To link to this article: http://dx.doi.org/10.1080/02772248609357151

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 Toxicological and Environmental Chemistry, 1986, Vol. 12, pp. 77-86
0277-2248/86/1202-0077 $18.50/0
© 1986 Gordon and Breach, Science Publishers, Inc.
Printed in Great Britain

Effect of Copper and Cadmium

on Carbon Assimilation and
Uptake of Metals by Algae
Downloaded by [University of Glasgow] at 21:53 06 January 2015

P. A. AZEEZ and D. K. BANERJEE

School of Environmental Sciences, Jawaharlal Nehru University, New
Delhi-110 067, India

(Received March 22, 1986)

Two species of blue green algae Spirulina platensis and Anacystis nidulans grown in
artificial aqueous media were treated with Cu and Cd in concentrations of 0.01, 0.1,
1.0 and 10 ppm to study carbon assimilation and Chlorophyll (Chl) A content. The
species were treated with concentrations of 0.1, 0.5, 1.0, 5.0 and 10.0 ppm to study the
uptake of metals with exposure time. Carbon assimilation and Chl A content showed
responses proportional to the concentration in the general form y = K + n ln C, where
C is the concentration of metal in ppm, while in case of uptake the relation was
y = KC" (where C is the molar concentration x 10 - 6 of the metal). The n values in case
of uptake was found to be < 1 indicating a non-Langmuir type of sorption. The
concentration factors of metals decreased with metal concentration in the medium.

KEY WORDS: Algae, copper, cadmium, spectrophotometry, AAS, carbon assimilation,

chlorophyll A content, metal uptake.

INTRODUCTION

Cu and Cd inhibit many biochemical activities like photosynthesis,

nitrogen fixation, nutrient uptake etc. in algal species. Decrease in Chi A
content, and photosynthesis with Cd exposure has been noted for many
algal species by various scientists.1"3 Kallquist and
Meadows, Steeman-Nielsen and Wium-Andersen,5 Saifulla,6 Rueter7
4

and Wu and Lorenzen8 demonstrated the inhibitory effect of copper

77
 78 P. A. AZEEZ AND D. K. BANERJEE
on different species of algae. Uptake of metals has been explored by
Jennette, 9 Dongmann and Nurnberg, 10 Rebhum and Ben-Amotz 11
and, Geisweid and Urbach. 12
In this study the inhibition induced by Cu and Cd on two species
of cyanobacteria Spirulina platensis, a filamentous form and Anacy-
stis nidulans, a unicellular form is reported. To explore and compare
the potentials of these two species for metal removal from the media
the metal uptake was determined.
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MATERIALS AND METHODS

The algae (Spirulina platensis and Anacystis nidulans) were grown in

aqueous artificial culture media, the constitution of which has been
reported earlier.13 Carbon assimilation was studied using labelled
carbon (as Na 2 CO 3 ) of activity 5/iCi. After 6hrs exposure the algae
(both light and dark tubes) were filtered through 2.5 cm GFC filter
papers, treated with cone. HC1 fumes for 20min, 14 dried and
counted using a Beckman liquid scintillation counter (model No.
LS7000). The effect of the metals on Chi A content was determined
both under illuminated and dark conditions to compare the toxic-
ities. The chlorophyll content was estimated after 6hrs expo'sure to
the metal by extraction with 80% acetone. The optical density of
acetone extract was determined by a "Spectronic 1001" spectro-
photometer at 663 and 645 nm. Optical density of the extract is
correlated with Chi A content by the following equation.

Chi A (mg/ml)=(0.127 xA 6 6 3 )-(0.00269 xA 6 4 5 )

and the values are expressed in mg/gm. 15 Metal contents in the algal
samples at intervals of 12, 24, 36 and 48hrs were estimated by
atomic absorption spectrophotometers GBC 902 with air acetylene
flame, after acid digestion (HNO 3 : HC1O4: 2:1 ratio).

RESULTS

The alteration in Chi A content induced by Cu and Cd exposure on

Anacystis nidulans and Spirulina platensis are shown in Table I. The
 TABLE I
Chlorophyll A content (per cent of control) in Anacystis nidulans and Spirulina platensis under different
concentrations of Cu and Cd. K and n are constants of the best fitting curve (Chi A content 10~ 3 gm/gm vs.
metal concentration). L = illuminated samples, D = samples kept under dark.

Metal concentration in ppm Constants

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ChlA
(control) Intercept Slope M
mg/gm (control) 0.01 0.10 1.0 10.0
a
Cu 7.4 ± 100.0 100.0 ± 88.5+ 70.9+ 59.5 ± 5.385 -0.4473
9
0.2 3.2 2.4 3.2 1.5 o
o
o
D 6.6 ± 100.0 106.1 ± 98.5+ 83.3+ 75.8 ± 5.650 -0.3040
•a 0.22 6.1 2.4 3.6 3.4 M

I Cd L 7.4 + 100.0 94.6+ 85.1 ± 76.1+ 67.6 + 5.649 -0.2897

0.2 5.4 3.2 2.4 1.4 O

D 6.6 ± 100.0 100.0± 95.5 + 87.9 ± 81.8 + 5.820 -0.1781 i

0.22 3.6 3.9 2.4 2.1

Cu L 4.20 + 100.0 97.6+ 85.7± 71.4+ 61.9 ± 3.070 -0.2215

o
0.10 2.5 4.8 2.9 2.1
>
4.00 ± 100.0 100.0 ± 90.0 + 80.0 ± 67.5 ± 3.160 -0.1867 5
0.06 3.0 2.8 2.3 1.5
>
I Cd 4.20 ± 100.0 97.6± 83.3 ± 71.4 + 54.8 + 2.930 -0.2562
CO
0.10 2.4 2.4 2.4 1.9
J

4.00 ± 100.0 102.5 + 90.0 + 74.0 ± 62.0 + 3.010 -0.2389

0.06 2.3 2.3 - 2.5 3.0
 80 P. A. AZEEZ AND D. K. BANERJEE
decrease in Chi A content shows a general relation Chi A = K + n In C,
where C — concentration of metal in ppm, K and n are constants.
Under illuminated conditions the metals were more inhibitory than
under dark conditions. The slope of the curve for Cu was higher
than for Cd in case of Anacystis sp. while the reverse was the case
for Spirulina sp. The lower slope for the experiments under dark con-
ditions shows higher toxicity of both the metals under illumination.
In case of carbon assimilation the results of the experiments are
shown in Table II. Carbon assimilation also followed a similar trend
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as the Chi A content, with higher slopes indicating a higher

sensitivity for carbon assimilation compared to Chi A content. Chi A
content of Spirulina sp. under illuminated conditions, was reduced by
38.1% with 10.0 ppm Cu, while the reduction of carbon assimilation
was 58.1% with the same concentration of the metal (Tables I and
II). Cu was somewhat more inhibitory than Cd for Anacystis sp.
carbon assimilation. In case of Spirulina sp. even though a higher
slope of the equation was obtained, the net reduction was higher in
case of cadmium. The higher slope obtained is due to the enhanced
carbon assimilation at 0.01 ppm Cu. For both species 0.01 ppm
concentration of copper resulted in enhanced carbon assimilation. In
case of Anacystis sp. the net carbon uptake value increased to
129.5%, for Spirulina sp. the increase was upto 161.3%.
The general form of the equation of uptake was y = KC with n
always <1.0 (C=concentration in micromole) (Table III). The
concentration factor of metal (cone, in algae/conc. in medium)
showed a 10 to 20 fold decrease with increase in concentration of
metal in the medium (Table IV). In case of Anacystis sp. and
Spirulina sp. for lower metal concentration in the medium higher
concentration factors were obtained. The concentration factors do
not show any correlation with exposure times either for single metals
or combinations.

DISCUSSION

The unicellular species Anacystis shows a higher Chi A content and

higher capacity of carbon assimilation than the filamentous species.
In case of Anacystis the Chi A content observed in this study was
^0.74% of the total dry wt. and for Spirulina sp. it was ~0.42%. A
 Downloaded by [University of Glasgow] at 21:53 06 January 2015

w
3

<3

o S
O
§1

°
O o

is;
5. S o
to <a \s

•I 3 1
w
I 8
-S 1

IMS
tj
O

d
p
o
o
o

§ 1g

—
+1
+1
+1

til
1.71 1.46 1.03

3
1.6 + 921.2 + 68.8 ± 47.4 ±

+1
d
3.6 1.9 1.9 1.9

rt
'
1.79 1.62 1.37 1.06

p
8
1.3 + 122.6 + 75.8 + 41.9 +

+1
>o d
4.9 8.2 1.7 1.7

tq
~
2.51 2.11 1.57 1.00
EFFECT OF COPPER AND CADMIUM ON ALGAE

3
5.5 + 67.7 ± 46.8 + 29.0 +

+1

vuijtiuidg
6.6 8.2 1.7 3.3
81

1.20 0.97 0.78

Downloaded by [University of Glasgow] at 21:53 06 January 2015

TABLE III y
Anacystis nidulans and Spirulina platensis: Constants (K and n) of the metal concentration (10~ 6 mol) in >
the medium vs. metal concentration (10~ 4 mol/gm) in algal biomass plot at different time intervals. >
N
fO
12hrs 24hrs 36hrs 48hrs N
(K) (») (K) (») (K) (") (K) (»)
o
p
:ystis

Cu 0.3849 0.3147 0.4797 0.2864 0.4956 0.3126 0.4642 0.3227

•x Cd 0.1555 0.5980 0.1366 0.6074 0.1304 0.6150 0.1165 0.6084 a

Z
Spirulina

Cu 0.5092 0.3196 0.5118 0.4669 0.3770 0.5007 0.3153 0.5112 m

2w
Cd 0.1431 0.5935 0.1840 0.5026 0.1846 0.4860 0.1676 0.4523 en
 EFFECT OF COPPER AND CADMIUM ON ALGAE 83

- TABLE IV
Anacystis nidulans and Spirulina platensis: Concentration factors (103) at
varying time intervals for Cu, and Cd added in different concentrations.

Cone, (ppm) 12hrs 24hrs 36hrs 48hrs

Cu
0.1 28.31 36.01 35.39 31.18
0.5 9.37 11.02 12.00 11.47
1.0 5.76 6.22 7.81 8.58
Downloaded by [University of Glasgow] at 21:53 06 January 2015

5.0 1.93 2.12 2.45 2.41

10.0 1.20 1.35 1.49 1.38
Amicy tis

Cd
0.1 16.76 14.02 13.04 13.41
0.5 8.56 7.64 7.36 6.50
1.0 6.13 6.00 6.16 4.13
5.0 3.38 3.08 3.03 2.63
10.0 2.63 2.31 2.21 2.20

Cu
0.1 36.65 39.51 31.20 25.38
0.5 12.50 17.00 12.70 11.40
1.0 8.13 12.20 9.53 8.13
5.0 2.60 4.99 4.27 3.68
10.0 1.46 3.40 3.07 2.68
c
-uli

Cd
'5. 0.1 14.61 18.30 18.00 16.10
0.5 7.81 8.77 8.56 7.42
1.0 6.22 7.04 7.14 6.77
5.0 3.06 2.79 2.63 2.10
10.0 2.42 1.85 1.69 1.55

slightly higher ratio of carbon assimilation to chlorophyll content

was also seen in case of Anacystis sp. Anacystis sp. showed higher
instantaneous growth coefficient and lower doubling time than
Spirulina sp. Piorreck et al.16 reported a higher value of Chi A
content for Anacystis sp. (0.92% total dry wt.) and for Spirulina sp.
(0.50% of total dry wt.) in media containing lower nitrogen content
(0.1%) than the media used in the present study (0.18%). Irmer
et al.1 noted significant decrease in Chi A content and carbon
assimilation in presence of Cd in case of Chlamydomonas sp. A
 84 P. A. AZEEZ AND D. K. BANERJEE

concentration ifiM (O.lmg/1) Cd led to a decrease of 35% with 48

hrs exposure while 3hrs exposure resulted in only 3% reduction.
20 iM (2.2mg/l) reduced the same by 89% and 9% for 48 hrs and
3 hrs exposures respectively. AfiM (0.1mg/l) Cd reduced the photo-
synthetic oxygen production by 13% after 3 hrs exposure. Kallquist
and Meadows4 observed that Cu in the concentration range 0.1-
0.5 ppm showed sharp reduction in productivity of algae and a
decrease in inhibitory effect with further increase in metal con-
centration. Rueter et aV reported a 50% reduction in carbon
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assimilation by 10~10M Cu in case of Oscillatoria sp. In the present

study 0.01 ppm (1.57 x 10"7M) of Cd and Cu individually, was
found to reduce the Chi A content to 99.6% of the control by 6 hrs
exposure in case of Spirulina sp. In case of Anacystis sp. 0.01 Cu
(1.57xlO~7M) did not make any reduction while with the same
concentration (ppm) of Cd it resulted in 4% reduction of Chi A
content. An enhancement in the net carbon assimilation with metal
concentration of 0.01 (8.89xl0~ 8 M) ppm was observed in all the
cases except in case of Cd added to Spirulina sp.
A number of studies have reported the enhancement of the
inhibitory effect of heavy metals on the photosynthesis with increas-
ing light intensity.8'17>18 The formation of photosynthetic pigments
also are found to be dependent on the illumination. Many enzymes
like protochlorophyllide reductase important in the photosynthetic
structure developments are activated only under illumination.19 The
synthesis of mRNAs encoded for the enzymes necessary in the
development of photosynthetic system are also illumination depend-
ent. So one of the possible reasons for the high inhibition of Chi A
content in Spirulina and Anacystis under illumination may be that the
conformational changes in enzyme structure occurring during the
activation process expose many functional groups to which metal
ions show high affinity and the metal ions binding with these groups
make the enzyme denatured or inactivated. However, this process
alone is not sufficient to explain the situation observed in the present
study. In the present study a difference in the reduction in the Chi A
content under illuminated and dark conditions were observed in case
of both metals. A possible alteration of the metabolic turnover of
Chi A can be effected by the metal exposure.
The present study shows that the uptake of metals at different
exposure times followed y = KC. Similar relations have been re-
 EFFECT OF COPPER AND CADMIUM ON ALGAE 85

ported for Chlorella stigmatophora by Rebhum and Ben-Amotz 11

and for Chlorella vulgaris, Ankistrodesmus braunii and Eremosphera
viridis with Cd by Geisweid and Urbach. 12 Rebhum and Ben-
Amotz 11 reported a value >1.0 for the slope of the equation
indicating that with increase in the metal concentration the rate of
uptake increases. But Geisweid and Urbach 12 reported «<1.0 in-
dicating a non-Langmuir type of sorption isotherm. The values
reported in the present study are much less than those of Geisweid
and Urbach. 12 For this wide variation from the general trend of
Downloaded by [University of Glasgow] at 21:53 06 January 2015

uptake the high ionic strength (/ = 0.287) of the medium used in the
present study may be one of the important reasons. 20 These uptake
processes follow Freundlich's isotherm. However, Dongmann and
Nurnberg 10 and Davies 21 reported that Cd, Ni and Z are
accumulated by Thalassiosira rotula and Pheaodactylum sp. in a
Langmuir adsorption isotherm form. Variation in the concentration
factor with different metal concentrations was reported by Les and
Walker, 22 Geisweid and Urbach 12 and Dongmann .and Nurnberg 10
in different species of algae and different metals. This phenomena
indicates a saturation effect of the absorption of metal species by
algae.

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2. B. A. Hart and B. D. Scaife, Environ. Res. 14, 401 (1977).
3. J. Overnell, Mar. Biol. 29, 99 (1975).
4. T. Kallquist and J. Meadows, Water Res. 12, 711 (1978).
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6. S. M. Saifulla, Mar. Biol. 44, 299 (1978).
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 86 P. A. AZEEZ AND D. K. BANERJEE

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