FEMS Microbiology Letters 98 (1992) 45-50
© 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00
Published by Elsevier
45
FEMSLE 05104
Effect of nutritional regime on accumulation of cobalt,
manganese and zinc by green microalgae
G e o f f r e y W. G a r n h a m , G e o f f r e y A. Codd and G e o f f r e y M. G a d d
Department of Biological Sciences, Uniuersity of Dundee, Dundee, UK
Received 18 June 1992
Revision received 24 July 1992
Accepted 27 July 1992
Key words: Cobalt; Manganese; Zinc; Microalgae; Accumulation; Chemoheterotrophy; Photoautotrophy
1. SUMMARY
Accumulation of cobalt, manganese and zinc
by the algae Chlorella emersonii, Chlamydomonas
reinhardtii and Scenedesmus obliquus has been
characterized under photoautotrophic, photoheterotrophic and chemoheterotrophic nutritional regimes. All three species accumulated
smaller amounts of Co 2+, Mn ~+ and Zn 2+ under
chemoheterotrophic and photoheterotrophic conditions than under photoautotrophic conditions
except in the case of cobalt accumulation by C.
reinhardtii where there was little difference in the
amount of cobalt accumulated under any of the
nutritional regimes. Decreased accumulation of
the three metals by C. emersonii and C. reinhardtii largely resulted from a decrease in the
initial biosorptive phase of uptake whereas the
decrease in Mn 2+ and Zn 2+ accumulation by C.
reinhardtii under chemoheterotrophic and photo-
Correspondence to: G.M. Gadd, Department of Biological
Sciences, University of Dundee, Dundee DD1 4HN, UK.
heterotrophic conditions was due to a decrease in
the slow energy-dependent phase of uptake.
2. INTRODUCTION
Cobalt (Co2+), manganese (Mn 2+) and zinc
(Zn 2+) are essential metal ions required for normal growth of microalgae [1]. However, they also
occur in the aquatic environment as the neutron
activation products 6°Co, 54Mn and 65Zn arising
from the waste streams of nuclear installations as
well as other sources [2]. The fate of these
radionuclides after release and the possibility of
ultimate transfer to humans have become matters
of concern [3]. Accumulation of these metals has
been studied in microalgae [3,4] but little is known
of the influence the nutritional mode may have
on the accumulation of these metals. Algae usually employ a photoautotrophic mode of nutrition, although under certain conditions in natural
environments, e.g. in densely populated algal
communites where light and CO 2 are growthlimiting or in effluents with an assimilable carbon
content, microalgae may show a photohetero-
46
trophic or chemoheterotrophic mode of nutrition
{5,6]. Avery et al. [7] have shown that accumulation of caesium was significantly greater in
Chlorella emersonii under a photoautotrophic nutritional regime c o m p a r e d to a c h e m o heterotrophie regime. The possibility that nutritional regime may affect accumulation of Co 2+,
Mn 2+ and Zn 2+ in green microalgae was investigated in this study.
3. M A T E R I A L S AND M E T H O D S
3.1. Organisms and growth conditions
Axenic cultures of Chlamydomonas reinhardtii
11 32a, Scenedesmus obliquus 276 3a and Chlorella
emersonii 211 8b were photoautotrophically grown
at 23°C in 100 ml B G l l medium [8] adjusted to
pH 8 with tetramethylammonium hydroxide and
autoclaved (120°C, 15 min) before being inoculated to approximately 2.5 × 105 ceils ml-~. Cultures were incubated at 23°C in 250-ml conical
flasks with rotary incubation (150 cycles min -~)
and with photon fluence incident on the surface
of the flasks of 12/xE m -2 s -~ provided by white
fluorescent light tubes. For chemoheterotrophic
growth of S. obliquus and C. emersonii 100 ml of
B G l l medium with 1% (w/v) glucose was used,
but for C. reinhardtii the medium was 100 ml
B G l l with 1% (w/v) sodium acetate since this
organism cannot use glucose as a carbon source
[5]. All were inoculated and incubated as above
except that black polythene was used to eliminate
incident light. Photoheterotrophic growth was obtained by adding 10 /xM 3,3,4-dichlorophenyl1,1-dimethylurea (DCMU) [6] to cultures incubated in the light with either 1% (w/v) glucose or
acetate present in the media as described above.
3.2. Uptake of Co e+, Mn 2+ and Zn 2+
Cultures in the exponential phase of growth
(approx. 20-day incubation) were harvested by
centrifugation (1000 × g, 5 min). The supernatant
was removed and the cells washed once with 10
mM N-tris (hydroxymethyl)methyl-3-amino-propane sulphonic acid (Taps) buffer, which has
negligible metal-binding properties [9], adjusted
to pH 8 with 1 M NaOH. Photoautotrophically
grown C. emersonii, C. reinhardtii and S. obliquus
were again centrifuged (1000 × g , 5 rain) and
resuspended in fresh buffer to the following respective cell densities of 5 × 106 , 1 × 107 and
5 × 107 cells ml ~ (equivalent to cell densities of
the mid-exponential phase of growth). Chemoheterotrophically and photoheterotrophically
grown cells were treated as above except C. emersonii and S. obliquus were resuspended in 10 mM
Taps buffer, pH 8, with 1% (w/v) glucose while
C. reinhardtii was resuspended in 10 mM Taps
buffer, pH 8, with 1% (w/v) sodium acetate.
Resuspension of chemoheterotrophic cells was
carried out in the dark. For photoheterotrophic
cells 10/xM DCMU [6] was included in the buffer
solution. For uptake experiments 10 ml of cell
suspensions were incubated at 23°C in a 25-ml
plastic vial on a magnetic stirrer either in the
light (30 /xE m 2 s-~) for photoautotrophic and
photoheterotrophic cells or in the dark for
chemoheterotrophic cells. As a control, photoautotrophic cells were set up as above but treated
24 h before resuspension in buffer with 10 /xM
DCMU [6]. Uptake experiments were performed
using 25 /xM concentrations of the metal chlorides spiked with 6°Co, 54Mn or 65Zn as described
in Garnham et al. [3]. Samples were taken at 0, 5,
15 and 30 rain and then every hour up to 4 h.
4. RESULTS
Uptake of cobalt, manganese and zinc by all
three microalgal species was typically biphasic
under photoautotrophic, photoheterotrophic and
chemoheterotrophic conditions. The majority of
each metal was generally accumulated in a rapid
initial phase, followed by a slower phase of accumulation (Figs. 1-3). In some cases, negligible
accumulation was evident after the initial phase
of uptake and in photoautotrophic cells incubated with 10 /xM DCMU the second phase of
accumulation was absent. Under all nutritional
regimes the three algal species showed the following orders of metal accumulation; Co2+> Mn 2+
> Zn 2+ by C. emersonii and C. reinhardtii and
Zn2+> Mn2+> Co 2+ by S. obliquus. Cobalt accumulation by C. emersonii and S. obliquus was
47
decreased under chemoheterotrophic growth
c o n d i t i o n s by 77 a n d 57% respectively, as comp a r e d with p h o t o a u t o t r o p h i c u p t a k e , a n d u n d e r
p h o t o h e t e r o t r o p h i c c o n d i t i o n s by 50 a n d 57%
respectively. T h e s e d i f f e r e n c e s in cobalt a c c u m u lation o c c u r r e d d u r i n g t h e r a p i d initial p h a s e
( 0 - 3 0 min) of m e t a l a c c u m u l a t i o n (Fig. 1). H o w ever, t h e r e was no significant r e d u c t i o n in cobalt a c c u m u l a t i o n by C. reinhardtii after 4 h
u n d e r c h e m o h e t e r o t r o p h i c or p h o t o h e t e r o t r o p h i c
regimes. C o b a l t a c c u m u l a t i o n was also r e d u c e d
b u t to a lesser e x t e n t in p h o t o a u t o t r o p h i c cells
with 10 ~ M D C M U . A c c u m u l a t i o n was r e d u c e d
~r~
r ( 8 )~
_ .o-
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Time ( h )
Fig. 2. Uptake of manganese by (a) Chlorella emersonii at a
cell density of 5 x 10 6 m l - 1, (b) Chlamydomonas reinhardtii at
,=,
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~-------.
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l0 T ml x and (c) Scenedesmus obliquus at 5 x 107 ml-i in 10
mM Taps buffer (pH 8), with a MnCI2 concentration of 25
~M at 23°C containing either 1% (w/v) glucose for chemoheterotrophic and photoheterotrophic cultures of C. emersonii
and S. obliquus or 1% (w/v) sodium acetate for chemoheterotrophic and photoheterotrophic C. reinhardtii. (o) photoautotrophic cultures, (e) chemoheterotrophic cultures, ([])
photoheterotrophic cultures and (11) photoautotrophic cultures with 10 /iM DCMU. Each point is a mean of three
replicates, bars indicate SEM and, when not shown, were
smaller than symbol dimensions.
0
0
i
i
i
i
(c)
Time (h)
Fig. 1. Uptake of cobalt by (a) Chlorella emersonii at a cell
density of 5 × 106 m l - l, (b) Chlamydomonas reinhardtii at 107
ml i and (e) Scenedesrnus obliquus at 5x107 m1-1 in 10 mM
Taps buffer (pH 8), with a CoC12 concentration of 25 /~M at
23°C, containing either 1% (w/v) glucose for chemoheterotrophic and photoheterotrophic cultures of C. emerSonii
and S. obliquus or 1% (w/v) sodium acetate for chemoheterotrophic and photoheterotrophic C. reinhardtii. (o) photoautotrophic cultures, (e) chemoheterotrophic cultures, ([])
photoheterotrophic cultures, ( I ) photoautotrophic cultures
with 10/xM DCMU. Each point is a mean of three replicates,
bars indicate SEM and, when not shown, were smaller than
symbol dimensions.
in C. emersonii, S. obliquus a n d C. re&hardtii by
a p p r o x i m a t e l y 33, 25 a n d 25% respectively, as
c o m p a r e d with p h o t o a u t o t r o p h i c u p t a k e . T h e s e
r e d u c t i o n s w e r e d u e to t h e a b s e n c e of t h e s e c o n d
p h a s e o f u p t a k e . T h e r e was a d e c r e a s e in m a n ganese accumulation under chemoheterotrophic
a n d p h o t o h e t e r o t r o p h i c c o n d i t i o n s in all t h r e e
algal species (Fig. 2). T h e d e c r e a s e s o b s e r v e d in
c h e m o h e t e r o t r o p h i c C. emersonii, S. obliquus and
C. reinhardtii after 4 h i n c u b a t i o n were a p p r o x i m a t e l y 92, 50 a n d 27% respectively; in p h o t o h e t e r o t r o p h i c C. emersonii, S. obliquus a n d C.
reinhardtii r e d u c t i o n s w e r e 47, 50 a n d 27% respectively. T h e d e c r e a s e d a c c u m u l a t i o n was d u e
to a r e d u c t i o n in t h e initial r a p i d p h a s e of u p t a k e
in C. emersonii a n d S. obliquus, b u t d u e to a
48
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creased in C. emersonii and S. obliquus by 87 and
33% respectively and again was due to decreased
accumulation during the initial rapid phase of
uptake. Zinc accumulation after 4 h was reduced
by 55% in C. reinhardtii under both chemoheterotrophic and photoheterotrophic conditions,
and as with manganese, this was due to reduced
accumulation during the second slow phase of
uptake. Zinc accumulation was also reduced in
photoheterotrophic cells with 10 ~ M DCMU,
which was due to the absence of a second phase
of uptake as with cobalt and manganese. The
reductions in accumulation recorded were less
than those with chemoheterotrophic or photoheterotrophic cells, with reductions in C. emersonii, S. obliquus and C. reinhardtii of 25, 35, and
35% respectively.
- - - - - - - -
Time (h)
Fig. 3. Uptake of zinc by (a) Chlorella emersonii at a cell
density of 5 × 106 m l - l, (b) Chlamydomonas reinhardtii at 107
m l - l and (c) Scenedesmus obliquus at 5 × 107 ml 1 in 10 mM
TAPS buffer (pH 8), with a ZnC12 concentration of 25 # M at
23°C containing either 1% (w/v) glucose for chemoheterotrophic and photoheterotrophic cultures of C. emersonii
and S. obliquus or 1% (w/v) sodium acetate for chemoheterotrophic and photoheterotrophic cultures of C. reinhardtii. (©) photoautotrophic cultures, (o) chemoheterotrophic cultures, ( D ) photoheterotrophic cultures and ( i )
photoautotrophic cultures with 10/xM DCMU. Each point is
a mean of three replicates, bars indicate SEM and, when not
shown, were smaller than symbol dimensions.
decreased second phase of uptake in C. reinhardtii with little difference between the two
nutritional regimes during the initial phase of
uptake (Fig. 2). As with cobalt, manganese accumulation was also reduced in photoautotrophic
cells with 10 FzM DCMU due to the absence of a
second phase of uptake. The reductions recorded
in C. emersonii, S. obliquus and C. reinhardtii
were 21, 37 and 47% respectively. Zinc accumulation, like that of cobalt and manganese was reduced in all three algal species under chemoheterotrophic and photoheterotrophic conditions
compared with photoautotrophic cells (Fig. 3).
Zinc accumulation after 4 h incubation was de-
5. DISCUSSION
Heavy metal/radionuclide accumulation by
microalgae is often described as consisting of two
phases; a rapid phase of metabolism-independent
binding to the cell wall ('biosorption') followed by
a slower phase due to the simultaneous effects of
growth and surface adsorption, active uptake or
intracellular uptake by passive diffusion [3,10,11].
Accumulation of Co 2+, Mn 2+ and Zn 2+ by all
three species under p h o t o a u t o t r o p h i c and
chemoheterotrophic regimes showed both phases
of uptake, although under our experimental conditions, the slow phase of uptake was not marked
in some organisms, i.e. cobalt accumulation by C.
emersonii and S. obliquus and was absent in photoautotrophic cells in the presence of DCMU. C.
emersonii and S. obliquus showed decreased accumulation of Co 2+, Mn 2+ and Zn 2+ under chemoheterotrophic and photoheterotrophic conditions
which was largely determined by a decrease in
the initial biosorptive phase. Such biosorption
can be highly dependent on cell-wall structure
and composition [12,13] although alterations in
the wall microenvironment as a result of metabolic
activity, metabolite excretion, etc. may also affect
interactions of metal ions with microbial cell walls
[11]. Martinez et al. [14] have reported changes in
the cell-wall structure and composition of
49
Chlorella vulgaris when grown in heterotrophic
conditions consisting of cell-wall thickening with
the inclusion of sporopollenin into the cell wail.
Under
chemoheterotrophic
and photoheterotrophic conditions there may also be a
change in the nature and amount of extracellular
polysaccharide(s) produced by the cells; these
substances can play an important role in biosorption of metals [11].
Only Mn 2+ and Zn 2+ accumulation was red u c e d in C. reinhardtii u n d e r c h e m o heterotrophic and photoheterotrophic conditions.
The difference observed between photoautotrophic and c h e m o h e t e r o t r o p h i c / p h o t o h e t erotrophic nutritional regimes here was largely
due to a decreased slow phase of energy-dependent uptake in chemoheterotrophic cells. Such
reduced uptake could reflect the reduced respiratory rate of chemoheterotrophic and photoheterotrophic cells; the respiration rate of
chemoheterotrophically grown C. emersonii was
shown to be as much as 72% lower than that of
photoautotrophic cells [7]. Furthermore, the requirement for Mn 2+ and Zn 2+ by chemoheterotrophic Chlorella pyrenoidosa has been described as being smaller than for photoautotrophic cells [15] perhaps indicating that
lower amounts of the metals need to be actively
accumulated.
Thus, under chemoheterotrophic and photoheterotrophic conditions, microalgae accumulate
smaller amounts of Co 2+, Mn 2+ and Zn 2+ than
under photoautotrophic conditions. These findings support those of Avery et al. [7] who described reduced Cs + accumulation by chemoheterotrophically grown C. emersonii. It was suggested that different K + (Cs +) uptake systems
may operate according to the mode of nutrition
and that increased K + / C s + accumulation in
photo-autotrophic cells may be due to the K +
requirements of the extra enzymes required for
the catalysis of photoautotrophy that are not necessary for chemoheterotrophy. This could also be
true of Zn 2+ and Mn 2+ since both are required
for enzymes involved in photoautotrophy; Mn 2+
is known to be a constituent of superoxide dismutase and is also associated with the Oz-evolving
centre of the photosynthetic electron transport
chain [16] while Zn 2+ is a constituent of carbonic
anhydrase and may also enhance photosynthetic
CO z fixation under conditions of low CO z concentration [17]. In most cases there was little
difference in the accumulation of metals in the
initial phase of uptake in photoautotrophic cells
with or without DCMU. With photoautotrophic
cells with D C M U , the reduced metal accumulation observed was due to the absence of the
second energy-dependent phase of uptake. However, accumulation of the metals in the initial
p h a s e by p h o t o h e t e r o t r o p h i c and c h e m o heterotrophic C. emersonii and S. obliquus was
less than that of photoautotrophic C. emersonii
and S. obliquus incubated with DCMU, indicating that there may be a change in the cell-wall
structure or polysaccharides when these cells are
grown chemoheterotrophicaIly and photoheterotrophically leading to reduced accumulation of
the metals. In C. reinhardtii there were no differences in the initial phases of uptake between the
different nutritional regimes, and it was only the
second phase of uptake that was affected. The
nutritional regime of microalgae has rarely been
considered in physiological studies on metal
transport or when assessing their role in the
accumulation of radionuclides/heavy metals in
natural environments particularly where they occur in blooms, sediments, waters below the photic
zone and soils where chemoheterotrophic growth
may be exhibited. The results presented here
indicate that the mode of nutrition, in terms of
energy and principle carbon source, can have a
m a j o r influence on algal heavy m e t a l /
radionuclide accumulation.
ACKNOWLEDGEMENT
G.M.G. and G.A.C. gratefully acknowledge financial support from the Natural Environmental
Research Council (GR3/7292).
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50
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