Limnol. Oceanogr.

, 26(l), 1981, 67-73

The interaction between zinc deficiency and copper

toxicity as it affects the silicic acid uptake
mechanisms in Thalassiosira pseudonana l
John G. Rueter, Jr.,2 and F. M. M. Morel
Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge 02139

Abstract
Zinc-deficient cultures of Thalassiosira pseudonana exhibited reduced silicic acid uptake
rates. Copper toxicity decreased the silicic acid uptake rate at any zinc concentration. This
resulted in the uptake rate being a function of the ratio in the medium of the cupric ion
activity to the zinc activity rather than of either metal activity separately. These results are
consistent with a proposed mechanism for the interaction between silicic acid and cupric ion
activity involving a zinc-dependent active site.

In work to be published elsewhere suspected initially from circumstantial

(Rueter and Morel in prep.) we found a observations and evidence in the litera-
competitive relationship between cupric ture. Our original observation was that
ion activity and silicic acid concentration zinc-limited diatoms, Thalassiosira
as they affect growth, silicic acid uptake, weissjlogii (Grun.) (= T. jluviatilis Hust.)
and copper uptake in the marine diatom (Anderson et al. 1978), morphologically
Thalassiosira pseudonana (3H) (Hust.) resembled the copper-inhibited or sili-
Hasle and Heimdal. The result of this cate-starved T. pseudonana (Rueter and
competitive interaction was that relative- Morel in prep.). This suggested that the
ly high cupric ion activities resulted in symptoms of copper toxicity might be
higher rates of copper uptake and lower equivalent in some cases to Zn deficien-
rates of silicic acid uptake. Conversely, cy, due to the inactivation of zinc sites by
high silicic acid concentrations could re- copper. Copper has been shown to dis-
verse the effect of copper toxicity, caus- place zinc from high molecular weight
ing lower copper uptake rates and near compounds and the amount of displace-
maximal silicic acid uptake rates. We ment is directly related to the toxic effect
have considered a mechanism for this in- of either Cu or Cd to many organisms
teraction in which an active site for silicic (Brown 1978). Similarly the ratio of cop-
acid uptake is inhibited by the cupric ion per to zinc in the medium can determine
activity and protected by high substrate the growth response in Euglena (Price
(silicic acid) concentrations. This form of and Quigley 1966).
inhibition is similar to nonreversible We investigate here our primary hy-
competitive binding to the active site that pothesis that zinc was involved in a silic-
has been studied in other enzyme sys- ic acid uptake site by examining the si-
tems (Zeffren and Hall 1973). We also licit acid uptake rate in silicon-limited
thought that the binding of copper at this cultures grown in a range of zinc concen-
active site was one possible pathway for trations and also a secondary hypothesis
copper to enter the cell. that the zinc was exchangeable for cop-
In order to explain this proposed mech- per.
anism, we considered that zinc could be We thank S. Chisholm for advice and
involved in silicic acid uptake. Zinc was help on the manuscript and R. Collier for
developing the methods used for copper
’ This research was supported by NSF grant OCE analysis.
77-0900 to F. M. M. Morel and NOAA grant
NA79AA-D-00077. Methods
2 Present address: P.O. Box 751, Department of
Biology, Portland State University, Portland, Ore- Thalassiosira pseudonana (3H) was
gon 97207. obtained from R. R. L. Guillard and
67
68 Rueter and Morel

Table 1. Zinc ion activity in Aquil for given total Table 2. Cupric ion activity in Aquil for given
added zinc concentrations. pZn’+ computed for total copper concentrations. pCu* calculated for
5x lo-” M and 5x 10 B M EDTA. 5x 10-fi M EDTA.

I Zn concn pcu* z Cu
pZn’+ 5x10 B 5x10 a
13 none
13.4 none (assume 11.2 10-ti M
5x 10 .I’)) 10.1 4x10 fi M
13.1 none 8.9 5x10-” M
12.5 4x10 L,
12.1 10-l)
11.6 3x 10-g
11.5 4 x 10-g 4x10 H
Si(OH), was determined by centrifug-
11.1 10-H ing the sample (5 min at 1,600 x g) to re-
10.5 4x 10-H 4x10 7 move the cells and using a reduced vol-
9.5 4x 10-7 ume modification of the method described
8.5 4 x 10-G by Strickland and Parsons (1972). Alka-
line phosphatase activity was measured
by following the evolution of nitrophenol
maintained in f/2 (Guillard and Ryther from p -nitrophenylphosphate at 4 10 nm
1962) with synthetic ocean water (Morel (Kuenzler and Perras 1965). The acid-sol-
et al. 1979). For experimental work the uble copper was determined by digesting
cultures were grown at 22”C, illumi- the centrifuged pellet from about 50 ml
nated continuously from below at 95 of culture with 40% freshly distilled (Vy-
PEinst. rn-“. s-l, and grown in variations car) nitric acid for 1 h and then reading
of the medium Aquil (Morel et al. 1979). directly on a model 360 Perkin-Elmer
For the present study we used 3 x lo-” atomic absorption spectrophotometer
M NO,, 2 x lo-” M P04, 2 x lo-” M with a HGA-2100 graphite furnace. Cells
Si(OH)4, and 5 x lo-” M EDTA plus were counted with a Fuchs Rosenthal
trace metals with no zinc: zinc was added hemocytometer.
in the amounts shown in the figures. The The silicic acid uptake data were ob-
normal preparation of Aquil includes tained from two types of experiments. In
sterilization through a 0.4-pm Nuclepore the first we grew cells to stationary phase
filter and then aseptic transfer to auto- due to silicic acid limitation at four zinc
claved polycarbonate flasks of distilled concentrations, spiked them with 10 PM
water. The special preparation of Aquil Si(OH), and copper to pCu* = 8.9, and
used here to reduce zinc contamination followed the uptake over the next 4 h.
included the following precautions: me- The cell specific uptake rate could be
dium was passed through a Teflon col- computed from the velocity and cell
umn containing Chelex 100 (instead of number since the cell concentrations did
the glass column normally used); the not vary greatly over the uptake interval.
flasks and pipette tips were washed in In the second type of experiment we fol-
distilled nitric acid and rinsed with Corn- lowed the uptake of silicic acid during
ing distilled water immediately before the exponential phase of growth. The ini-
use; all transfers were made in a laminar tial Si(OH), in these cultures was 20 PM.
flow hood; no part of the labware was The velocity of uptake could be comput-
ever autoclaved or exposed to a flame (i.e. ed directly for any interval of uptake, or
standard sterile techniques could not be the cell specific uptake rate could be
used); and the cultures were grown in computed as the growth rate times the
closed flasks wrapped in plastic bags and final average cell quota. These computa-
only opened in the laminar flow hood. tions depend on the value of the growth
Most of these precautions were the result rate since the cell number changes rap-
of a search for contamination sources idly during the uptake interval.
with measurements on the atomic ab- Two other metabolic activities were
sorption spectrophotometer. examined in silicic acid-limited cultures
 Cu:Zn to silicate uptake 69

pCu*= 8a9

o(Cu”: Zn**)

a. Fig. 2. Uptake rate of silicic acid as a function

II ]
Q.
of ratio of cupric ion to zinc ion activities. Data are
same as in Fig. 1 plotted against minus log of ratio
I I I I I of copper to zinc ion activities. To calculate zinc
IO 9 8 7 6 5 ion activity we assumed that there was no signifi-
PZn TOTAL cant depletion of zinc from medium. On this figure,
higher activities of copper and lower activities of
Fig. 1. Uptake rate of silicic acid as a function zinc are toward left.
of log total zinc concentration at two cupric ion ac-
tivities. Silicic acid-limited cultures in stationary
phase of cell density (2.46 & 0.2) x lo* cells-liter-’
that were grown in four concentrations of zinc were 5 x lo-” M. This indicates that the back-
then spiked with two concentrations of copper to ground con tamination i n the normal
equal pCu* = 13 and pCu* = 8.9. Uptake rate was
determined by disappearance from media over 4 h
preparation of Aquil mi nus zinc (de-
after a spike of 10 PM Si(OH),. Background zinc signed to be very low in all trace metals)
concentration was assumed to be lo-l0 M. pZn’+ was sufficient for maximum growth rate.
ranges from 13.1 to 8.1 (Table 1: 5 x lo-” M EDTA). Although neither the growth-rate during
exponential phase nor the plateau den-
sity was limited by zinc, the potential up-
at stationary phase that had been grown take rate of these stationary phase cul-
in different concentrations of zinc. The tures was higher in the higher zinc
cultures were spiked with 10 PM Si(OH),, cultures (Fig. 1, 0). These stationary
and then with either pNPP or with cop- phase cultures were silicic acid-limited
per (pCu* = 8.9), to determine alkaline and the uptake rate was determined by
phosphatase activity over 20 h or copper disappearance of a 10 PM Si(OH), spike.
uptake over 4 h. This means that the growth rate in
The chemical speciation of the medi- these cultures was not related to the max-
um Aquil was calculated with the com- imum silicic acid uptake rate at stationary
puter program MINEQL (Westall et al. phase. When copper was added to give
1976). The zinc ion and copper ion activ- a cupric ion activity of 1O-s.g (pCu* =
ities in these experiments are the result 8.9), the uptake rate of silicic acid was
of the total zinc and copper concentra- again a direct function of the original zinc
tions added to the media, as shown in added to the medium, but Si(OH), uptake
Tables 1 and 2. rates were uniformly inhibited by Cu for
all values of Zn (Fig. 1, 0). This indicates
Results that zinc concentrations up to 5 x lo-” M
In the first set of experiments there was can ameliorate the effect of copper tox-
no difference in the growth rate or final icity. In this experiment for the least fa-
cell yield of 7’. pseudonana cultures in vorable conditions -high copper and
response to total added zinc from 0 to lowest zinc-there was a net negative up-
 Rueter and Morel

IO!

i
r
-i II
>

01

E IO’ o No Zn
+ IO+ Zn i
-v) A 3x10dgZn L!-L-L----A- I
u 0 lOme Zn 0 I 3 IO
0 Zn TOTAL x109M

Fie. 4. Silicic acid uDtake velocities for cultures

groin in four differeni zinc concentrations. Cells
were in the middle of exponential phase (from day
1.5 to 2.5: Fig. 3) increasing from an average of
1.3 x lo4 cells *ml- to 10.2 x lo4 over the 26.5-h
interval. Uptake velocity calculated by change in
external silicic acid concentration. Absolute value
of concentration of zinc at an extraDolated zero UP-
I I I
10: take velocity is used as an estimate for background
I 2 3 4
contamination of zinc in media.
DAYS
Fig. 3. Cell concentration of T. pseudonancl as
a function of time for four zinc concentrations. A
very clean preparation technique was used to less- acid uptake rates in this experiment in
en background zinc contamination (see methods). the 26.5-h interval from 1.5 to 2.5 days
after inoculation into media of different
zinc concentrations and activities. This
take of Si(OH), (fl ux of silicic acid out of experiment was designed to have lower
the cell). cell numbers than before to allow the as-
Our hypothesis predicts that the inhi- sumption of as small as possible a change
bition by copper should be related to the in the zinc concentration due to cellular
ratio Cu:Zn. As predicted, if the data uptake. The uptake rate shows a hyper-
from Fig. 1 are transformed there is a lin- bolic relationship to the original zinc
ear relationship between the silicic acid concentration in the media (Fig. 4). The
uptake rate and the log of the ratio of the uptake rates per cell for exponential
copper to zinc activities (Fig. 2). phase cultures are higher than for station-
Using extraordinary care in media ary phase cells (Fig. l)-2540 x 10-l”
preparation (see methods), we could re- pmol . cell-’ *h-l for stationary phase.
duce the zinc contamination to such a The background contaminant zinc con-
level that Zn was actually limiting the centration can be estimated by extrapo-
growth rate and cell yield of T. pseudo- lating the relationship between the up-
nana (Fig. 3). We also determined silicic take and the total zinc concentration to
 Cu:Zn to silicate uptake 71

l,5r

p (Cu*+: ZnZ+ )

z- Fig. 6. Copper per cell as a function of minus

log of ratio of copper to zinc ion activities. ‘4 silicon-
limited culture was spiked with copper to pCu* =
8.9 and zinc to concentrations of lo-‘, lo-” M, and
none added, and then with silicic acid. Cultures
with an average cell density of 19.9 x 10” cells. ml- ’
-Ll-Lld were harvested after 4 h and analyzed for acid-sol-
IO II 12 13 14 uble copper per cell. Zinc activities were computed
and background concentration of 5 x 10-l” M was
p Zn*+ assumed. The no copper control (a) is plotted as a
Fig. 5. Silicic acid uptake rate and alkaline background copper estimate.
phosphatase activity in same culture as a function
of zinc activity in media. Silicon-limited cultures
grown to same stationary phase density (2 x 1Oj
cells -ml-‘) in different zinc concentrations and 5 x tion of zinc that corresponds to this in-
lo-” M EDTA. Silicic acid uptake rate per cell de-
termined from growth phase of cultures and alka-
tercept is (0.7 2 0.2) x lo-” M.
line phosphatase activity determined at stationary Zinc seems to be strongly implicated
phase by spiking with pNPP and sampling after 20 in the silicic acid uptake mechanism.
h. All values are compared to uptake or utilization There are two additional supporting
rate of normal Aquil activity of zinc (which equals pieces of evidence that the uptake system
10-10.” M).
for silicic acid is zinc-dependent. The
first is the demonstration that silicic acid
uptake behaves in a way similar to that
the abscissa to calculate the additive in- of other zinc-dependent systems. Alka-
tercept (Fig. 4). Simply, it is assumed that line phosphatase is a zinc metalloenzyme
if this amount of zinc could be removed present in T. pseudonana and has a con-
from the medium a zero uptake rate venient assay. The activity of alkaline
would be expected. The total concentra- phosphatase and the silicic acid uptake
72 Rueter und Morel

rate show the same trend with respect to seems to be conserved even for the low-
the original zinc activity in the medium est Zn activities (Fig. 2).
(Fig. 5). Additional supporting evidence The negative uptake rates that were
is from the uptake of copper. If copper seen in stationary phase cultures under
uptake depends on the displacement of the least favorable copper and zinc com-
zinc by copper at a zinc-active site (as bination (Figs. 1 and 2) can be related to
proposed by our model), then the rate of two previous observations. First, the ac-
displacement should depend on the ratio tual dissolution rate may depend on trace
of copper to zinc activities. Copper per metal absorption onto the frustule, as
cell decreased with increased activity of shown by Lewin (1961) for other divalent
zinc in the medium at pCu* = 8.9 (Fig. trace metals. Second, the net uptake rate
6, X) and, in addition, the copper per cell for all the other copper to zinc ratios may
at pCu* = 13 (with no added copper) include a dissolution component that is
(Fig. 6, 0) also gives a reasonable value less than the uptake rate, as suggested by
if plotted according to its Cu:Zn value. Nelson et al. (1976). Trace metal condi-
tions that result in higher dissolution
Discussion rates are just as important in the biogeo-
All of the results presented here are chemical cycle of silicon as the effect on
consistent with the hypothesis that silicic the silicic acid uptake rate.
acid uptake is mediated by a zinc-depen- Our results also show that T. pseudo-
dent system which is inactivated by cop- nana can achieve maximum growth at
per. The biochemical isolation of the en- lower zinc activities than can other
zyme responsible for this uptake will be species. An addition of lo-” M zinc with
necessary to prove the hypothesis. How- 5 x lo-” M EDTA resulted in a pZrP+ of
ever, the fact that zinc is related to the 12.1 and fully restored the cells to maxi-
activity of the enzyme may facilitate its mal growth rate (Fig. 3). Using Aquil me-
isolation and characterization. dia, Anderson et al. (1978) found that T.
Although the evidence suggests that weissflogii showed growth limitation
the site of the Cu:Zn interaction is at the starting at pZn 3+ = 11. Thus it takes an
Si transport step, interactions at many order of magnitude less zinc activity for
steps in the cell metabolism might ulti- full growth of T. pseudonana than it takes
mately be manifested in the silicic acid to limit the growth of T. weissjlogii. Al-
uptake rate. The net result would be the though cell size may play a role in this
same, but until the actual mechanism is difference, the mechanism is not ob-
determined this interaction is most sim- vious. This variation between similar
ply modeled as a single zinc-dependent species in their response to zinc activi-
system for silicic acid uptake. ties adds another dimension to the inter-
The ratio of copper activity to zinc ac- action between phytoplankton and the
tivity in the medium seems to be an im- metals in their environment and, in par-
portant parameter for determining silicic ticular, illustrates the complications in-
acid uptake. Although our experiments troduced by the interaction between
were designed to minimize zinc uptake toxic and nutritional metals. Although
into the cells, at the low zinc concentra- previous work with copper toxicity (Sun-
tions added slight uptake could reduce da and Guillard 1976; Morel et al. 1978;
the zinc activity drastically, resulting in Rueter and Morel in prep.) did not ad-
a higher susceptibility to copper than dress the zinc interaction, as long as the
predicted from the ratio of cupric ion to zinc concentrations were uniform in any
original zinc ion activity. The effect of study the responses observed should be
such a mechanism was minimal in these due only to copper.
experiments, as seen from the linearity of Zinc is involved in the uptake of silicic
the uptake rate as a function of the ratio acid in T. pseudonana. This can be mod-
of the activities of Cu and Zn which eled as a zinc-active site that has three
 Cu.-Zn to silicate uptake 73

characteristics: cells show reduced up- AND R. R. GUILLARD. 1979. Aquil: A chemi-
take of silicic acid with less zinc, Cu com- cally defined phytoplankton media for trace
metal studies. J. Phycol. 15: 135-141.
petes with Zn for the active site and dis- MOREL, N. M.,J. G. RUETER, AND F. M. MOREL.
ables the site, and copper enters the cell 1978. Copper toxicity to Skeletonema costatum.
as a function of the ratio of copper to zinc J. Phycol. 11: 4348.
ion activities. These results are consis- NELSON, D. M.,J.J. GOERING, S. S. KILHAM,AND
tent with our model for a silicic acid up- R. R. GUILLARD. 1976. Kinetics ofsilicic acid
uptake and rates of silica dissolution in the ma-
take mechanism (Rueter and Morel in rine diatom Thalassiosira pseudonana. J. Phy-
prep.). col. 12: 246-256.
PFUCE,~. A., AND J. W. QUIGLEY. 1966. A method
References for determining quantitative zinc requirements
ANDERSON, M. A., F. M. MOREL, AND R. R. GUIL- for growth. Soil Sci. 101: 11-16.
LARD. 1978. Growth limitation of a coastal dia- STRICKLAND, J. D., AND T. R. PARSONS. 1972. A
tom by low zinc ion activity. Nature 276: 70- practical handbook of seawater analysis, 2nd
71. ed. Bull. Fish. Res. Bd. Can. 167.
BROWN, D. A. 1978. Toxicology of trace elements: SUNDA, W. G., AND R. R. GUILLARD. 1976. Rela-
Metallothionein production and carcinogenesis. tionship between cupric ion activity and the
Ph.D. thesis, Univ. British Columbia. 226 p. toxicity of copper to phytoplankton. J. Mar. Res.
GUILLARD, R.R., AND J. H. RYTHER. 1962. Studies 34: 51 l-529.
on marine planktonic diatoms. 1. CycZoteZZa WESTALL, J.C.,J.L. ZACHARY,AND F.M. MOREL.
nana Hustaedt and Dentonula confervacea 1976. MINEQL, a computer program for the
(Cleve) Gran. Can. J. Microbial. 8: 229-239. calculation of chemical equilibrium composi-
KUENZLER, E. J., AND J. P. PERRAS. 1965. Phos- tion of aqueous systems. Mass. Inst. Technol.
phatases of marine algae. Biol. Bull. 128: 271- Water Quality Lab. Tech. Note 18.
284. ZEFFREN, E., AND P. L. HALL. 1973. The study of
LENIN, J. C. 1961. The dissolution of silica from enzyme mechanisms. Wiley.
diatom walls. Geochim. Cosmochim. Acta 21:
182-195. Submitted: 11 October 1979
MOREL, F. M., J. G. RUETER, D. M. ANDERSON, Accepted: 3 July 1980