Planta (Berl.) 121,205--212 (1974) �9 by Springer-Verlag 1974

Silicic-acid Uptake in Diatoms Studied with [68Ge]Germanic Acid as Tracer

Farooq Azam

Institute of Marine Resources, University of California/San Diego, La Jolla, California 92037, USA

Received August 23 / October 11, 1974

Summary. Uptake of silicic acid in the diatoms Navicula pelliculosa and Nitzschia alba was studied, using the natural isotope, ~ssi, or a radioisotope, 31Si. The rate of uptake of silicic acid was also determined by using [SSGeJgermanie acid as a tracer of silicie acid. At a given silieic- acid concentration in the growth medium, the fractions of [6SGe]germanic acid taken up followed closely the fraction of silicie acid taken up in the same time period. When the initial concentration of silieic acid was varied at a constant (trace) concentration of [6SGe]-germanic acid, the uptake of 6SGe followed the fraction of silieic acid removed, and not the absolute amount removed from the medium, at all silicie-acid concentrations. The usefulness of this approach in studying silicic-acid uptake is discussed.

Introduction

Diatoms have an absolute requirement for silicon, in the form of o-silicic acid, Si(OH)4 (Richter, 1906; Lewin, 1955) for growth. Germanic acid, Ge(OH)4, at high Ge/Si ratios, inhibits diatom growth (Lewin, 1966), silicic-acid uptake (Lewin, 1966; Azam etai., 1973), chlorophyll synthesis and the synthesis of NADP +- dependent glyceraldehyde-3-phosphate dehydrogenase (Werner, 1966, 1967), and photosynthetic carbon fixation (Thomas and Dodson, in press). Using a radio- isotope of germanium, 6SGe, Azam et al. (1973) found that when present at or below Ge/Si ratios of 0.01, Ge(OH)a is not inhibitory to diatom growth; however, 6SGe is taken up by the cells and is incorporated into the silica of the cell wall, presumably by copolymerization. I t appears that at sub-inhibitory concentrations, Ge(OH)4 might take part in the normal metabolism of diatoms as an analogue of Si(OH)4.

Studies on Si(OH)4 uptake and metabolism in diatoms are hampered by the fact that the only available radioisotope of silicon, alSi, is short-lived (half life = 156 rain). Germanium-68, on the other hand, has a half life of 282 days, and is available commercially in carrier-free form. The purpose of this paper is to examine the possibility that a trace amount of 6SGe(OH)4 added to Si(OH)4 might act as a tracer for the uptake of Si(OH)4 by diatoms.

Materials and Methods Cultures. Nitzschia alba (Lewin and Lewin; Strain LTP-1), originally isolated in the

laboratory of Professor B. E. Volcani, from rotting Macroeystis pyri/era collected on the beach near Scripps Institution of Oceanography, La Jolla, Calif., USA, was kindly provided by Professor Volcani. It was grown at 30 ~ in a synthetic-seawater medium, SSW (Hemmingsen, 1971), with 0.2 % D-glucose as the carbon and energy source. Navicula pelliculosa (Breb.) Hilse;

14 Planta (Berl.), Vol. 121

206 F. Azam

Strain No. 668, Indiana University Culture Collection, Bloomington, Ind., USA) was cultured in a synthetic salts medium (Darley and Volcani, 1971) at 20-22 ~ on a reciprocal shaker under continuous overhead illumination of 5 000 lx from Sylvania "cool white" and ~ warm white" fluorescent lamps (Sylvania, Salem, Mass., USA).

Cell Counts. Samples were fixed with Lugol's iodine and the cell numbers determined by a Coulter Model B Particle Counter (Coulter Electronics Hialeaha, Fla., USA) according to Coombs et al. (1967).

Silicic Acid Analyses were done by the speetrophotometric method of Strickland and Parsons (1972).

JRadioehemieals. 6SGeCl~ (carrier-flee, 6.8 Ci/mg; half-life 282 days) was purchased from New England Nuclear (Boston, Mass., USA). I t was converted to 6SGe(OH)a by the addition of NaOH. [a~Si]Silicic acid was prepared by neutron activation, as described earlier (Azam et al., 1974).

Silicon Starvation. Late-exponential-phase Nitzschia alba cultures were harvested by centrifugation at 1600 • for 1 rain. The cell pellet was resuspended in SSW containing no Si(OH)4, in a polypropylene flask, and stirred magnetically at 30 ~ for 2 h.

Radloassay. The radioactivity of the samples containing alSi or 6SGe was determined in a Beckman LS100C Liquid Scintillation System, using "Aquasol" (New England Nuclear) as the scintillator.

Uptake o] Si(OH)4 and ~SGe(OH)a by N. pellieulosa. An exponentially growing culture was used. The concentration of Si(OH)a in the cell-flee growth medium was determined just before starting the experiment, and was found to be 210 ~M. Aliquots (100 ml) of the culture were placed in four 250-ml Pyrex-glass Erlenmeyer flasks. A neutralized stock solution of Si(OH)a was added to 3 flasks to obtain final concentrations of 400, 600, and 700 y3I; the fourth flask received no Si(OH)~, so that the concentration remained at 210 izM. The flasks were placed on a reciprocating shaker with overhead illumination of 5000 lx (as above), at 19 ~ To each flask, 0.2 ~Ci 6SGe(OH)a were added. Aliquots were withdrawn periodically over 22 h to de- termine the cell number, and the amount of Si(OH)4 and 6SGe remaining in the cell-free medium (obtained by centrifuging 3 ml in 12-ml centrifuge tubes at 1600• for 1 min).

Uptake o/slSi(OH)4 and 6SGe(OH)4 by N. alba. In two 125-ml polypropylene flasks, 25 ml of silicon-starved cultures of h r. alba (8.7• l0 s cells/ml) were placed. One flask received slSi(OH)a at a final concentration of 71 tzM, while the second flask received 71 tzM unlabelled Si(OH)a containing 0.1 ~Ci 6SGe(OH)4. The cultures were magnetically stirred at 30 ~ Aliquots were removed periodically, centrifuged at 1600 x g for 1 mill, and the cell-flee supernatant radioassayed to determine ~SGe or slSi remaining in the medium.

Short-Term Uptake by hr. alba. Silicon-starved h r. alba cultures were used. The uptake of 6SGe was determined in 3 different experiments: (1) To 8 polypropylene flasks, each containing 55 ml culture, different amounts of Si(OH)4, mixed with a constant amount of ~SGe(OH)4 (0.1 ~Ci) were added. The final concentrations of Si(OH)a thus obtained varied from 0.89 ~M to 89 ~ , whereas the final concentration of 6SGe(OH)4 in all 8 flasks was the same. (2) To a stock solution of Si(OH)a, 6SGe(OH)4 was added, so that there was 0.072 ~zCi ~SGe per fzmol Si(OH)a. A range of concentrations from 0.89 ~zM to 89 ~M was obtained by the addition of this st~ek solution to 55 ml culture in each of 6 polypropylene flasks. (3) A dilution of tracer- free 6SGe(OH)4 was made in distilled water so that the concentration of 6SGe(OH)4 was the same as in the stock solution in (2). To 6 polypropylene flasks, this 6SGe(OH)a solution was added to obtain the 6SGe(OH)a concentrations as in the flasks in (2).

In all experiments 5-ml aliquots were filtered rapidly, washed once with iced SSW con- taining no Si(OH)4, resuspended in 1 ml SSW, and radioassayed in aquasol (New England Nuclear) to determine 6SGe uptake.

Results Uptake o/Si(OH)4 and 6SGe(OH)~ by •. pelliculosa

I n this experiment, four equal aliquots derived from the same exponent ia l ly growing culture of N. pelliculosa were exposed to 210, 400, 600, or 700 ~M Si(OH)4 and a cons tant trace a m o u n t (10 -11 M) of carrier-free 6SGe(OH)a, and growth per-

Silieic-acid Uptake in Diatoms with [6SGe] as Tracer 207

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Fig. 1A--D. Uptake of Si(OII)4 (.) and ~SGe (o), and the increase in cell number (A) in exponentially growing cultures of AT. l)elliculosa grown at different concentrations of Si(OH)t (210, 400, 600, 700 ~M) and a constant amount (10-11M) ~SGe(OH)~. The uptake of Si(OH)4 and 6aGe is expressed as percent of the total amount added in the growth medium.

The increase in cell number is expressed as percent of the total increase

mitred to continue. Uptake of Si(0H)a and 6SGe, and the cell numbers were de- termined over a period of 22 h, as described in Materials and Methods. The rationale of the experiment was as follows: Since even the lowest concentration of Si(OH)4 used was considered to be at least 10 times greater than the half- saturation constant (K~) for uptake, it was expected tha t the rate of uptake of Si(OH)4 will be equal at all four concentrations (" Vmax" ) ; conseqnently, the per- cent of the total Si(OH)4 taken up in a given t ime ("percent up take") will de- crease from A to D. Also, since the four cultures had the same initial cell density and approximately the same growth rate, the cell number and the rate of increase of biomass will be similar in all cultures (until Si(OH)4 becomes rate-limiting in A). I f 6SGe(OH)4 is taken up at the same rate at all four Si(OH)4 concentrations, it will indicate tha t 6SGe(OH)4 uptake is related to the cell density and the growth rate, and not directly to Si(OH)4 uptake. However, if 6SGe(OH)4 uptake decreases from A to D and follows the percent ul)tal~e of Si(OH)4 , it will mean tha t 6SGe(OH)4 up- take is related to Si(OH)4 uptake.

The results of this experiment (Figs. 1 A-D) show tha t the rates of 68Ge(OH)4 uptake decerase from A to D, and fairly closely follow the percent uptake of Si(OH)4. Thus, when 50 % of 6SGe(OH)4 had been assimilated in A, the correspond- ing values for B, C and D were 31, 26 and 19.5, respectively; when 90% of 6SGe(OH)4 had been assimilated in A, the corresponding values for B, C and D

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l~ig. 2. Uplbake of slSi(OI-I)~ or 6SGe(OI-I)4 by a silicon-starved culture of 1u cdba at 30 ~ C. The radioaetivit~y of 31Si or 6SGe remaining in the cell-free medium is plotted, o 6SGe(OI-I)~, �9 Si(OI-I)~

were 60, 45 and 37, respectively. The increase in cell number, plotted as percent of the total increase (at 22 h, at which time 97-99% of Si(OH)4 had been as- similated in all cultures) follows the percent uptake of Si(OIt)4 and ~SGe(OI-I)a. I t , therefore, appears tha t in N. pellieulosa SSGe(OtI)4 acts as a tracer for Si(OH)a uptake.

Short-Term Uptake Experiments with IV. alba I t was considered of interest to see if the similarity in Si(OI-I)a and ~SGe(OH)4

uptake kinetics can also be demonstrated in short-term experiments (15 s to 3 rain) since such studies might be used in determining kinetic parameters, k s and Vmax. The apoehlorotic (non-photosynthetic) diatom 2i. alba was selected for these ex- periments since dense cultures could be used without the problem of light limita- tion; moreover, a detailed s tudy of the short-term uptake kinetics using 81Si- labelled silicic acid has been done with this diatom (Azam et al., 1974) and could serve as a reference point.

81Si(0H)4 and SSGe(OH)4 Uptake by 37. alba Fig. 2 shows the disappearance of 31Si from 71 ~M 31Si(0I-I)4 added to the

culture at zero t ime; also shown is the disappearance of 6SGe when added in trace amounts to 71 ~M unlabelled Si(OH)4 in the culture. The rates of removal of 31Si and 6SGe are very similar for the first one hour of the incubation; however, when the concentration of 31Si in the medium is reduced to about 5 ~M, the fraction of the 6SGe remaining in the medium was significantly higher than 3~Si. This may be due to a preferential dissolution of 6SGe from dying cells, since there is indication tha t the 6SGe pool may be more easily extractable with water than ~lSi pool (Mehard et at., 1974), at least in Cylindrotheea ]usi/ormis.

Dependence o] ~SGe(OH)4 Uptake on Si(OH)4 Concentration Two short-term uptake experiments were done with N. atba to examine if the

relationship between 6SGe(Ott)4 and Si(OH)4 uptake observed for N. pelliculosa in

Silicie-aeid Uptake in Diatoms with pSGe] as Tracer 209

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Fig. 3.6SGe(OH)t uptake by a silicon-starved culture of _hr. alba. �9 Si(OH)t concentration was varied at a constant (10 -11 M) 6SGe(OH)t concentration. 0 Si(OH)t concentration was varied

at a constant 6SGe/Si ratio of 5 • 10 - s

long-term growth experiment will hold for instantaneous uptake measurements. In one experiment, a fixed amount of ~SGe(OH)4 (1 • 10 -11 M) was added, while the concentration of Si(Ott)4 in the medium was varied from 0.89 to 89 txM. In another experiment, the ratio Ge/Si was kept constant by adding a trace amount of SSGe(Ott)4 to a stock solution of Si(OIt)~; this stock solution was then used to obtain final Si(OH)4 concentrations over the range 0.89-89 BM, and the short- term uptake was measured, as described in Materials and Methods.

The results of these experiments are shown in Figs. 3 and 4. Fig. 3 shows that when Si(OH)4 concentration is raised, maintaining 68Ge(OH), concentration con- stant, the rate of SSGe(OH)4 uptake does not change in the concentration range 0.89-3.57 ~M; at higher Si(OH)4 concentrations, the uptake of SSGe(OH)~ drops gradually. These results can be explained by assuming that SSGe(OH)4 acts as a tracer of Si(OH)4. The apparent K 8 for Si(OIt)4 uptake by N . alba is 4.5 ~M (Azam et al., 1974). The rate of uptake of Si(OtI)~ would be expected to increase as its concentration is increased from a non-saturating 0.89 tzM; but the concomitant decrease in the "specific act ivi ty" will tend to counterbalance the increase in tracer uptake rate. As the system approaches saturation the net tracer uptake rate would be expected to decrease, which is consistent with the observations. On the same assumption, when Ge/Si is kept constant (no decrease in specific activity) the tracer uptake should exhibit a hyperbolic relationship with the Si(OH)4 con- eentration, conforming with Miehaelis-Menten kinetics. This also is consistent with the observations (Fig. 3).

The initial rates of uptake from these two experiments are plotted against Si(OH)4 concentrations, in Fig. 4A and B. The rates for the first experiment have been adjusted to account lo t " i so tope dilution" (Fig. 4A), since in this experiment the Si(OH)~ concentration was varied at a fixed 6SGe(OtI)4 concentration. The Mnetie parameters K s and Vmax determined from these data for the two experi- ments are in good agreement (K 8 = 6.8 ~M; Vmax -= 13.6 ~zmol g-1 rain-l).

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Fig. 4A and B. Lineweaver-Burk plot of Si(OH)4 uptake, using ~SGe(OH)4 as tracer, by N. alba. (A) 6SGe(OtI)~ concentration was kept constant and Si(OIt)4 concentration varied. The initial rates of uptake were corrected for "isotope dilution". (B) Initial rates of tracer uptake, measured at different Si(OH)4 concentrations at a constant 6SGe/Si ratio, are plotted

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Fig. 5. Uptake of 6SGe(OH)4 by a silicon-starved culture of 2V. alba, in the absence of Si(OH) 4

A control experimen~ was done to determine the tracer uptake kinetics when SSGe(OH)4 was added at concentrations used in the second experiment, but without adding any Si(Ott)4. The tracer uptake rate increased linearly without any tendency towards saturation (Fig. 5).

Discussion The d~ta presented here are consistent with the interpretation that eSGe(0tt)4,

when present in trace concentrations relative to Si(0H)4, acts as a tracer of Si(0H)4 uptake and assimilation in the diatoms N. ~elliculosa and ~ . alba. This conclusion appears to be valid for long-term growth experiments as well as for uptake measured over the first few minutes.

Silicic-aeid Uptake in Diatoms with [eSGe] as Tracer 211

Silicie acid is rapidly metabolized intracellularly. Thus, Azam et al. (1974) found that when silicifying N. alba cells are pulse-labelled with 31Si(0H)4, radio- chemical equilibrium in the soluble pool is attained within 2 min. I t is therefore not clear whether transport of silicic acid across the plasma membrane sets the pace of the net silicon metabolism, or whether a step in the intermediary metabo- lism is rate-limiting. Consequently, a treatment of 6SGe(0H)4 ~- Si(OH)a uptake in terms of trans-membrane transport would be partial and premature. On the other hand, the sensitivity provided by the use of eSGe(0H)4 as a tracer of Si(0H)4 should be of great value in future attempts to elucidate the steps involved in the transport and metabolism of silicic acid in diatoms and possibly in other plant and animal systems.

The use of 6SGe(OH)4 as a tracer of Si(0H)4 should be of potential value in the studies of diatom ecology, particularly in marine environments. Due to the low concentration of diatom cells in the sea it is not possible to directly determine the rates of utilization of silicie acid. l~ecently, however, Goering et at. (1974) have proposed a method using stable isotopes of silicon (29Si and 3~ and mass spectro- metry. If 6SGe(OH)4 can be used as a tracer for Si(0H)4 uptake by natural phyto- plankton populations, it will offer experimental simplicity and greater sensitivity.

I thank Dr. Richard W. Eppley, Scripps Institution of Oceanography, La Jells, Calif., USA, for stimulating discussions and for critically reading the manuscript. This work was supported by the U.S. Atomic Energy Commission, Contract No. AT(ll-1)GEN 10, P.A.20, and in part by grant GM 08229-10-12 from the National Institutes of Health to Prof. B. E. Volcani.

References

Azam, F., ttemmingsen, B. B., Volcani, B.E.: Germanium incorporation into the silica of diatom cell walls. Arch. Mikrobiol. 92, 11-20 (1973)

Azam, F., ttemmingsen, B. B., Voleani, B. E. : Role of silicon in diatom metabolism. V. Silieie acid transport in the heterotrophie diatom Nitzschia alba. Arch. Mikrobiol. 97, 103-114 (1974)

Azam, F., Volcani, B.E.: Role of silicon in diatom metabolism. VI. Active transport of germanic acid in the heterotrophic diatom Nitzschia alba. Arch. Mikrobiol., in press

Coombs, J., Halicki, P. J., Holm-Hansen, O., Volcani, B. E.: Studies on the biochemistry and fine structure of silica shell formation in diatoms. II. Changes in concentration of nueleoside triphosphates in silicon-starvation synchrony of Navicula pelliculosa (Br6b.) Hilse. Exp. Cell Res. 47, 315-328 (1967)

Darley, W. M., Volcani, B. E. : Synchronized cultures: Diatoms. In: Methods in enzymology, vol. XXIIIA, p. 85-96, A. San Pierre, ed. New York-London: Acad. Press 1971

Goering, J. J., Nelson, D.M., Carter, J.A.: Silieic acid uptake by natural populations of marine phytoplankton. Deep-Sea Res. 20, 777-789 (1973)

Hemmingsen, B.B.: A mono-silieie acid stimulated adenosinetriphosphatase from proto- plasts of the apoehlorotic diatom Nitzschia alba. Doer. dissert., Univ. of Calif., San Diego 1971

Lewin, J. C. : Silicon metabolism in diatoms. II. Sources of silicon for growth of Navlcula 19elliculosa. Plant Physiol. 30, 129-134 (1955)

Lewin, J. C.: Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phycologia 6, 1-12 (1966)

Mehard, C. W., Sullivan, C. W., Azam, F., Volcani, B. E. : Role of silicon in diatom metabolism. IV. Sub-cellular localization of germanium in Nitzschia alba and Cylindrotheca fusiformis. Physiol. Plantarum (Cph.) 30, 265-272 (1974)

212 F. Azam

Richter, O.: Zur Physiologic der Diatomeen (I. Mitt.). S.-B. Akad. Wiss. Wien, math.-nat. K1. 115, 27-119 (1906)

Strickland, J. D. H., Parsons, T. R. : A practical handbook of seawater analysis. Fish. Res. Bd. Canada, Bull. :No. 167 (1972)

Thomas, W.H. , Dodson, A. :N.: Inhibition of diatom photosynthesis by germanic acid: separation of diatom productivity from total marine primary productivity. Mar. Biol., in press

Werner, D.: Die Kiesels~Lure im Stoffwechsel yon Cyclotella cryptica Reimann, Lewin und Guillard. Arch. Mikrobiol. 55, 278-308 (1966)

Werner, D.: Hemmung dcr Chlorophyllsynthese und der NADP+-abhgngigen Glycerinaldehyd- 3-phosphat-dehydrogenase durch Germaniumsgure bei Cyclotella cryptica. Arch. Mikro- biol. 57, 51-60 (1967)