Arch. Mikrobiol. 92, 11--20 (1973) 9 by Springer-Verlag 1973

Germanium Incorporation into the Silica of Diatom Cell Walls Farooq Azam, Barbara B. Hemmingsen, and Benjamin E. Voleani University of California (San Diego), La Jolla, California 92037 Received March 16, 1973 Summary. 1. The diatoms, Nitzschia alba, 2gavicula pelliculosa, Cylindrotheca /usi/ormis, and CycloteUa nana, took up radioisotopically labelled germanic acid, ~SGe(OH)~, from their growth media and incorporated up to 80~ of it into the silica of their cell walls. Siticifieation appeared to be required for germanium incorporation. 2. The uptake and incorporation of germanic acid was dependent upon the relative concentrations of Ge(OH)4 and Si(OH)~, i.e., the [Ge]/[Si]. 3. At [Ge]/[Si] of 0.01, no inhibition of growth or of silicic acid uptake by hr. alba was observed. The cell morphology was also normal. 60 to 800/0 of the ~SGe(OH)4 taken up was incorporated. 4. At [Ge]/[Si] of 0.1, silicic acid uptake and growth of N. alba were inhibited by about 950/0. Concomitantly, striking morphological aberrations occurred. 10 to 20~ of the 6SGe(OH)4taken up was incorporated. 5. The possible use of 6SGe(OH)4for the study of silicon metabolism is discussed. Diatoms have an absolute requirement for silieie acid, Si(OH)4, in the growth medium for the formation of the cell wall (Richter, 1906; Lewin, 1955) as well as for some other important cellular processes such as the synthesis of DNA (Darley and Voleani, 1969) and possibly the synthesis of DNA polymerase and thymidylate kinase (Sullivan and Voleani, 1973). Most of the silieie acid taken up by the cell is polymerized and deposited intracellularly within the siliealemma, a membrane-limited structure (Stoermer et al., 1965; Reimann et al., 1965), to form the siliceous part of the cell wall. However, little is known about the biochemical processes involved in the metabolism of silicic acid partly because of the unavailability of a convenient radioisotope of sitieon. Germanium, which occurs just below silicon in Group IV-A of the periodic table of the elements, has chemical properties similar to those of silicon (Jolly, 1966) and is known to substitute for silicon in a number of silicate minerals (Goldschmidt, 1958). This similarity prompted Lewin (1966) to suggest that germanic acid, Ge(OI-I)4, might act as an antimetabolite or as a competitive inhibitor of silicon utilization in organisms which require silicon as an essential nutrient. Indeed, she found that germanic acid inhibited the growth of 10 diatom species. The inhibition

12

F. Azam et al. :

could be reversed b y increasing the silicic acid c o n c e n t r a t i o n in the growth m e d i u m t h e r e b y suggesting a c o m p e t i t i o n of Ge(OtI)4 with Si(OH)~. Lewin concluded t h a t g e r m a n i c acid interfered with silicification processes i n diatoms. W e r n e r (1966, 1967) came to a similar conclusion in his studies of the effect of g e r m a n i c acid on silicic acid requiring processes in the d i a t o m Cyclotella cryptica. The p r e s e n t s t u d y is concerned with the metabolic fate of germanic acid itself. W e h a v e used a radioisotope of g e r m a n i u m , 6SGe (half-life, 282 days), to e x a m i n e the possibility t h a t , i n diatoms, Ge(OH)4 m i g h t follow the same metabolic p a t h w a y as Si(OH)4. Specifically we have e x a m i n e d the u p t a k e a n d i n c o r p o r a t i o n of 6SGe(OtI)a a t low, noni n h i b i t o r y c o n c e n t r a t i o n s a n d also a t the higher c o n c e n t r a t i o n s f o u n d i n h i b i t o r y b y previous workers. A t these higher c o n c e n t r a t i o n s striking a b e r r a t i o n s i n the cell wall m o r p h o l o g y occur. Since these a b e r r a t i o n s h a d gone u n n o t i c e d i n the earlier studies, t h e y are described in this report. Materials and Methods Cultures. The non-photosynthetic diatom Nitzschia alba (Strain LTP-1) was grown at 30~ in a synthetic seawater medium (Hemmingsen, 1971) with 0.20/o D-glucose as the carbon and energy source and 0.71 miVi Si(OIt)4 as the source of silicon. Fifty ml cultures in 125 ml Erlenmeyer flasks were agitated by a magnetic stirrer.

The marine photosynthetic diatoms, Cylindrotheca/usi/ormis (Watson's Strain 13) and Cyclotella nana (Guillard's Sargasso Sea Strain) were grown in synthetic seawater media (Darley and Volcani, 1969; Hemmingsen, 1971). The fresh water, photosynthetic diatom, Navicula pelliculosa (Strain No. 668, Indiana University Culture Collection) was cultured in a synthetic salts medium (Darley and Volcani, 1971). Fifty ml cultures in 125 ml Erlenmeyer flasks were grown at 20--22~ on a reciprocal shaker under continuous overhead illumination of 5000 lux from Sylvania "cool white" and "warm white" fluorescent lamps. Cell Counts. The number of cells in the cultures was found by use of a Coulter Model B Particle Counter (Coulter Electronics). Two ml aliquots of each culture were killed by the addition of one drop of Lugol's iodine and treated as previously described (Coombs et al., 1967b). Viability Determinations. The viability of cells of N. alba which had been exposed to various [Ge]/[Si] was assessed at the end of the experiments by the end-point dilution technique (Meynell and Meynell, 1970). Germanic acid-free growth medium was used and 5 tubes were inoculated from each 10-fold dilution of the experimental culture. Radiochemical. Carrier-free ~sGeC]4 (specific activity, 6.8Ci/mg; half-life, 282 days) was obtained from New England Nuclear. 6SGe(OIt)t was prepared by addition of NaOH. Uptalce and Incorporation o/Germanic Acid. When the culture entered the first half of the exponential phase of growth, and the concentration of Si(Ott)4 in the growth medium was reduced from 0.71 mM to 0.64 mM, 0.2 ~Ci 6SGe(OH)r together with enough non-radioactive Ge(OH)4 to obtain [Ge]/[Si] of 0.01, 0.1 or 1.0 was added. Two 2 ml samples were removed periodically and filtered through membrane filters (0.45 fzm pore size, Millipore). The ceils were washed on the filter with about

Germanium Incorporation in Diatoms

13

20 ml of ice-cold growth medium free of Si(OH)4 and Ge(OH)~. One filter, with its adhering cells, was glued to an aluminum planchet, dried under an infrared lamp and radioassayed in a Sharp (Beckman) Low-beta System, connected to an Anton G-M thin-end window radiation detection tube (1001 TA) to obtain the total germanic acid uptake by the cells. Incorporation of germanic acid into the silica shell was found by measuring tile radioactivity remaining in the sample on the second filter after removal of all organic material by acid digestion (Reimann et al., 1965). The filter, with the adhering cells, was transferred to a 15 ml test tube. After addition of 2 ml conc. H2SO~, the tube was placed in a boiling water-bath for 5 rain. A few crystals of KNO a were added and heating continued until the liquid turned pale yellow (3 to 5 min). After cooling, the contents of the tube were transferred to about 20 ml cold distilled water and then filtered through a membrane filter. The material on the filter, i.e., the silica component of the diatom cell wall, was washed with five 5 ml portions of distilled water at room temperature and radioassayed as described above. Isotope Discrimination. Cells of hr. alba in the early exponential phase of growth were concentrated by centrifugation at 2000 • g for 2 min and resuspended in two 50 ml portions of the growth medium to a density of about 5 • l0 Gcells/ml. After addition of Ge(OH)4 to a [Ge]/[Si] of 0.1, 0.2 ~zCi~SGe(OH)~ was immediately added to one culture and the uptake of the radioisotope followed as described above. The disappearance of the unlabelled Ge(OI-I)~ from the other culture was followed by a colorimetric method (Sorrentino and Paul, 1970) after removal of the cells by centrifugation at 2000 • g for 2 rain. Silicic Acid Uptake. Cultures free of the radioisotope but identical in all other respects were used to follow Si(OH)4 disappearance from the medium. Cells were removed by centrifugation at 2000 • g for 2 min and the Si(OH)~ concentration in the supernatant fluid determined by a slight modification of the colorimetric method of Tfima (1962). Electron Microscopy. Cells of N. alba were fixed 2 h at 5~ in 2 ~ glutaraldehyde buffered with 0.1 M phosphate buffer (pH 6.4) containing 3.3 ~ NaC1 and post-fixed for 1 h at room temperature in 1 ~ OsO4 in the same buffer-NaC1 mixture (Matsudo, 1967). After dehydration in an ethanol-propylene oxide series, the cells were embedded in Epon 812. Sections were stained with uranyl acetate-lead citrate (Reynolds, 1963) and observed in an Elmiskop I electron microscope (Siemens).

Results Since t h e i n h i b i t o r y effects of g e r m a n i c acid on d i a t o m g r o w t h are d e p e n d e n t u p o n t h e r e l a t i v e a m o u n t s of g e r m a n i u m an d silicon in t h e g r o w t h m e d i u m (Lewin, 1966; D a r l e y , 1969), in t h e p r e s e n t e x p e r i m e n t s t h e initial silicon c o n c e n t r a t i o n was held c o n s t a n t an d increasing a m o u n t s of g e r m a n i u m were a d d e d to o b t a i n t h e desired m o l a r ratios of Ge to Si. I n addition, silieie acid u p t a k e a n d increase in cell n u m b e r of t h e n o n - p h o t o s y n t h e t i c d i a t o m , N. alba, were d e t e r m i n e d in order to relate g e r m a n i u m m e t a b o l i s m to silieifieation an d cell growth. E[/ect o] [Ge]/[Si] o/ 0.01. A t this m o l a r r at i o n e i t h e r t h e r a t e of increase in cell n u m b e r n o r t h e r a t e of Si(OH)4 u p t a k e b y N. alba w a s i n h i b i t e d (Fig. 1 a a n d b). I n fact, t h e final cell n u m b e r was s l i g h t l y higher t h a n in t h e control c u l tu r e w i t h o u t Ge(OIt)~. T h e cells t o o k up Ge(Ott)4 f r o m t h e m e d i u m a n d 60 t o 800/0 of t h a t t a k e n up was i n c o r p o r a t e d i n t o

14

F. Azam et al. : I 6.2

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Fig. l a - - d . Increase in cell number, silicic acid uptake, germanic acid uptake and germanic acid incorporation by cells of N . alba. For determination of a eel1 number and b silieie acid uptake, the cultures contained no germanic acid (o o), [Ge]/[Si] of 0.01 (. .), [Ge]/[Si] of 0.1 (, A). Germanic acid uptake ( X - - X ) and incorporation (= 9) were determined at c [Ge]/[Si] of 0.01 and d [Ge]/[Si] of 0.1. For purposes of comparison, the data for silicic acid uptake, germanic acid uptake and germanium incorporation at [Ge]/[Si] of 0.1 were normalized to the same number of cells/ml as in the control culture

t h e silica of t h e cell wall d u r i n g t h e e x p o n e n t i a l p h a s e of g r o w t h (Fig. 1 e). As t h e c u l t u r e e n t e r e d t h e s t a t i o n a r y p h a s e of g r o w t h due t o d e p l e t i o n o f S i ( 0 t t 4 in t h e g r o w t h m e d i u m , g e r m a n i u m i n c o r p o r a t i o n slowed as d i d Si(OH)4 u p t a k e . H o w e v e r , G e ( O H 4 u p t a k e continued, resulting in t h e b u i l d u p of a large i n t r a c e l l u l a r pool of G e ( O t t 4 or its i n t e r m e d i a t e s .

Germanium Incorporation in Diatoms

15

IO0 o

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Fig. 2. U p t a k e of g e r m a n i c acid labelled w i t h t h e radioisotope 6SGe ( X - -

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natural isotope 7~Ge(. .), by cells of N. alba at a [Ge]/[Si] of 0.1. The equations for the lines were computed by the method of least squares regression. F-test applied to the data showed that the lines are not significantly different (/9 ~ 0.6)

I n a control experiment, ceils which had been lysed in distilled water before the start of the experiment did not take up or incorporate any significant amount of Ge(OH)4. Thus, uptake and incorporation are a result of the metabolic activity of the cells and are not due to absorption onto the cells. Another control experiment was carried out to demonstrate that uptake of radioactive 6SGe(OH)4 is a valid measure of Ge(OH)4 uptake. Early exponential phase cells took up 6SGe(OH)4 and Ge(OH)4 at rates which differed by less than 40/0 (Fig.2). Therefore, the diatom N. alba does not discriminate significantly for or against this radioisotope of germanium. After 24 h of growth in the presence of a [Ge]/[Si] of 0.01, the cells were examined in both the phase contrast and the electron microscope and found to be morphologically indistinguishable from the control cells. From these observations we conclude that a [Ge]/[Si] of 0.01 is noninhibitory to growth or to silicifieation and that, under these conditions, Ge(OH)4 is transported into the cells and incorporated into the silica of the cell wall. E[[ect o[ [Ge]/[Si] o[ 0.1. When the [Ge]/[Si] was increased to 0.1, the results were quantitatively as well as qualitatively different from those described above. The rates of inerease in cell number and of silicic acid uptake were greatly reduced (Fig. 1 a and b). Although the rate of germanic acid uptake was about 5 times greater than that at a [Ge]/[Si] of

16

F. Azam et al. :

Fig.3a--d. Electron micrographs of cells of N . alba grown in a the absence of germanic acid (• 7880); b the presence of [Ge]/[Si] of 0.1 (• 5200); c and d the presence of [Ge]/[Si] of 1.0 (• 6500 and • 28200, respectively). I n b the cell pairs have multiple wall-like structures ( M W ) between them. In c the cytoplasm contains electron-opaque granules (arrows) which are at a higher magnification in d. N nucleus; M mitochondrion; O W old wall; N W new wall; M W multiple wall-like structures. The lines in a, b and c represent 1 ~m; in d 0.5 ~m

0.01, g e r m a n i u m i n c o r p o r a t i o n after 12 h was 14~ of t h a t t a k e n u p (Fig. i d) as c o m p a r e d to 72 ~ i n cells exposed to the lower ratio (Fig. 1 c). E x p o s u r e to [Ge]/[Si] of 0.1 also h a d a d r a m a t i c effect on the morphology of the cells. A p p a r e n t l y m o s t of the cells u n d e r w e n t a division, b u t the d a u g h t e r cells did n o t separate completely. Multiple wall-like struc-

Germanium Incorporation in Diatoms

17

tures were laid down between the daughter cells (Fig. 3 b) at about the rate of one every 5 h. This is close to the division time of 2/. alba under normal growth conditions (Fig. 1 a). Since the rate of silicic acid uptake by the cells in the experimental culture was less t h a n 5 ~ of t h a t in the absence of Ge(OH)4, it appears t h a t the cells can support the continued formation of the "multi-walls" with only a fraction of the normal intracellular complement of silica. Detailed fine structural and micro-analytical studies, now in progress, indicate t h a t germanium and silicon are present in the inorganic component of the "multi-walls".

E/]ect o/ [Ge]/[Si] o] 1.0. At equimolar concentrations of germanic acid and siticic acid, increase in cell number is slight. Germanic acid is slowly taken up (0.02 nanomoles/ml/h) but very little is incorporated into acid-insoluble material. Silicic acid uptake was not determined because of the interference of such high concentrations of germanic acid with the colorimetric assay. "Multi-walls" were not numerous but the layers which did form (Fig. 3 c) are probably composed mostly of organic material since t r e a t m e n t with HC1 (which removes Ge02) or with I-IF (which removes SIO2) did not alter their appearance in the electron microscope. I n N. alba, clusters of oval to round, electron-opaque granules within vesicles were found in the cytoplasm (Fig. 3c and d). The significance of these granules is not known at this time. E//ect o/ Various [Ge]/[Si] on Cell Viability. The viability of cells of N. alba was assessed at the end of the 24 h experiments. All of the cells in the culture exposed to [Ge]/[Si] of 0.01 and 80~ of the cell pairs formed during exposure to [Ge]/[Si] of 0.1 were viable. Preliminary experiments indicated t h a t a substantial proportion of the cells were not killed b y exposure to [Ge]/[Si] of 1.0. Thus, the inhibition of cell growth, normal division and normal cell wall formation at the higher [Ge]/[Si] appear to be reversible.

E//ect of Various [Ge]/[Si] on Photosynthetic Diatoms. Silicic acid uptake and increase in cell number in the photosynthetic marine diatoms, C. nana and C. ]usi/ormis, and in the photosynthetic fresh water diatom, N. pelliculosa, were affected by [Ge]/[Si] of 0.01 and 0.1 in a fashion similar to N. alba. Rates of germanic acid uptake and germanium incorporation at these ratios are summarized in Table 1. Darley (1969) found t h a t a [Ge]/[Si] greater than 0.1 was required for complete inhibition of the growth of C. ]usi/ormis; we found t h a t a [Ge]/[Si] of 0.5 was required for complete inhibition of silieie acid uptake and cell division. Thus, this diatom seems to be less sensitive to germanic acid than the other species tested. Morphological changes were found in all species tested at the higher [Ge]/[Si]; the characteristics of these alterations, which are different from those seen in N. alba will be described in a separate report. 2

Arch. Mikrobiol., ]3d. 92

18

F. Azam et al. : Table 1. Uptake and incorporation of germanic acid by some diatom species

Species

Ge(OH)4 uptake (nanomoles/ml/h) [Ge]/[Si] : 0.01 0.1

Nitzschia alba Navicula pelliculosa Cyclotella nana Cylindrotheca ]usi/ormis

0.140 0.080 0.060 0.045

0.375 1.000 0.150 0.390 a

Ge(OH)4 incorporation (nanomoles/ml/h) 0.01 0.1 0.084 0.062 0.022 0.031

0.040 0.156 0.020 0.030 a

[Ce]/[Si] was 0.5. The photosynthetic diatoms were grown in media which initially contained 0.71 mM Si(OH)4, 0.1 tzCi ~SGe(OH)~, and enough Ge(OH)~ to obtain the desired [Ge]/[Si]. Other details as described in the text. The rates were normalized to l0 s cells/ml. The rates for N. alba were computed from Fig. 1.

Discussion These studies have demonstrated, for the first time, that diatoms are able to take up germanic acid and incorporate it into the silica of the cell wall. I t is not known how the germanium, presumably in the form of GeO2, is deposited. I t is very likely that it is polymerized with the silica, Si02, since any surface adsorbed GeO 2 would be removed during the hot tt~SO a and ItNO 3 treatment of the shells. The present evidence suggests that silicifieation of the newly forming cell wall is necessary for germanium incorporation. At the non-inhibitory [Ge]/[Si] of 0.01, silicic acid uptake, considered to be a measure of the rate of silicification (Coombs et al., 1967 a, b), and germanium incorporation are linear processes during the exponential increase in cell number (Fig. 1). When the cells enter the stationary phase of growth due to depletion of silicic acid in the medium, germanium incorporation slows down and stops as does silieic acid uptake. But germanic acid uptake continues, resulting in the build-up of a large germanium pool (Fig. 1). At the inhibitory [Ge]/[Si] of 0.1, silieic acid uptake is only about 5 ~ of that in the control culture. Nevertheless, this appears sufficient to permit some silicification and substantial germanium deposition (Fig. 1). The inhibition of cell growth, normal division and normal cell wall formation at [Ge]/[Si] of 0.1 to 1.0 may be due to the high levels of unincorporated germanium within the cell as well as to the reduced level of silicon. Indeed, metabolic processes in diatoms, other than those directly associated with wall formation, have been shown to be dependent on or stimulated by silicic acid and inhibited by germanic acid (Werner, 1967 ; Darley and Voleani, 1969; Itemmingsen, 1971). The polymerization and deposition of silica are the last steps in the metabolism of silicon in diatoms. The participation of germanium at this point in a number of diatoms (Table 1) thus suggests that it is metabo-

Germanium Incorporation in Diatoms

19

lized as a n a n a l o g u e of silicon. S t u d i e s of silicic acid a n d g e r m a n i c a c i d t r a n s p o r t b y N. alba ( u n p u b l i s h e d d a t a ) s u p p o r t this view. This a p p a r e n t s i m i l a r i t y of g e r m a n i c a c i d a n d silicic acid m e t a b o l i s m m a y p r o v e to be a v a l u a b l e tool in t h e s t u d y of silicon m e t a b o l i s m n o t o n l y in d i a t o m s , b u t also in o t h e r organisms which r e q u i r e silicon, i.e., Equisetum arvense (Chen a n d Lewin, 1969), r a t s (Carlisle, 1970; Schwarz a n d Milne, 1972) a n d chicks (Carlisle, 1972). The finding t h a t d i a t o m s are able to i n c o r p o r a t e a n d t h e r e b y insolubilize g e r m a n i u m m a y h a v e geochemical implications. The c o n c e n t r a t i o n of g e r m a n i c acid in t h e ocean is r e p o r t e d to be a t a c o n s t a n t level of 0.06 p p b d e s p i t e t h e i n p u t o f r e l a t i v e l y g e r m a n i u m - r i c h r i v e r w a t e r s (Goldschmidt, 1958). Since p r e l i m i n a r y e x p e r i m e n t s h a v e shown t h a t several d i a t o m s (N. pelliculosa, C. /usi/ormis, C. nana a n d Nitzschia angularis) a r e a b l e to t a k e u p a n d i n c o r p o r a t e ~SGe(OH)4 e v e n when t h e concent r a t i o n in t h e g r o w t h m e d i u m is as lou T as 0.03 p p b ( u n p u b l i s h e d d a t a ) , i t is possible t h a t these algae m a y p l a y a h i t h e r t o unrecognized role in t h e geochemical cycle o f g e r m a n i u m .

Acknowledgements. We thank Miss M. L. Chiappino for making the electron micrographs. This work was supported by grants GM-1065 and GM-08224-10-11 from the National Institutes of Health. References Carlisle, E. M. : Silicon : a possible factor in bone calcification. Science 167, 279--280 (1970). Carlisle, E. M.: Silicon: an essential element for the chick. Science 178, 619--621 (1972). Chen, C. H., Lewin, J. : Silicon as a nutrient element for Equisetum arvense. Canad. J. Bot. 47, 125--131 (1969). Coombs, J., Halicki, P. J., Holm-Hansen, 0., Volcani, B. E.: Studies on the biochemistry and fine structure of silica shell formation in diatoms. Changes in concentration of nucleoside triphosphates during synchronized division of Cylindrotheca/usi/ormis Reimann and Lewin. Exp. Cell Res. 47, 302--314 (1967a). Coombs, J., galicki, P. J., Holm-ttansen, O., Volcani, B. E.: Studies on the biochemistry and fine structure of silica shell formation in diatoms. II. Changes in concentration of nucleoside triphosphates in silicon-starvation synchrony of Naviculapelliculosa (Brdb.) Hilse. Exp. Cell Res. 47, 315--328 (1967b). Darley, W. M. : Silicon requirement for growth and macromolecular synthesis in synchronized cultures of the diatoms, Navicula pelliculosa (Br~bisson) tIilse and Cylindrotheca/usi/or,mis geimann and Lewin. Ph.D. thesis, Univ. Calif. 1969. Darley, W. M., Volcani, B. E.: Role of silicon in diatom metabolism. A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca /usi/ormis Reimann and Lewin. Exp. Cell Res. 58, 334--343 (1969). Darley, W. M., Volcani, ]3. E.: Synchronized cultl~res: Diatoms. In: Methods in enzymology, Vol. X X I I I A, pp. 85--96, A. San Pietro, Ed. New York-London: Academic Press 1971. Goldschmidt, V. M. : Geochemistry. London: Oxford Univ. Press 1958. 2*

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F. Azam et al. : Germanium Incorporation in Diatoms

Hemmingsen, B. B. : A mono-silicic acid stimulated adenosinetriphosphatase from protoplasts of the apochlorotie diatom Nitzschia alba. Ph.D. thesis, Univ. Calif. 1971. Jolly, W. L. : The chemistry of the non-metals, 1st edition. Englewood Cliffs, New Jersey: Prentice-Hall, Inc. 1966. Lewin, J. C.: Silicon metabolism in diatoms. II. Sources of silicon for growth of Navicula pelliculosa. Plant Physiol. 80, 129--134 (1955). Lewin, J. C.: Silicon metabolism in diatoms. V. Germanium dioxide, a specific inhibitor of diatom growth. Phyeologia 6, 1--12 (1966). Matsudo, H. : On the ultrastrueture and morphogenesis of a marine chonotrichous ciliate protozoan Lobochona prorates. Ph.D. thesis, Univ. Southern Calif. 1967. Meynell, G. G., Meynell, E. : Theory and practice in experimental bacteriology, 2nd edition. London: Cambridge Univ. Press 1970. Reimann, B. E. F., Lewin, J. C., Volcani, B. E. : Studies on the biochemistry and fine structure of silica shell formation in diatoms. I. The structure of the cell wall of Cylindrotheca/usi/ormis Reimann and Lewin. J. Cell Biol. 24, 39--55 (1965). Reynolds, E. S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963). Richter, O.: Zur Physiologic der Diatomeen (I. Mitteiinng). S.-B. Akad. Wiss. Wien, math.-nat. K1. 115, 27--119 (1906). Sehwarz, K., Milne, D. B.: Growth-promoting effects of silicon in rats. Nature (Lond.) 289, 333--334 (1972). Sorrentino, F. A., Paul, J. : Simultaneous determination of arsenic, germanium, phosphorus, and silicon. Microchem. J. 15, 446--451 (1970). Stoermer, E.F., Pankratz, H.S., Bowen, C.C.: Fine structure of the diatom Amphipleura pellucida. II. Cytoplasmic fine structure and frustule formation. Amer. J. Bot. 52, 1067--1078 (1965). Sullivan, C. W., Volcani, B. E.: Role of silicon in diatom metabolism. III. The effects of silicon on DNA polymerase, T ~ P kinase and DNA synthesis in Cylindrotheca/usi/ormis. Biochim. biophys. Acta (in press). Tfima, J. : Optimum conditions for the colorimetric microdetermination of silicon. Mikrochim. Acta 8, 513--523 (1962). Werner, D.: Die Kiesels~ure im Stoffwechsel yon Cyclotella cryptica Reimann, Lewin und Guillard. Arch. Mikrobiol. 55, 278--308 (1966). Werner, D.: Hemmung der Chlorophyllsynthese und der NADP+-abh~ngigen Glycerinaldehyd-3-phosphat-dehydrogenase durch Germaniums/~ure bei Cyclotella cryptica. Arch. Mikrobiol. 57, 51--60 (1967). Dr. Farooq Azam Institute of Marine Resources Scripps Institution of Oceanography University of California La Jolla, California 92037, U.S.A.