EFFECTS OF COPPER TOXICITY ON SILICIC ACID UPTAKE AND GROWTH IN THALASSIOSIRA PSEUDONANA11

The toxicity of Cu to Thalassiosira pseudonana (Hustedt) Hasle and Heimdal was investigated by examining both short and long term effects of Cu on cellular processes. Toxic levels of Cu (pCu* < 13) were found to inhibit short term Si(OH)₄ uptake rates with kinetics characteristic of irreversible inhibition at a hypothetical Cu-sensitive Si(OH)₄ transport site. Residual Si(OH)₄ concentrations (those below which no uptake could occur) were found to increase with increasing levels of Cu, and the toxic effects of Cu could be reversed by increasing the concentrations of Si(OH)₄ in the medium. The actual uptake of Cu by the cells was found to vary inversely with the ambient Si(OH)₄ concentration. Copper did not inhibit the uptake of NO₃^? or PO₄^3-. The long term inhibition of growth rate by Cu in this species was shown not to be a result of Si deficiency caused by the inhibition of Si(OH)₄ uptake. Cu inhibited cells were found to have higher Si cell quotas (including a sizable soluble pool) than the control cultures. They were, however, observed to have aberrant frustules, significantly larger than the control cells, suggesting interference with the silification process as a possible mechanism for inhibition of growth by Cu. A conceptual model is proposed for the Cu-Si(OH)₄-growth relationship. It includes a Cu sensitive Si(OH)₄ transport site that may also serve to transport Cu into the cell, and growth inhibition mediated by intracellular Cu concentrations which may block cell division or cause general cellular disfunction.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited, at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a Junction of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

DIATOM MINERALIZATION OF SILICIC ACID. VIII. METABOLIC REQUIREMENTS AND THE TIMING OF PROTEIN SYNTHESIS1

We have examined the influence of temperature, protein synthesis, and energy metabolism on the process of silicon biomineralization in synchronized cultures of the diatom Navicula saprophilia Lange-Bertalot & Bonik (1976). Temperature effects on silicon polymerization were compared in vitro and in vivo. In vivo incorporation was very temperature dependent with a Q₁₀ of 7.53. In contrast, the Q₁₀ for in vitro polymerization was 1.42, indicating much lower temperature dependence. This difference in Q10 values suggests that in vivo polymerization involves more than autopolycondensation. Cycloheximide addition to synchronized cultures up to, but not later than one hour after the addition of silicic acid depressed total uptake, incorporation, but not pool size. Developing valves demonstrated morphological abnormalities with cycloheximide additions from 0 to 2 h following silicic acid addition. These data suggest that de nova proteins are required in biomineralization and that they are synthesized during or just after cytokinesis. Biomineralization is not coupled to energy derived directly from photosystem II or photosynthesis, since neither darkness nor DCMU had an effect on any aspect of silicification.     

KINETICS OF SILICIC ACID UPTAKE AND RATES OF SILICA DISSOLUTION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA1,2

Tracer techniques using the stable isotope ³⁰Si were used to measure rates of silicic acid uptake and silica dissolution in silicon replete and silicon depleted populations of 2 clones of the marine diatom Thalassiosira pseudonana Hasle & Heimdal. Uptake kinetics were describable using the Michaelis-Menten equation for enzyme kinetics, and no threshold concentration for uptake was evident. The maximum specific uptake rate of the estuarine clone 3H (0.062–0.092 · h^?1) was higher than that of the Sargasso Sea clone 13-1 (0.028–0.031 · h^?1), but half-saturation constants for uptake by the 2 clones were not measurably different (0.8–2.3 μM for 3H; 1.4–1.5 μM for 13-1). There was little or no light dependence of uptake in populations grown under optimal light conditions prior to the experiment. Exponentially growing populations released silicic acid to the medium by dissolution of cellular silica at rates ranging from 6.5 to 15% of the maximum uptake rate.     

DIATOM MINERALIZATION OF SILICIC ACID IV. KINETICS OF SOLUBLE Si POOL FORMATION IN EXPONENTIALLY GROWING AND SYNCHRONIZED NAVICULA PELLICULOSA1

Silicic acid taken up from the growth medium by Navicula pelliculosa (Bréb.) Hilse was shown to enter at least two compartments: i) soluble pools; ii) insoluble fraction comprised predominantly of the silica frustule. Soluble Si pools were extracted by a variety of agents from cells uniformly labeled for ten generations in medium containing ⁶⁸Ge-Si(OH)₄. 100 C water soluble and 0 C perchloric acid (PCA) soluble Si pools of 680 mM Si·l^?1 and 490 mM Si·l^?1 cell water represented 13 and 9%, respectively, of total, cell Si in exponential growth phase cells. Uniformly labeled cells synchronized by the combined synchronization technique accumulate at the cell cycle stage where silica frustule development is initiated. These cells contain water and PCA soluble pools of 10 nmol Si·10⁶ cells^?1 and-8.8 nmol Si·10⁶ cells^?1, respectively. On addition of Si(OH)₄, a rapid uptake ensues allowing the Si pool to expand 2.5-fold, apparently to provide precursors of the silica frustule.     

DIATOM MINERALIZATION OF SILICIC ACID. VI. THE EFFECTS OF MICROTUBULE INHIBITORS ON SILICIC ACID METABOLISM IN NAVICULA SAPROPHILA1

The role of microtubules in silicon metabolism leading to valve formation was investigated in the pennate diatom Navicula saprophila Lange-Bertalot & Bonik. By using synchronized cells blocked after mitosis and cytokinesis but prior to cell wall formation, effects due to inhibition of mitosis were eliminated. Cells were treated with three anti-microtubule drugs to assess the role of microtubules. Chemical analogs to two of the drugs provided controls for inhibition not related to microtubule disruption. Although all three anti-microtubule drugs reduced cell separation at high concentrations (1 × 10^?3 M), podophyllotoxin was the only drug which reduced cell separation at concentrations lower than 1 × 10^?5 M. None of the drugs at any concentration tested affected cell viability. There was no differential inhibitory effect between the active and inactive drugs on silicic acid transport, total uptake, incorporation, or pool formation. There was no qualitative difference between silica incorporated in treated and untreated cells. A colchicine binding component was isolated from N. saprophila. The characteristics of colchicine binding suggest this component may be tubulin. Microtubules do not appear to be involved in any of the steps of silicon metabolism leading to valve formation and yet they have profound influence on the symmetry and pattern of the mineralized product, the siliceous valve.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C. protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a function of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

SILICON METABOLISM IN DIATOMS. VI. SILICIC ACID UPTAKE BY A COLORLESS MARINE DIATOM,NITZSCHIA ALBA LEWIN AND LEWIN12

Cells of the colorless, heterotrophic diatom Nitzschia alba, after removal from the culture medium, were able to absorb silicic acid from a salt solution lacking carbon, nitrogen, and phosphorus. Silicic acid uptake continued for approximately 12 hr. At low cell densities (ca. 2.5×10⁵ cells/ml), the cell number doubled under these conditions. At high cell densities (ca. 2 ×10⁶ cells/ml) no cell division resulted. When such cells were washed with a salt solution, their ability to absorb silicic acid was somewhat impaired. The degree of impairment became progressively more pronounced after subsequent washing treatments. A heat-stable factor washed from the cells and present in the first “wash water” was able to restore completely the ability of washed cells to absorb silicic acid. The factor was not identified. Aspartic acid (5 ± 10^-4 M) or glutamine (5 ±10^-4 M) when added to the saline solution similarly promoted complete recovery. A t such concentrations, these substances had only a slight effect on unwashed cells. A solution of 5 ±10^-4 M Na glutamate (or aspartic acid plus glutamine) had an men more pronounced effect, and in addition promoted cell division or growth" of unwashed cells. Several other amino acids and other compounds tested were apparently without effect.     

Patterns and regulation of silicon accumulation in Synechococcus spp.

Six clones of the marine cyanobacterium Synechococcus, representing four major clades, were all found to contain significant amounts of silicon in culture. Growth rate was unaffected by silicic acid, Si(OH)₄, concentration between 1 and 120 μM suggesting that Synechococcus lacks an obligate need for silicon (Si). Strains contained two major pools of Si: an aqueous soluble and an aqueous insoluble pool. Soluble pool sizes correspond to estimated intracellular dissolved Si concentrations of 2–24 mM, which would be thermodynamically unstable implying the binding of intracellular soluble Si to organic ligands. The Si content of all clones was inversely related to growth rate and increased with higher [Si(OH)₄] in the growth medium. Accumulation rates showed a unique bilinear response to increasing [Si(OH)₄] from 1 to 500 μM with the rate of Si acquisition increasing abruptly between 80 and 100 μM Si(OH)₄. Although these linear responses imply some form of diffusion‐mediated transport, Si uptake rates at low Si (~1 μM Si) were inhibited by orthophosphate, suggesting a role of phosphate transporters in Si acquisition. Theoretical calculations imply that observed Si acquisition rates are too rapid to be supported by lipid‐solubility diffusion of Si through the plasmalemma; however, facilitated diffusion involving membrane protein channels may suffice. The data are used to construct a working model of the mechanisms governing the Si content and rate of Si acquisition in Synechococcus.     

INFLUENCE OF FERULIC ACID ON MINERAL DEPLETION AND UPTAKE OF 86Rb BY PAUL'S SCARLET ROSE CELL-SUSPENSION CULTURES

The influence of 10^-4 m ferulic acid on mineral depletion and ion uptake in sterile cultures of Paul's Scarlet rose was examined. The effect of ferulic acid on the rate of depletion of Mg²⁺, Ca²⁺, K⁺, P, Fe³⁺, Mn²⁺, and Mo³⁺ from the medium during the 14-day growth cycle varied with the age of the cells and the ion under consideration. In general, rates of uptake were higher than control rates in older cells and less than control rates in cells 3–5 days old. The degree of inhibition of uptake of ⁸⁶Rb also varied with age. Young (4–5 day) cells showed approximately 50% inhibition at high concentrations of RbCl (system 2) and approximately 25% inhibition at low concentrations of RbCl (system 1). In contrast, the rate of ⁸⁶Rb uptake in 10-day cells was not significantly altered by incubation in ferulic acid.     

A stable isotope trace method to measure silicic acid uptake by marine phytoplankton.

A tracer method is described that uses the stable isotope ³⁰Si to measure rates of silicic acid uptake by diatom cultures and natural populations of marine phytoplankton. The method involves (i) incubation of organisms requiring silicic acid for growth in the presence of ³⁰Si-labeled silicic acid, (ii) collection of the resulting particulate silicon, (iii) conversion of the particulate silicon to BaSiF₆, (iv) determination of the ³⁰Si content of BaSiF₆ by solid sample mass spectrometry, and (v) calculation of the uptake rate from the ³⁰Si enrichment of the particulate matter during the incubation. The maximum overall error in the uptake rate measurement is ±10%.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. II. THE 14CO2-LABELING KINETICS OF CAROTENOIDS1

The biosynthesis and turnover of the pigments fucoxanthin, diadinoxanthin (DD), and diatoxanthin (DT) were studied in exponentially growing cultures of the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle to investigate the dependence of pigment turnover on algal growth rates and light intensity. ¹⁴C-bicarbonate was used as a tracer. The labeling kinetics of fucoxanthin and DT were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor and pigment turnover rate. At growth irradiances < 200 μE · m^?2· s^?1, labeling kinetics of DD indicated the presence of two kinetically distinct DD pools and at least one precursor pool. The average growth rate-normalized pigment turnover rate of fucoxanthin was 0. The growth rate-normalized turnover rate of DT, determined only at high light irradiances (> 200 μE·m^?2·s^?1), was 1.3. At high light irradiances, the growth rate-normalized turnover rate of DD was 1.8. At low light irradiances, the turnover rates of the two DD pools were 3.7 and 0, respectively. The corresponding pigment turnover times were on the order of days to weeks, depending on the growth rate of the cultures. A comparison of pigment pool sizes, pigment turnover rates, and precursor turnover rates suggests that fucoxanthin is synthesized from a pool of DD and that DD and DT are synthesized from a common precursor, possibly β-carotene. No evidence was seen for dynamic xanthophyll cycling. This suggests that the commonly known “xanthophyll cycle” is the simple unidirectional conversion of DD into DT, or of DT into DD, in response to rapid irradiance changes.     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

NITROGEN UTILIZATION AND METABOLISM RELATIVE TO PATTERNS OF N2 FIXATION IN CULTURES OF TRICHODESMIUM NIBB1067

We measured uptake kinetics for four combined N sources, ambient rates of N uptake and N₂ fixation, glutamine synthetase activity (transferase and biosynthetic), and concentrations of intracellular pools of glutamate (glu) and glutamine (gln) in cultures of Trichodesmium NIBB1067. N dynamics and metabolism were examined to assess the relative importance of N₂ fixation and N uptake to Trichodesmium nutrition. Comparisons were made between cultures grown on medium without added N, with excess NO, or with excess urea. Of the combined N sources tested, Trichodesmium NIBB1067 had the highest affinity for NH; high uptake capacities for NH, urea, and glu; and little capacity for NO uptake. In cultures grown on medium without added N, NH accumulated in the medium during growth, resulting in high NH uptake rates relative to rates of N₂ fixation. Glu uptake rates were low but consistent throughout the diel period. In cultures grown on excess NO or urea, uptake of these compounds supplied the majority of the daily N demand, although some N₂ fixation occurred during the light period. NO uptake rates were reduced when N₂-fixation rates were high. In all of the cultures, the highest gln/glu ratios and the lowest glutamine synthetase transferase/biosynthetic ratios were observed during the period when rates of total N uptake were highest. In cultures growing exponentially on medium without added N, N₂ fixation accounted for 14%– 16% of the total daily N uptake. Uptake of NH and glu, presumably regenerated within the culture vessels, represented 84%–86% of the daily N uptake. Because these systems were closed, net growth was constrained by the rate at which N₂ could be fixed into the system. However, total daily N turnover was greater than that necessary to accommodate the observed increase in culture biomass. The rapid N turnover rates observed in these cultures may support gross productivity and balance the high rates of C fixation observed in natural populations of Trichodesmium.     

Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed.     

CHANGES IN DOMOIC ACID PRODUCTION AND CELLULAR CHEMICAL COMPOSITION OF THE TOXIGENIC DIATOM PSEUDO-NITZSCHIA MULTISERIES UNDER PHOSPHATE LIMITATION1

Production of domoic acid (DA), a neurotoxin, by the diatom Pseudo-nitzschia multiseries (previously Nitzschia pungens f. multiseries) Hasle and its cellular chemical composition were studied in phosphate-limited chemostat continuous cultures and in subsequent batch cultures. Under steady-state chemostat conditions, DA production increased from 0.01 to 0.26 pg DA · cell^?1· d^?1 as the growth rate decreased. When the nutrient supply was discontinued (to produce a batch culture), DA production was enhanced by a factor of ca. 3. DA production was temporarily suspended upon addition of phosphate to the batch cultures but resumed 1 d later at a higher rate coincident with the decline of phosphate uptake. In both steady-state continuous culture and batch culture, more DA was produced when alkaline phosphatase activity (APA) was high. The association of high DA production with high levels of APA and high cellular N:P ratios strongly suggests that phosphate limitation enhances DA production. Also, DA production was high when other primary metabolism (e.g. uptake of carbon, nitrogen, phosphorus and silicon, and cell division) was low, but chlorophyll a and adenosine triphosphate were generally high. This suggests that the synthesis of DA requires a substantial amount of biogenic energy.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

CHANGE OF FATTY ACID COMPOSITION OF CHLORELLA ELLIPSOIDEA DURING ITS CELL CYCLE

The lipids extracted from Chlorella cells at different developmentalstages were separated by chromatography on silicic acid into"nonpolar" (chloroform-eluate) and "polar" (methanol-eluate)lipid fractions. The lipids were also subjected to florisilchromatography to fractionate neutral glycerides and free fattyacids. Gas-liquid chromatographic analysis of these fractions has revealeda marked difference in their fatty acid compositions which werefound to undergo characteristic changes during the course ofalgal cell cycle. It was found that the fatty acids in the "nonpolar"lipid (fat) fraction are synthesized during the growth phasein the light and consumed during the process of cellular division. (Received September 24, 1966; )     

Effects of copper and zinc on growth,morphology, and metabolism of Asterionella japonica (Cleve) 1

When exposed to elevated levels of copper or zinc, the diatom Asterionella japonica (Cleve) showed a reduced cell division rate and a marked increase in cell size. Metal-treated cells had greater cell volumes, dry weights, carbon, nitrogen, chlorophyll, and DNA contents, all in approximately the same proportion as control cells. Two protoplasts often appeared to be contained within one frustule. Metal-treated cells photosynthesized at near-normal rates on a per chlorophyll basis and above normal rates on a per cell basis. Excretion of photosynthetically fixed carbon was depressed by metal treatment; 10–22% of fixed carbon was excreted in control cells and typically less than 1% in treated cells. Thus, metal-treated cells showed an uncoupling of photosynthesis from cell division and continued to enlarge when fixed carbon could not be excreted or utilized in cell division.Uptake of sulphate and silicic acid proceeded at slower rates than other processes (e.g., nitrogen uptake or photosynthesis) in copper-treated cells. Free amino acids in copper-treated cells totalled ≈ 10% of control cell levels, with greatest proportional declines in methionine, cysteine, aspartic acid, valine, and isoleucine. Copper-treated cells resuspended in fresh medium shrank to normal size when exposed to methionine (which they accumulated), although cell division rates did not return to normal. These cells excreted 2–3 times as much fixed carbon as comparable EDTA-treated or untreated cells, neither of which decreased in size. Copper-treated cells appeared indistinguishable from silicon-limited cells (i.e., cells not dividing for lack of silicon) in a copper-free medium. Cells treated with the sulfhydryl binder PCMB divided at reduced rates and also swelled in a manner comparable to copper-treated cells. The results suggest that toxic metals may bind to sulfhydryl groups on cell membranes, impairing normal membrane function and reducing silicic acid uptake and amino-acid synthesis, thereby resulting in depressed cell division rates.