SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. II. STRONG pH DEPENDENCE OF UPTAKE KINETIC PARAMETERS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Silicon uptake kinetics of the diatom Phaeodactylum tricornutum (Bohlin) were examined at pH 8.8 ± 0.1 and pH 9.1 ± 0.1. Uptake follows hyperbolic saturation kinetics at both pH's, but at the higher pH the half-saturation constant for uptake is 11.8 μM, as opposed to 54.8 μM at the lower pH. When the uptake rate is examined as a function of the calculated concentration of the monovalent conjugate base, SiO(OH)₃^?, the half-saturation constant for uptake is 6.6 μM at either pH.     

LIGHT DEPENDENCE OF GROWTH AND PHOTOSYNTHESIS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Phaeodactylum tricornutum Bohlin was maintained in exponential growth over a range of photon flux densities (PFD) from 7 to 230 μmol·m^?2s^?1. The chlorophyll a-specific light absorption coefficient, maximum quantum yield of photosynthesis, and C:N atom ratio were all independent of the PFD to which cells were acclimated. Carbon- and cell-specific, light-satuated, gross photosynthesis rates and dark respiration rates were largely independent of acclimation PFD. Decreases in the chlorophyll a-specific, gross photosynthesis rate and the carbon: chlorophyll ratio and increases of cell- or carbon-specific absorption coefficients were associated with an increase in cell chlorophyll a in cultures acclimated to low PFDs. The compensation PFD for growth was calculated to be 0.5 μmol·m^?2s^?1. The maintenance metabolic rate (2 × 10^?7s^?1), calculated on the basis of the compensation PFD, is an order of magnitude lower than the measured dark respiration rate(2.7 × 10^?6mol O₂·mol C^?1s^?1). Maintenance of high carbon-specific, light-saturated photosynthesis rates in cells acclimated to low PFDs may allow effective use of short exposures to high PFDs in a temporally variable light environment.     

FIRST INSIGHTS INTO IMPROVEMENT OF EICOSAPENTAENOIC ACID CONTENT IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) BY INDUCED MUTAGENESIS1

A strain improvement program was initiated based on mutagenesis with the goal of commercial production of eicosapentaenoic acid (EPA)from EPA-overproducing microalgal strains. Two rounds of mutation and selection were conducted using Phaeodactylum tricornutum Bohlin UTEX #640 as the parent strain. After the first round of mutagenesis, a putative mutant (provisionally labeled 114) was obtained. The EPA content (% of dry weight) of this mutant strain was 37% higher than that of the wild type. 114 was further mutated and another putative mutant (provisionally called II242) was isolated, the EPA content of which was 44% higher than that of the wild type. When cultured with aeration in 1-L flasks, EPA content of the wild type and putative mutants 114 and II242 was, 17.3 mg · g^?1, 31.5mg · g^?1, and 38.6 mg · g^?1 dry biomass, respectively. EPA productivity was 3.48 mg · L^?1· d^?1 4.01 mg · L^?1· d^?1, and 4.98 mg · L^?1· d^?1 respectively. These figures compare favorably with many other promising EPA-producing microorganisms and suggest that the use of a single methodology such as mutation and selection is a way to improve the polyunsaturated fatty acid content of microalgae and other microorganisms.     

QUANTITATIVE GENETICS OF FATTY ACID VARIATION IN ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Fatty acid variation among culture collection strains and 40 new isolates of Isochrysis galbana Parke was analyzed by quantitative genetic methods. Fatty acid variation among strains and among isolates was highly significant for major fatty acids showing the existence of a genetic component in the determination of differences in fatty acid content. The heritabilities for the major fatty acids ranged between 0.68 and 0.99 among collection strains and between 0.31 and 0.43 among isolates. Eicosapentaenoic acid (EPA) had the highest heritability in I. galbana, but the majority of remaining fatty acids also showed high heritability values. A similar experiment with five UTEX strains of Phaeodactylum tricornutum also showed the presence of a genetic component in four out of seven major fatty acids. Nevertheless, the UTEX strains did not differ significantly in EPA content, although they showed a heritability of 0.40 for this fatty acid. An additional experiment culturing the same isolates of I. galbana in larger volumes of media showed that there was a high significant positive linear relation between EPA content in different volumes. Therefore, EPA content in small volume cultures was an unbiased indicator of EPA content in larger volume cultures. Our results provide support for the genetic determination of fatty acid content in microalgae and suggest that selection, and mutation and selection, are likely to improve EPA content in I. galbana and probably in many other microalgae. Such a selection program can be carried out in small-volume cultures with high confidence.     

PROTEINACEOUS AND IMMUNOCHEMICAL DISTINCTIONS BETWEEN THE OVAL AND FUSIFORM MORPHOTYPES OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Two morphotypes, fusiform and oval, were isolated from a single clone of the diatom Phaeodactylum tricornutum Bohlin and maintained as subclones by culturing in liquid and solid substrates, respectively. Salinity of the medium, from brackish to marine, had no effect on expression of the phenotypes. The oval cell is generated endogenously within a “transformed”fusiform cell upon transfer from liquid medium to agar plates. With the light microscope, normal and “transformed”fusiform cells, prior to giving rise to oval cells, can be discriminated by means of their staining response to toluidine blue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts from lysed cells revealed slight differences in polypeptide composition between fusiform and oval types. A phenotype-restoration experiment from oval to fusiform demonstrated that the oval type readily reestablished not only fusiform morphology but also the protein pattern characteristic for the fusiform type. Immunochemical analyses (western blots) using antisera raised against whole and lysed cells of both morphotypes revealed antigenic alterations of the oval morphotype. Several antigenic determinants restricted mainly to the surface of oval cells were detected. Results indicate that environmentally induced phenotypes of Phaeodactylum may be not only the consequence of specific gene expression but also the result of significant, general post-translational modifications.     

EFFECTS OF IRON DEFICIENCY ON ISOCHRYSIS GALBANA (CHRYSOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of Isochrysis galbana Parks and Phaeodactylum tricornutum Bohlin were grown in iron-limited chemostats. With increasing iron deficiency, photosynthetic rate per cell and assimilation number decreased. The pattern of photosynthesis was also altered; in Fe deficient cells the proportion of ¹⁴C fixed in glycine and serine decreased with an accompanying increase into alanine after 3 min assimilation. Although there was no significant effect of Fe deficiency on the proportion of ¹⁴C incorporated into total amino acids and amides, the percentage of total ¹⁴C fixed in protein increased with increasing Fe deficiency. Cellular levels of chlorophyll a, carotenoids, cytochromes and protein also decreased with increasing Fe deficiency. However, the reduction in chlorophyll a/cell was not as great as that of cytochrorne f₁ and Fe deficient cells therefore showed a marked increase in chlorophyll a:cytochrorne f₁ ratio.     

CHRONIC EFFECTS OF ULTRAVIOLET-B RADIATION ON GROWTH AND CELL VOLUME OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cultures of the temperate estuarine diatom, Phaeodactylum tricornutum Bohlin (NEPCC Clone 31), were grown under ambient intensities of ultraviolet-A radiation (UVAR), photosynthetically active radiation (PAR), and various intensities of ultraviolet-B radiation (UVBR; 290–320 nm). Growth rates and cell volumes were monitored for 36 d. UVBR decreased growth rates and increased cell volumes. Sensitivity of growth to UVBR increased with time. Growth rates of P. tricornutum decreased with increasing ratios of UVBR:UVAR + PAR.     

EFFECTS OF LIGHT INTENSITY AND TEMPERATURE INTERACTIONS ON GROWTH CHARACTERISTICS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Cell division rate, carbon fixation per cell, cell width and chloroplast length of Phaeodactylum tricornutum Bohlin were determined at 30 different combinations of light intensity and temperature. Division rate peaked at 23° C or less depending on light intensity. For each light intensity studied, carbon fixation increased directly with growth temperature from 14 to 25° C. The slope of this relationship was modified by light intensity. Cells grown at 23–25° C tended to be larger than those grown at lower temperatures, possibly due to increased carbon fixation per cell coupled with lower division rates. Chloroplasts were largest at a combination of temperatures above 21° C and low light intensities. This effect could cause cells to sink at a higher than normal rate due to reduced vacuole size and is presented as a possible mechanism affecting the distribution of P. tricornutum.     

VARIATION IN THE OCCURRENCE OF EXTERNAL CARBONIC ANHYDRASE AMONG STRAINS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Eleven different strains of Phaeodactylum tricornutum Bohlin were obtained from three culture collections and were examined for the presence of external and internal carbonic anhydrase (CA). Cells of all strains, grown in standing culture at alkaline pH and low, dissolved inorganic carbon had internal CA, but only eight were found to have external CA. External CA activity was reduced when cultures were bubbled with air and was completely repressed when they were grown on 5% CO₂. Expression of external CA activity appears to be regulated by CO₂ concentration in the growth medium, but within one species, there appears to be a variation in occurrence of external CA and consequently in the mode of inorganic carbon acquisition.     

EFFECTS OF ENVIRONMENTAL FACTORS ON PHOTOSYNTHESIS PATTERNS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE). I. EFFECT OF NITROGEN DEFICIENCY AND LIGHT INTENSITY1

Cultures of the marine diatom Phaeodactylum tricornutum Bohlin incorporated, a large proportion of the total fixed carbon (50% or more) into amino acids and amides during short periods of photo-assimilation of ¹⁴C-labelled carbon dioxide. Although increasing nitrogen limitation in a nitrate-limited chemostat had little significant effect on the proportion of C incorporated into amino acids and amides combined, it did affect the distribution of radioactivity within individual compounds of this group. In particular, increasing degrees of N deficiency reduced the proportion incorporated into amides to almost undetectable levels, reduced the proportion in alanine and increased the proportion in glutamic acid. Also, increasing N limitation decreased the relative synthesis of sugar phosphates and increased the proportion of C assimilated into intermediates of the tricarboxylic acid cycle. Reduced light intensity did not have any significant effect on the proportion of C incorporated into the total amino acids and amides, but did cause a decrease in the radioactivity     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

REVERSIBLE KINETIC MODEL FOR THE SHORT-TERM REGULATION OF METHYLAMMONIUM UPTAKE IN TWO PHYTOPLANKTON SPECIES,DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Methylammonium, an ammonium analog, was used to study the short-term kinetics of ammonium uptake in a diatom, Phaeodactylum tricornutum Bohlin, and a green alga, Dunaliella tertiolecta Butcher. Time courses of methylammonium disappearance were measured over a wide range of initial substrate concentrations for the two species. It was shown that feedback inhibition, described mathematically by a reversible enzyme kinetic model, can be used to explain the data. For the two species, there was good agreement between the kinetic parameters obtained from the analysis of the uptake versus substrate curve and those from the fit of the reversible kinetic model to the time-course data. All time courses of CH₃NH₃⁺ disappearance could be described by constants V_m and k_s. Ammonium time-course data show some similarities to its analog, methylammonium. Our study suggests that the apparent change in V_m and k_s with time measured after the addition of saturating ammonium concentrations reflects an uncoupling between transport and assimilation of the substrate rather than a real change in the kinetic parameters of the transport mechanism.     

EFFECT OF NITROGEN SOURCE ON PLATYMONAS (CHLOROPHYTA) CELL COMPOSITION AND AMINO ACID UPTAKE RATES1

The effect of nitrogen source (nitrate, ammonia and/or amino acids) on cell composition and amino acid uptake rates was examined. Substantial levels of free amino acids accumulated intracellularly with all nitrogen sources used. Ammonia accumulated only when provided in the medium. The presence of ammonia in the medium decreased the intracellular accumulation of free amino acids, especially arginine. Amino acid uptake rates were suppressed by the presence of excess nitrogen, especially ammonia. However, the suppression of uptake did not show any particular relation to the nitrogenous cell composition.     

EXTRACELLULAR MATRIX ASSEMBLY IN DIATOMS (BACILLARIOPHYCEAE). V. ENVIRONMENTAL EFFECTS ON POLYSACCHARIDE SYNTHESIS IN THE MODEL DIATOM,PHAEODACTYLUM TRICORNUTUM1

The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media‐soluble polymers (CL and cEPS) were enriched in 4‐linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3‐linked xylosyl residues, whereas HW polymers consisted mainly of 3‐linked glucosyl as well as terminal and 2,4‐linked glucuronosyl residues. The HB fraction was enriched in terminal and 2‐linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3‐ and 3‐linked mannosyl residues. The fusiform morphotype predominated in standard and P‐limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O‐methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5‐ to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O‐methylated sugars, uronic acids, and sulfate. The increased deoxy‐ and O‐methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross‐linking, suggesting that these changes could ameliorate the effects of salinity or P‐stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.     

EFFECTS OF LOWERING TEMPERATURE DURING CULTURE ON THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The composition of fatty acids and contents of eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) of the economically important marine diatom, Phaeodactylum tricornutum (Bohlin), were investigated to see whether reducing the culture temperature enhances the production of EPA and PUFAs. The contents of EPA and PUFAs of P. tricornutum were found to be higher at lower temperature when cultured at 10, 15, 20, or 25°C. When the cells grown at 25°C were shifted to 20, 15, or 10°C, the contents per dry mass of PUFAs and EPA increased to the maximal values in 48, 24, and 12 h, respectively. The highest yields of PUFAs and EPA per unit dry mass (per unit volume of culture) were 4.9% and 2.6% (12.4 and 6.6 mg·L^?1), respectively, when temperature was shifted from 25 to 10°C for 12 h, both being raised by 120% compared with the control. The representative fatty acids in the total fatty acids, when temperature was lowered from 25 to 10°C, decreased proportionally by about 30% in C_16:0 and 20% in C_16:1(n?7) but increased about 85% in EPA. It was concluded that lowering culture temperature of P. tricornutum could significantly raise the yields of EPA and PUFAs.     

INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH-CELL-DENSITY DIALYSIS CULTURE1

The consumption of inorganic macronutrients (NO₃^?+ NO₂^?, NH₄⁺, and PO₄^?3) and the composition of intra- and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous-reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 10⁷ cells · mL^?1 was obtained in stationary phase, which coincided with the NO₃^?+ NO₂^? diffusion limit (Nd_max) of the dialysis apparatus. The Nd_max for NH₄⁺ occurred much earlier, at the end of exponential growth, whereas Nd_max for PO₄^?3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β-GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N-conversion coefficient (Y_N). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.     

EVIDENCE FOR A COMMON EVOLUTIONARY ORIGIN OF LIGHT-HARVESTING FUCOXANTHIN CHLOROPHYLL a/c-PROTEIN COMPLEXES OF PAVLOVA GYRANS (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

A fucoxanthin-chlorophyll a/c-protein complex has been isolated from the prymnesiophyte Pavlova gyrans. Thylakoid membranes were treated with the mild anionic detergent sodium taurodeoxycholate followed by sucrose density gradient centrifugation. The brown fraction produced by this procedure was treated with Triton X-100 followed by a second sucrose density gradient centrifugation. A brown fraction isolated from this gradient was shown to be a light-harvesting complex nearly identical to that which is present in the diatom Phaeodactylum tricornutum. The complexes from the two organisms have nearly identical absorption and flourescence spectra, both complexes contain fucoxanthin and two other carotenoids, both contain four polypeptides of similar molecular weights, and polypeptides from both complexes cross react with antibodies raised to polypeptides of the Phaeodactylum tricornutum complex. Results suggest a common evolutionary origin for these light-harvesting complexes, in apparent contrast to the great differences in cell structure between prymnesiophytes and diatoms.     

UPTAKE OF NITRATE,AMMONIUM, AND UREA BY NITROGEN-STARVED CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE): TRANSIENT RESPONSES1

Nitrogen uptake rates were measured as a function of time following saturating additions (15 μMg-at N·^?1) of ¹⁵N-labelid ammonium, urea, and nitrate to N-starved cultures of the picoflagellate Micromonas pusilla Butcher. Uptake rates were estimated from both the accumulation of ¹⁵N into the cells and the disappearance of nitrogen from the medium. Transient elevated (surge) uptake rates of NH₄⁺ and urea were observed after enrichment. During the first 5 min the initial urea and NH₄⁺ uptake rates were 2- and 4-fold greater than the maximum growth rate (μM_max)observed prior to No₃^? depletion in the cultures. The elevated urea uptake rates declined quickly to a relatively constant value, whereas the initial rates of NH₄⁺ uptake declined rapidly but were followed by a subsequent increase prior to remaining roughly constant. Nitrate was not taken up as readily by N-starved M. pusilla as the reduced N forms. Although NO₃⁺ uptake commenced immediately after enrichment (i.e. no lag period) the N-Specific rate over the next 6 h averaged half the μM_max observed during NO₃^? replete conditions.     

EFFECTS OF Si:P CONCENTRATION RATIOS AND NUTRIENT LIMITATION ON THE CELLULAR COMPOSITION AND MORPHOLOGY OF ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The freshwater diatom Asterionella formosa Haas. was grown in semicontinuous culture at 20°C under continuous cool-white fluorescent light of ca. 20 μEin · m^?2· s ·^?1 in a medium containing Si: P in various concentration ratios. The cell quotas of P and Si changed in relation to the available concentrations of P and Si at constant μ= 0.11 and 0.16 d^?1. Under Si-limitation, the P cell quota increased by over an order of magnitude as the influent [Si:P] decreased. The Si cell quota increased with increase in [Si] in the influent medium, and it increased as [P] increased at a specific [Si]. Under P-limitation, the P cell quotas were fairly constant and low; the Si cell quotas were relatively high and decreased slightly as influent [P] and [Si] increased. Asterionella stored up to 28 times more P and 2 times more Si than needed. The number of Asterionella cells per colony varied as a function of the influent [Si:P] and nutrient limitation being usually less than or equal to 6 when P-limited, and greater than 10 when Si-limited.     

13C AND 15N UPTAKE BY MARINE PHYTOPLANKTON. I. INFLUENCE OF NITROGEN SOURCE AND CONCENTRATION IN LABORATORY CULTURES OF DIATOMS1

Two species of marine diatoms, Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin were grown in batch and continuous cultures on four different nitrogen compounds (nitrate, nitrite, ammonium, urea). Carbon and nitrogen uptake were measured simultaneously with the stable isotopes ¹³C and ¹⁵N. Nitrogen uptake generally increased with N concentration in the medium, but no clear difference existed between the N sources. Carbon fixation was decreased for up to 5 h following the addition of the N compound. Nitrite generally had the greatest inhibitory effect on C uptake. Carbon-to-nitrogen uptake ratios decreased with increasing dissolved N concentration, becoming lower than one in nutrient-limited cultures. In contrast, batch cultures exhibited C:N uptake ratios greater than one. These effects are essentially short-term and differ from long-term influences of the N source on the cellular chemical composition.