Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.     

Nutrient pulsing as a regulator of phytoplankton abundance and community composition in Galveston Bay, Texas

Galveston Bay, Texas, is a large shallow estuary with a watershed that includes 60% of the major industrial facilities of Texas. However, the system exhibits low to moderate (2-20 μg l⁻¹) microalgal biomass with sporadic phytoplankton blooms. Both nitrogen (N) and phosphate (P) limitation of phytoplankton growth have been proposed for the estuary. However, shifts between N and P limitation of algae growth may occur due to annual fluctuations in nutrient concentrations. The primary goal of this work was to determine the primary limiting nutrient for phytoplankton in Galveston Bay. Nutrient addition bioassays were used to assess short-term (1-2 days) phytoplankton responses (both biomass and community composition) to potentially limiting nutrients. The experimental bioassays were conducted over an annual cycle using natural water collected from the center to lower part of the estuary. Total phytoplankton biomass increased in the nitrate (10 μM) additions in 11 of the 13 bioassays, but no significant increases were detected in the phosphate (3 μM)-only additions. Bioassay results suggest that the phytoplankton community was usually not phosphate limited. All major groups increased in biomass following nitrate additions but diatoms increased in biomass at a faster rate than other groups, shifting the community composition toward higher relative abundance of diatoms. The results of this study suggest that pulsed N input events preferentially favor increases in diatom biomass in this estuary. The broader implications of this study are that N pulsing events, primarily due to river discharge, play an important role in structuring the phytoplankton community in the Galveston Bay estuary.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO₂ concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

Phosphate limitation stress induces xylitol overproduction by Debaryomyces hansenii

The physiological responses of xylose-grown Debaryomyces hansenii were studied under different nutritive stress conditions using continuous cultivation at a constant dilution rate of 0.055 h⁻¹. Metabolic steady-state data were obtained for xylose, ammonium, potassium, phosphate and oxygen limitation. For xylose and potassium limitation, fully oxidative metabolism occurred leading to the production of biomass and CO₂ as the only metabolic products. However, potassium-limiting cultivation was the most severe nutritional stress of all tested, exhibiting the highest xylose and O₂ specific consumption rates along with the lowest biomass yield, 0.22 g g⁻¹ xylose. It is suggested that carbon was mainly channelled to meet the cellular energy requirements for potassium uptake. For the other limiting nutritional conditions increasing amounts of extracellular xylitol were found for ammonium, phosphate and oxygen limitation. Although xylitol excretion is not significant for ammonium limitation, the same is not true for phosphate limitation where the xylitol productivity reached 0.10 g l⁻¹ h⁻¹, about half of that found under oxygen-limiting conditions, 0.21 g l⁻¹ h⁻¹. This work is the first evidence that xylitol production by D. hansenii might not only be a consequence of a redox imbalance usually attained under semi-aerobic conditions, but additional physiological mechanisms must be involved, especially under phosphate limitation. Cell yields changed drastically as a function of the limiting nutrient, being 0.22, 0.29, and 0.39 g g⁻¹ xylose for potassium, oxygen and phosphate limitation, respectively, and are a good indicator of the severity of nutritive stress.     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

Levels of Glycogen and Trehalose in Mycobacterium smegmatis and the Purification and Properties of the Glycogen Synthetase

The levels of glycogen, free trehalose, and lipid-bound trehalose were compared in Mycobacterium smegmatis grown under various conditions of nitrogen limitation. In a mineral salts medium supplemented with yeast extract and containing fructose as the carbon source, the accumulation of glycogen increased dramatically as the NH(4)Cl content of the medium was lowered. However, levels of free trehalose remained relatively constant. Cells were grown in low nitrogen medium and were then shifted to medium containing high nitrogen. Under these conditions, there was a rapid accumulation of glycogen in low nitrogen, and this glycogen was rapidly depleted when cells were placed in high nitrogen medium. Again the concentration of free trehalose remained fairly constant. However, when cells were grown in low nitrogen medium with [(14)C]fructose and then transferred to high nitrogen medium with unlabeled fructose, the specific radioactivity (counts per minute per micromole) of the free trehalose fell immediately, indicating that it was being synthesized and turned over continually. On the other hand, the specific radioactivity of the glycogen and bound trehalose declined much more slowly, suggesting that these two compounds were not turning over as rapidly or were being synthesized at a much slower rate. Experiments on the incorporation of [(14)C]fructose into glycogen and trehalose indicated that cells in high nitrogen medium synthesized much less glycogen than those in low nitrogen. However, synthesis of both free trehalose and bound trehalose was the same in both cases. The specific enzymatic activities of the glycogen synthetase and the trehalose phosphate synthetase varied somewhat from one growth condition to another, but there was no correlation between enzymatic activity and the amount of glycogen or trehalose, suggesting that changes in glycogen levels were not due to increased synthetic capacity. The glycogen synthetase was purified about 35-fold and its properties were examined. This enzyme was specific for adenosine diphosphate glucose as the glucosyl donor.     

Optimization of date syrup as a novel medium for lovastatin production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> ATCC 20542 and analyzing assimilation kinetic of carbohydrates

Lovastatin is a statin drug, which lowers cholesterol level in blood due to inhibition of (S)-3-hydroxy-3-methylglutaryl-CoA reductase. Date syrup is a rich medium for microbial growth and metabolite production. The main carbohydrates present in the date syrup are glucose and fructose. In this study, date syrup was used as a complex and bioresource medium for lovastatin production by Aspergillus terreus in the submerged cultivation. Optimization of the date syrup medium in order to achieve the highest titers of lovastatin and biomass was carried out. Four factors were studied by response surface methodology including concentration of date syrup carbohydrates, yeast extract concentration, pH, and rotation speed of the shaker. Optimal conditions for these factors found were as follows: concentration of date syrup carbohydrates, 64 g/l; yeast extract concentration, 15 g/l; pH, 6.5; and agitation speed, 150 rpm. It gave lovastatin concentration of 105.6 mg/l. Next, batch cultures in the optimal conditions were performed in a 2.5-l working volume bioreactor and led to the lovastatin titer of 241.1 mg/l during 12 days. Aspergillus terreus showed diauxic growth in the optimized medium with a shift from glucose to fructose assimilation during the run. Glucose and fructose assimilation kinetic parameters revealed that more lovastatin is produced during glucose assimilation, while more biomass was formed during fructose assimilation.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.     

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his⁺ DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 × 10³ to 2 × 10⁷ cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.     

Short-term feedback inhibition of photosynthesis in wheat leaves supplied with sucrose and glycerol at two temperatures

The inhibition of photosynthesis by reduced sink demand or low rates of end product synthesis was investigated by supplying detached wheat (Triticum aestivum L. cv. Tauro) leaves with 50 mM sucrose, 50 mM glycerol or water through the transpiration stream for 2 h, either at 23 or 12 °C. Lowering the temperature and sucrose and glycerol feeding decreased photosynthetic oxygen evolution at high irradiance and saturating CO₂. The decrease in temperature reduced the pools of sucrose and starch, and the ratio glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P), while it increased the concentrations of G6P and F6P (hexose phosphates). Sucrose feeding, in contrast to glycerol feeding, increased sucrose, glucose and fructose contents and the G6P/F6P ratio. Sucrose and glycerol incubations at 23 °C, as well as decreasing the temperature in leaves incubated in water, increased the concentration of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) and decreased the glycerate 3-phosphate (PGA) content, thus increasing the TP/PGA ratio; they also tended to increase the ribulose 1,5-bisphosphate (RuBP) content and the RuBP/PGA ratio. Sucrose and glycerol feeding at 12 °C and the decrease in temperature of leaves incubated in these solutions decreased TP and RuBP contents and the TP/PGA and RuBP/PGA ratios. The results suggest that the phosphate limitation caused by accumulation of end products, restriction of their synthesis and sequestration of cytosolic phosphate can inhibit photosynthesis through decreased carboxylation of RuBP or, with increased phosphate limitation, through lowered supply of ATP. This revised version was published online in August 2006 with corrections to the Cover Date.     

Utilizing high-throughput experimentation to enhance specific productivity of an <Emphasis Type="Italic">E.coli</Emphasis>T7 expression system by phosphate limitation

Background  

The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Biosynthesis of cephamycin by resting cells of Streptomyces lactamdurans L 2/6

In order to investigate the nutritional conditions of cephamycin biosynthesis independently of the biomass growth process, the nutrient limited-resting cell system was used. A replacement medium eliminating cell multiplication was developed. The presence of Mg2+, carbon source and nitrogen source was necessary for cephamycin production by resting cells of Streptomyces lactamdurans L 2/6. Maximum antibiotic production was obtained when maltose, saccharose, and fructose were used as carbon source, and L-asparagine as nitrogen source. An inhibitory effect on the process was exerted by the calcium ions. There was no visible inhibition of cephamycin biosynthesis by inorganic phosphate ions in concentration up to 100 mM.     

Development of a fructose based medium for biosynthesis of cyclosporin-A byBeauveria nivea

Summary A new fructose based medium was developed to growBeauveria nivea ATCC 34921 for the production of Cyclosporin A (CyA). Two different sugars and three different nitrogen sources were used for CyA production. Different pH levels in the broth revealed that the best pH for CyA production was 5.5. Maximum concentration of 170 mg CyA/L of broth was obtained with the new medium in 8 days corresponding to yields of 14.8 mg CyA/g dry weight biomass and 5.67 mg CyA/g fructose supplied.     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

Macronutrients requirements of the dinoflagellate Protoceratium reticulatum

The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217 μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217 μM, an increase in nitrate concentration over the range of 882–8824 μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764 μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth.