Effect of glucocorticoids on fetoprotein production by an established cell line from Morris hepatoma 8994

Glucocorticoids at concentrations equal to or higher than 10^?7M lead to an increase of alpha-fetoprotein production by an established cell line from Morris hepatoma 8994. These cells also secreted alpha_M-fetoprotein into the culture medium but only after addition of at least 4×10^?7M hydrocortisone or 5×10^?8M dexamethasone. The effects on both fetoproteins were observed in spite of a decrease of cell multiplication and an increase of cell detachment.     

Biphasic toxicity of diethyldithiocarbamate, a metal chelation, to T lymphocytes and polymorphonuclear granulocytes: reversal by zinc and copper.

A new type of toxicity biphasically dependent on concentration was observed with diethyldithiocarbamate, a metal chelator utilized in medicine. As judged by cell survival and [³H]Urd incorporation, diethyldithiocarbamate was maximally toxic to T lymphocytes and polymorphonuclears at 2.5×10^?5 M (first phase) and at higher than 2.5×10^?3 M (second phase), but was not toxic at intermediate concentrations around 2.5×10^?4 M. The response of chelator treated T lymphocytes to phytohemagglutinin was also biphasic. The first toxic phase was partially reversed by 2.5×10^?5 M ZnCl₂, while the second phase was partially reversed by 10^?2 M CuCl₂. This suggests that inhibition of Zn-metalloenzymes in the first phase and of Cu-metalloenzymes in the second may play a crucial role in the mechanism of toxicity. The second toxic phase may be in part due to the observed inhibition of superoxide dismutase rendering the cells susceptible to oxygen toxicity, like obligate anaerobes.     

The cAMP signal from Dictyostelium discoideum amoebae.

Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10^?9 to 3.5 × 10^?7M. Populations of 10⁶ cells could amplify an initial cAMP concentration of 2.5 × 10^?9M with a low probability, while an initial cAMP concentration of 5 × 10^?8M always induced a response. An initial cAMP concentration of 1.2 × 10^?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 10⁷ molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10^?7M or more. The amplification of a 10^?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 10⁴ cells/cm², both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10^?13–10^?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation.     

Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line

The potential of spent medium to support the growth and recombinant protein production of High-Five? cells was investigated. Growth in medium consisting of three parts fresh and one part spent medium was comparable to that in fresh medium (maximal specific growth rates of 0.028 and 0.029 h^?1, and maximal cell densities of 4 and 4.5 × 10⁶ cells ml^?1, respectively). Glucose exhaustion coincided with an abrupt decrease of viability. Of 15 amino acids analyzed, not a single one was completely exhausted at the end of the growth phase. Growth in medium consisting of equal parts spent and fresh medium led to lower maximal cell concentration (2.9 × 10⁶ cells ml^?1) with a smoother death phase. Glucose supplementation at the beginning of the culture or at the end of the growth phase did not lead to an increase of either the maximal cell density or the specific growth rate. Infection of High-Five? cells at three different densities (1.4, 2.5 and 4.2 × 10⁶ cells ml^?1) without medium change led to monotonically decreased specific productions for β-galactosidase. Partial (75%) or total medium replacement at the higher infection density restored the specific production at the levels of the intermediate density infection (321, 292 and 389 U.(10⁶ cells)^?1, respectively).     

Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells

Murine pre-B-cells grown in the presence of lower (1 μM) or higher (5 μM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 μM cadmium chloride concentration showed that a. at 5×10⁵ cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl₂ concentration (1 μM) at higher cell culture density (>5×10⁵ cell/ml) did not change significantly the average cell diameter. At 5 μM cadmium concentration and higher cell culture densities (>5×10⁵ cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0–2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.     

Exogenous salicylic acid stimulates physiological and biochemical changes to improve growth, yield and active constituents of fennel essential oil

Fennel (Foeniculum vulgare Mill) is a high-value medicinal and essential oil bearing plant used extensively in pharmaceutical, food and cosmetic industries. A pot experiment was carried out in the natural conditions of net house to resolve whether the foliar application of salicylic acid (SA) might enhance the growth, yield and essential oil production of fennel. Plants were sprayed three times with SA. The first spray was carried out at 40?days after sowing (DAS); the second and third sprays were applied one and 2?weeks later, the plants were sprayed with deionised water (control) and different concentrations of SA (10^?5, 10^?4 and 10^?3?M). The foliar spray of SA at 10^?4?M significantly enhanced the vegetative growth (shoot and root lengths, fresh and dry weights), physiological and biochemical characteristics (chl ??a??, chl ??b??, total chlorophyll and carotenoids contents, nitrate reductase activity, carbonic anhydrase activity, leaf-N, -P and -K contents), yield characteristics (number of umbels and fruits, 1,000-seed weight and seed yield) and essential oil yield of fennel. GLC analysis revealed the significant increase in the components of essential oil, viz. trans-anethole (80.4?C84.7?%), methyl chavicol (2.3?C2.5?%) and fenchone (5.6?C7.9?%). It was concluded that foliar spray of SA at 10^?4?M might be employed for enhancing the plant growth as well as yield and quality of essential oil of fennel.     

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of α-amino-γ-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of grampositive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10^?5?10^?3 M HSL or 10^?5?10^?4 M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10^?4?10^?3 M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10^?4 to (1.5?2.5)×10^?4 M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10^?4 M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1 cm² disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075–0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions at the microbial community level.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Identification and structure–activity relationship study of carvacrol derivatives as Mycobacterium tuberculosis chorismate mutase inhibitors

Abstract

In the present study, we identified carvacrol, a major phenolic component of oregano oil as a novel small molecule inhibitor of Mycobacterium tuberculosis (MTB) chorismate mutase (CM) enzyme with IC₅₀ of 1.06?±?0.4?µM. Virtual screening of the BITS-Pilani in-house database using the crystal structure of the MTB CM bound transition state intermediate (PDB: 2FP2) as framework identified carvacrol as a potential lead. Further various carvacrol derivatives were evaluated in vitro for their ability to inhibit MTB CM enzyme, whole cell MTB and cytotoxicity as steps toward the derivation of structure–activity relationships (SAR) and lead optimization.     

Effects of density on growth rates of four benthic diatoms and variations in biochemical composition associated with growth phase

Diatom cell quantity and their biochemical composition vary among species and are greatly affected by harvest stage or culture conditions. This study compares growth pattern, cell attachment, and biochemical composition of four diatoms suitable for abalone post-larvae: Navicula incerta, Proschkinia sp., Nitzschia sp., and Amphora sp. The four diatoms were grown in F/2 medium at 28.5?±?1.4°C, under 62?±?8?μmol?photons?m^?2?s^?1, at different original inoculating densities (0.05?×?10⁶, 0.10?×?10⁶, and 0.25?×?10⁶?cells?mL^?1) and were harvested in log and stationary phase of growth for biochemical analysis. Total protein, carbohydrate, lipid, and ash composition, as well as fatty acid composition, were determined. All diatoms grew better when inoculated at 0.10?×?10⁶?cell?mL^?1 with Proschkinia sp. reaching the highest cell density of 6.56?×?10⁶?cells?mL^?1 in log phase. Amphora sp. had the highest cell attachment capacity when inoculated at 0.10?×?10⁶?cell?mL^?1 (11,580?cells?mm^?2), whereas N. incerta had the lowest (7,750?cells?mm^?2). Protein and lipid (percent dry weight) contents were generally highest in cells during log phase of growth; Amphora sp. in log phase of growth had the highest lipid content of 9.74% DW, whereas significant differences in carbohydrate between the two growth phases were only observed for Proschkinia sp. Besides, all diatoms had higher energy contents in log phase of growth. There were no significant differences in ash content among the four diatoms. Polyunsaturated fatty acid (PUFA) content ranged from 23.25% to 38.62% of the total fatty acids, and the four diatoms tested were richer in n-3 PUFA than in n-6 PUFA. All the diatoms had significant quantities of 20:5n-3 (EPA) (between 12.69% and 17.68% of TFA), and Proschkinia sp., in log phase of growth, had the highest quantity of arachidonic acid (20:4n-6; ARA). The results highlight the influence of culture conditions and harvest protocols on diatom nutritive value and enabled a preliminary approach towards the selection of novel diatom species.     

A possible modulation of acetylcholine receptors of embryonic chick muscle cells by α-bungarotoxin

Acetylcholine receptors were assayed with α-bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D-tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 × 10^?9M and 2.7 × 10^?7M at 25°C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high-affinity receptors is about 200/μm². A time course of toxin binding to receptors at 37°C vs 25°C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was in-activating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

A perfusion-control strategy based on cellular consumption rates of oxygen and glucose was established for the production of single-chain urokinase-type plasminogen activator (scu-PA). Employing this strategy, the influences of microcarrier types and the culture media on culture performances were evaluated. In the control perfusion culture, which used a solid microcarrier and a 1% fetal bovine serum (FBS) medium, viable cell density reached 3.1?×?10⁷?cells?ml^?1. However, formation of large, heterogeneous aggregates (500–1,000?μm) resulted in a gradual decrease in viable cell density to less than 1.0?×?10⁷?cells?ml^?1. Accordingly, declines in the production of urokinase-type plasminogen activator (u-PA) and in the scu-PA portion of u-PA were observed. In the serum-free media, cell growth and u-PA production were suppressed 2–3?times, but were significantly enhanced when a porous microcarrier, Cultispheer G, was used. The cell-growth profile showed a continuous increase in cell density, reaching 5.1?×?10⁷?cells?ml^?1, and the production of u-PA remained stable throughout the culture (1586?±?247?IU?ml^?1). The values of all the parameters associated with cell growth and u-PA production were fairly comparable to or even higher than those in the control culture. Moreover, a 13% higher scu-PA portion of u-PA was observed in the serum-free culture, regardless of the microcarrier type, compared with scu-PA portion of u-PA in the control culture.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Production of phenolic compounds in callus cultures of tea plant under the effect of 2,4-D and NAA

The effect of hormone-like compounds at different concentrations: 2,4-D (2 × 10^?6; 2 × 10^?5; and 2 × 10^?4M) and 1-NAA (2 × 10^?7; 2 × 10^?6; 2 × 10^?5; 4 × 10^?5, and 6 × 10^?5 M) on the growth and production of phenolic compounds, including flavans and lignin, was investigated in callus culture of tea plant (Camellia sinensis L., a highly productive strain IFR ChS-2). The growth of the culture was vigorous, and production of phenolic compounds therein was efficient in the medium containing 2 × 10^?5 M 2,4-D. Substitution of 1-NAA for 2,4-D in all the cases decelerated the growth of the culture. These changes were more pronounced when 2 × 10^?7 and 2 × 10^?6 M 1-NAA was used; in this case, biomass accumulation decreased by 1.5–2.0 times as compared with control material growing on the medium with 2 × 10^?5 M 2,4-D. In the presence of 1-NAA, the content of total soluble phenolic compounds and flavans in the calli rose by 30% on the average as compared with control material. Accumulation of lignin remained essentially the same. Therefore, the replacement of 2,4-D with 1-NAA in the nutrient medium used for the growing of highly productive strain of tea plant callus did not induce considerable changes in its ability to produce phenolic compounds.     

Anomalous enhancing effect of hydroxyurea on DNA synthesis

$\text{In vitro}$ cultures of $\text{Crithidia}$ sp. were exposed to various concentrations of hydroxyurea (HU) during the logarithmic phase. In the presence of 5 × 10^?2M HU, cell division was completely blocked after an initial increase in cell numbers by about 20%. Inhibition of incorporation of ³H-thymidine into acid-insoluble material was effective within 1 hr of exposure to the drug (5 × 10^?2M) and it reached a level of 80% after 8 hr. At lower concentrations (5 × 10^?4M ? 1 × 10^?3M), however, incorporation of ³H-thymidine was remarkably increased while cell division remained unaffected indicating that the increase in incorporation was not due to increased DNA synthesis in preparation for cell division.     

Effect of trace elements and vitamins on the growth of Methanosarcina barkeri

Growth of Methanosarcina barkeri on methanol as energy source was found to be dependent on cobalt and molybdenum. In the presence of 10^?6 M Co and 5 × 10^?7M Mo optimal growth occurred. Furthermore it could be demonstrated that nickel and selenium each in a concentration of 10^?7 M stimulated the growth of this methanogenic bacterium while the following elements tested in the range of 10^?7 M to 10^?3 had no influence: B, Cr, Cu, Mn, Pb. The requirement of Co and Ni for optimal growth are in accordance with the results that the cells contain the Co containing corrinoid Factor III (0.1 – 0.2 mg 5-hydroxylbenzimidazolylcyanocobamide per g wet cells) and Factor F₄₃₀, a nickel component. Studies on the vitamin dependency of M. barkeri showed that this strain needs only the vitamin riboflavin for the growth in a defined medium. Under these conditions a cell density of 2.6 g dry cells/l could be obtained in a fed batch culture.     

A paradoxical effect of hydrated C60-fullerene at an ultralow concentration on the viability and aging of cultured Chinese hamster cells

The effect of an aqueous solution of hydrated C₆₀-fullerene (HyFn) on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during the slowing down of their proliferation within a single passage and the subsequent “aging” in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells was studied. The final calculated concentration of HyFn in the growth medium was 10^?19 M. A paradoxical result contrasting the available data on the absence of HyFn cytotoxicity at higher concentrations was obtained in our experiments: namely, HyFn decelerated cell proliferation (estimated by the growth of mass culture, as well as by the efficiency of colony formation) and accelerated the “stationary phase aging” of the cell culture. Moreover, repeated addition of an aqueous solution of HyFn (to the final calculated concentration of 10^?19 M) to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. The observed effect of HyFn at ultralow concentration is supposed to arise from the alterations in the properties of the water surrounding the fullerene molecule: namely, water becomes a donor and acceptor of electrons and regulates redox processes (especially those involving oxygen) in aqueous systems. This effect of HyFn at an ultralow concentration may be specific for transformed cells, and, therefore, experiments on normal fibroblasts with limited mitotic potential are planned as a continuation of the present study. It is also possible that the reported antiaging effect of HyFn in experimental animals is due to its anticancer, immunostimulatory, antiviral, and antibacterial properties manifested only at the whole-organism level.     

Revealing binding interaction between seven drugs and immobilized β2‐adrenoceptor by high‐performance affinity chromatography using frontal analysis

The development of new approaches to study the affinity between ligands and G‐protein‐coupled receptors proves to be of growing interest for pharmacologists, chemists, and biologists. The aim of this work was to determine the binding of seven drugs to β₂‐adrenoceptors by frontal analysis using immobilized receptor stationary phase. The dissociation constants (K_d) were determined to be (3.16 ± 0.09) × 10^?4 M for salbutamol, (4.29 ± 0.12) × 10^?4 M for terbutaline, (6.19 ± 0.16) × 10^?4 M for methoxyphenamine, (2.11 ± 0.07) × 10^?4 M for tulobuterol, (1.82 ± 0.11) × 10^?4 M for fenoterol, (9.75 ± 0.24) × 10^?6 M formoterol, and (9.84 ± 0.26) × 10^?5 M for clenbuterol. These results showed a good correlation with the data determined by radioligand binding assay. Further investigations revealed that the dissociation constant mainly attributed to the number of hydrogen bonds in the structures of ligands. This study indicates that affinity chromatography using immobilized receptor stationary phase can be used for the direct determination of drug‐receptor binding interactions and has the potential to become a reliable alternative for quantitative studies of ligand–receptor interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.