A CONTINUOUS CULTURE STUDY OF PHOSPHATE UPTAKE,GROWTH RATE AND POLYPHOSPHATE IN SCENEDESMUS SP.1

The kinetics of phosphate uptake and growth in Scenedesmus sp. have been studied in continuous culture with particular reference to the shifts in the cellular P compounds as a function of growth rate. Uptake velocity is a function of both internal and external substrate concentrations and can be described by the kinetics of noncompetitive enzyme inhibition. The concentrations of polyphosphates (alkali-extractable or 7-min) can he substituted as inhibitors in the kinetic equation. The apparent half-saturation constant of uptake. K_m, is 0.6 μM. The apparent half-saturation concentration for growth is less than K_m, by 1 order of magnitude. Growth is a function of cellular P concentrations, and the polyphosphates (alkali-extractable or 7-min) appear to regulate growth rate directly or indirectly. To understand P limitation, therefore, it is necessary to measure both external P and internal polyphosphate levels. Evidence indicates that alkali-extractable polyphosphates, which can be quantitatively determined by a simple method of measuring surplus P, are involved in cell division process find that a maintenance concentration of functional phosphate exists in the form of poly phosphates. Alkaline phosphatase activity has an inversely linear relationship to growth rate and to the reciprocals of both polyphosphates and surplus P. Changes in lipid P, RNA P, and presumably all other forms except DNA are related to changes in growth rate.     

PHOSPHATE‐LIMITED GROWTH OF PAVLOVA LUTHERI (PRYMNESIOPHYCEAE) IN CONTINUOUS CULTURE: DETERMINATION OF GROWTH‐RATE‐LIMITING SUBSTRATE CONCENTRATIONS WITH A SENSITIVE BIOASSAY PROCEDURE1

The relationship between steady‐state growth rate and phosphate concentration was studied for the marine prymnesiophyte Pavlova lutheri (Droop) J. C. Green grown in a chemostat at 22°C under continuous irradiance. A bioassay procedure involving short‐term uptake of 10 picomolar spikes of ³³P‐labeled phosphate was used to estimate the concentration of phosphate in the growth chamber. The relationship between growth rate and phosphate was well described by a simple rectangular hyperbola with a half‐saturation constant of 2.6 nM. The cells were able to take up micromolar spikes of phosphate at rates two to three orders of magnitude higher than steady‐state uptake rates. The kinetics of short‐term uptake displayed Holling type III behavior, suggesting that P. lutheri may have multiple uptake systems with different half‐saturation constants. Chl a:C ratios were linearly related to growth rate and similar to values previously reported for P. lutheri under nitrate‐limited conditions. C:N ratios, also linearly related to growth rate, were consistently lower than values reported for P. lutheri under nitrate‐limited conditions, a result presumably reflecting luxury assimilation of nitrogen under phosphate‐limited conditions. C:P ratios were linearly related to growth rate in a manner consistent with the Droop equation for growth rate versus cellular P:C ratio.     

Short-term phosphate uptake kinetics in Zostera noltii Hornem: a comparison between excised leaves and sediment-rooted plants

Short-term phosphate uptake by excised leaves of Zostera noltii Hornem. as well as by leaves of sediment-rooted plants were characterized and compared in a kinetic framework. Time courses of phosphate disappearance were measured over a wide range of initial substrate concentrations. Phosphate uptake determined by this perturbation method did not follow Michaelis-Menten kinetics. Both excised leaves and sediment-rooted plants exhibited a biphasic uptake pattern as a function of phosphate concentration. However, rooted plants showed higher uptake rates and accumulated higher amounts of phosphate than excised leaves. The results point out the importance of the structural and functional coupling between shoots and underground parts during the nutrient foliar uptake processes. Our study also indicates that Zostera noltii leaves function as a phosphate sink in the water column.A second objective of this work is to compare the perturbation and the multiple flask methods in determining the uptake kinetic parameters. The obtained results support that both methods provide valuable and complementary information in determining the uptake rates.     

An error estimation of Michaelis-Menten (Monod)-type kinetics

Due to research on biochemistry and genetic engineering, mathematical models of microbial growth have become more complicated but Michaelis-Menten or Monod type expressions have still been used for conversion rates, uptake rates, etc. It is worth examining the error that can be caused by these quasi-steady-state-hypotheses. This paper presents a simple but very effective rationale function that describes the error of the quasi-steady-state hypothesis in enzyme kinetics. A simplified fermentation kinetic model was used for comparison of microbial growth but no analytical error function has been found for batch cultivation. In the case of continuous fermentation the error can be given in an analytical form. Many simulations, based on real SCP experiments, show a significant effect of the quasi-steady-state hypothesis. Since the rate constants of intracellular events are not really known, we have to be very careful when taking into account Michaelis-Menten type expressions in the building of complicated models. Correspondence to: L. Szigeti     

Kinetics of nitrate uptake by freshwater algae

The kinetics of nitrate (NO₃ ^–) uptake, the maximum uptake velocity (V_m) and the half-saturation constant (K_s), were determined for 18 species of batch-cultured freshwater algae grown without nitrogen limitation. Values of K_s ranged from 0.25 to 6.94 µM l^–1 Chlorella pyrenoidosa Chick, and Navicula pelliculosa (Breb.) Hilse, respectively. Values of V_m ranged from 0.51 to 5.07 µM l^–1 h^–1 for Anabaena A7214 and Nitzschia W-32 O'Kelley, respectively. The mean positive values of K_s for Chlorophyta, Cyanophyta and Chrysophyta were 1.89, 3.67 and 4.07 µM l^–1, respectively. The mean values of V_m for the same phyla were 1.61, 1.02 and 2.97 µM l^–1 h^–1 10⁵ cells^–1, respectively. The ranges of these kinetic parameters encompass values of kinetic parameters for marine and freshwater species in batch culture, for freshwater algae grown in N-limited chemostats and for natural populations of freshwater phytoplankton. Thus, in spite of variability between species, uptake parameters for both marine and freshwater algae are identical.     

Environmental change and copper uptake by Bacillus subtilis subsp. niger and by Pseudomonas fluorescens

The passive uptake of copper by B. subtillis subsp. niger ATCC 9372 and by a strain of Ps. fluorescens, isolated from polluted soil, has been determined. Prior to exposure to the metal ions the strains were grown to steady state in a wide variety of nutrient-limited chemostats operated at a dilution rate equal to one-half of their respective maximum growth rates. Carbon-limited organisms had the lowest uptakes and the uptakes increased as the limiting nutrient was changed in the order carbon < magnesium < nitrogen (NH₄ ⁺) < potassium, with sulphur (SO₄ ^2-) - and phosphate (glycerol 2-phosphate)-limited organisms occupying different positions with the two strains.     

Phosphate‐limited growth of the marine diatom Thalassiosira weissflogii (Bacillariophyceae): evidence of non‐monod growth kinetics1

The marine diatom Thalassiosira weissflogii (Grunow) G. A. Fryxell & Hasle was grown in a chemostat over a series of phosphate‐limited growth rates. Ambient substrate concentrations were determined from bioassays involving picomolar spikes of ³³P‐labeled phosphate, and maximum uptake rates were determined from analogous bioassays that included the addition of micromolar concentrations of unlabeled phosphate and tracer concentrations of ³³P. The relationship between cell phosphorus quotas and growth rates was well described by the Droop equation. Maximum uptake rates of phosphate spikes were several orders of magnitude higher than steady state uptake rates. Despite the large size of the T. weissflogii cells, diffusion of phosphate through the boundary layer around the cells had little effect on growth kinetics, in part because the cellular N:P ratios exceeded the Redfield ratio at all growth rates. Fitting the Monod equation to the experimental data produced an estimate of the nutrient‐saturated growth rate that was ~50% greater than the maximum growth rate observed in batch culture. A modified hyperbolic equation with a curvature that is a maximum in magnitude at positive growth rates gave a better fit to the data and an estimate of the maximum growth rate that was consistent with observations. The failure of the Monod equation to describe the data may reflect a transition from substrate to co‐substrate limitation and/or the presence of an inducible uptake system.     

A possible mechanism of metabolic regulation in Gibberella fujikuroi using a mixed carbon source of glucose and corn oil inferred from analysis of the kinetics data obtained in a stirrer tank bioreactor

A nonstructured model was used to study the dynamics of gibberellic acid production in a stirred tank bioreactor. Experimental data were obtained from submerged batch cultures of Gibberella fujikuroi (CDBB H‐984) grown in varying ratios of glucose‐corn oil as the carbon source. The nitrogen depletion effect was included in mathematical model by considering the specific kinetic constants as a linear function of the normalized nitrogen consumption rate. The kinetics of biomass growth and consumption of phosphate and nitrogen were based on the logistic model. The traditional first‐order kinetic model was used to describe the specific consumption of glucose and corn oil. The nitrogen effect was solely included in the phosphate and corn oil consumption and biomass growth. The model fit was satisfactory, revealing the dependence of the kinetics with respect to the nitrogen assimilation rate. Through simulations, it was possible to make diagrams of specific growth rate and specific rate of substrate consumptions, which was a powerful tool for understanding the metabolic interactions that occurred during the various stages of fermentation process. This kinetic analysis provided the proposal of a possible mechanism of regulation on growth, substrate consumptions, and production of gibberellic acid (GA₃) in G. fujikuroi. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1169–1180, 2013     

The Influence of Extremely High Concentrations of Inorganic P at Varying pH on the Growth and Photosynthesis of Unicellular Algae

An increase in the phosphate concentration due to severe pollution is without real toxic effect for the phytoplankton in eutrophic lakes, although the various species have a variable tolerance against high concentrations. Experiments on photosynthesis and growth were made with cultures of two algae, Chlorella pyrenoidosa and Nitzcshia palea, using phosphate concentrations much higher than ever found in nature. The tolerance decreases with rising pH. This seems not to be due to a higher ratio of secondary phosphate at a high pH. Nor is the rise in the osmotic pressure apparently the cause of the decrease in the growth rate at high phosphate concentrations.     

Effect of glycollate on phosphate uptake in Chlorella pyrenoidosa

Summary A study was carried out on a strain of Chlorella pyrenoidosa known to excrete glycollic acid to see whether the presence of this acid had any stimulatory effect on phosphate uptake. Contrary to the results of a previous author working on Ankistrodesmus, no stimulation was found at any glycollate or phosphate concentration. However, 2×10^-4 M glycollate caused a marked inhibition of phosphate uptake during the initial period of uptake, but not on a subsequent phase of linear uptake.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

PREDICTING THE KINETICS OF DISSOLVED INORGANIC CARBON LIMITED GROWTH FROM THE SHORT-TERM KINETICS OF PHOTOSYNTHESIS IN SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA)1

The blue-green alga (Cyanobacterium) Synechococcus leopoliensis (Racib.) Komarek was grown in dissolved inorganic carbon [DIC]-limited chemostats over the entire range of growth rates. At each growth rate, the kinetics of photosynthesis with respect to [DIC] and the maximal rate of photosynthesis (P_max) were determined. The half-saturation constant for [DIC]-limited photosynthesis (K_1/2^DIC) for cells growing below 1.7 d^?1 was constant (4.7 μM) whereas for growth rates between 1.7 d^?1 and 2.1 d^?1 (μ_max) the kinetics of photosynthesis were multiphasic with an apparent K_1/2^DIC between 1.5–2.0 mM. P_max increased in a linear fashion with growth rate for growth rates below 1.7 d^?1. No trend in P_max was apparent for growth rates greater than 1.7 d^?1. These kinetic parameters were used to predict a growth rate versus [DIC] relationship. Results show that the Monod relationship is a physiologically valid expression of growth as a function of [DIC] provided (K_1/2^DIC) remains constant. The major change in (K_1/2^DIC) as μ approaches μ_max results in the conclusion that two separate and distinct Monod equations must be used to describe growth as a function of DIC over the entire growth range. These results point to a major discontinuity in the μ vs. [DIC] curve at 1.7 d^?1 which corresponds to the change from high to low affinity photosynthetic kinetics. We believe these results account for the previously described deficiencies of the Monod equation in describing [DIC]-limited algal growth.     

Kinetics for the transition between cis- and trans-helices in poly-L-proline

The kinetics for the cis-trans isomerization of long-chain poly-L -proline has been studied as a function of pressure, temperature, and solvent composition in the acetic acid + n-propanol solvent system. Our complete kinetic curves were fitted by Monte Carlo techniques, and rate constants for nucleation, growth, and termination were estimated. It was found that for the formation of a cis-helix, high pressure, low temperature, and increased acetic acid content of the solvent, lowered the rate of nucleation relative to growth. The inverse seems to be t rue for the formation of a trans-helix. Molecular models suggest that this behavior of the kinetic constants can be due to the exposure of peptide units to solvent in the transition state for trans nucleation, and the burying of peptide units in the transition state for cis nucleation. It is further suggested from our analysis of complete kinetic curves that at least one of the assumptions usually made in the analysis of relaxation kinetics is invalid for poly-L -proline.     

Regulation of phosphate uptake kinetics inOscillatoria agardhii

In order to study phosphate uptake kinetics the cyanobacteriumOscillatoria agardhii was grown in continuous culture under a phosphorus limitation. The affinity of the uptake system reflected in the initial slope of the uptake rate versus external substrate concentration curve (dV/ds) was found to be unaffected by the growth wate.The maximum phosphate uptake rate (V _m ) decreased as the growth rate was increased. Attempts were made to relate the decrease ofV _m to the increase in phosphorus content of the cells that occurred a higher growth rates. Accumulation of phosphate during pulse experiments indeed resulted in a decrease ofV _m . However feedback regulation ofV _m by accumulated phosphorus was found to occur only to a small extent in steady state growing cells. The main part of the regulation of the activity of the phosphate uptake system seemingly is determined by a long term process that is, at least longer than 2 h. The presence of short term feedback inhibition by accumulated phosphorus on the activity of the uptake system provides an explanation of the phenomenon thatOscillatoria agardhii is not able to grow at near _max growth rates under a phosphorus limitation.     

Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture

Summary Anabaena flos-aquae was grown in chemostats with phosphate-limiting growth and dilution rate of 0.015–0.03 h^-1. The yields of cells were dependent on dilution rate and a two-fold increase obtained by growth in the presence of 15 mM KNO₃. Alkaline phosphatase activity varied 20-fold, lowest activity with excess phosphate light-limited cells and the highest activity with cells grown in the presence of 15 mM KNO₃. There was no correlation between hot water soluble phosphate of cells and alkaline phosphatase activity.     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN ON LIGHT/DARK CYCLES.' I. SYNCHRONIZED BATCH CULTURES1

The characteristics of phosphate uptake in synchronized populations of Euglena gracilis Klebs (Z) were studied. The cells were grown autotrophically in batch culture and synchronized with a cycle of 14:10 LD. Incorporation of P was nonlinear with time for the first 2 h of incubation over a wide range of P concentrations and completely inhibited by darkness. The kinetics of P uptake as a function of P concentration were triphasic between 0 and 100 μM PO₄, obeying Michaelis-Menten kinetics over the 0–3 μM PO₄ range-only. Uptake velocity increased linearly with, concentration above 3 μM PO₄. The kinetics of P uptake varied with stage in the cell cycle. The half-saturation constant for uptake at the lower concentrations oscillated between 0.7 and 2.8 μM PO₄, reaching a peak immediately before the onset of cell division (beginning of the dark period). V_max was largest in the middle of the light period, as was the slope of the linear portion of the kinetic pattern. Further analysis of the kinetics suggests that changes in this slope are responsible for the oscillation in K_s values calculated for the lower concentrations. This analysis assumes 2 uptake mechanisms, one which saturates at low concentrations of phosphate, and one which is nonsaturable over the entire concentration range examined.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.