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

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

COMPETITION FOR PHOSPHORUS BETWEEN DESMIDS1

During competition for phosphate in continuous cultures, Cosmarium subcostatum Nord. routinely displaced Staurastrum paradoxum Meyer. The rate of displacement was independent of cell density between 100 and 6000 cells mL^?1. This suggests that competition for nutrients is important over a wide range of naturally occurring cell densities. C. subcostatum had higher saturated rates of phosphate uptake but also higher half saturation values for uptake. As a result, the two desmids were similarly able to take up phosphate at low concentrations. The competitive advantage of C. subcostatum lay in its greater yield per unit of phosphorus. Growth of the two algae in shared medium in a dual-chamber chemostat had no effect on uptake or yield characteristics.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

EFFECT OF VITAMIN CONCENTRATIONS ON GROWTH AND DEVELOPMENT OF VITAMIN-REQUIRING ALGAE1

Vitamin-requiring marine algae, Cyclotella nana, Monochrysis lutheri, and Amphidinium carterae, were grown in batch culture with limiting concentrations of vitamin B₁₂, thiamine, and biotin, respectively. Cell numbers, average cell volumes, biomasses, ¹¹CO₂ uptake rates, and chlorophyll a contents were determined daily. Maximum ¹⁴CO₂ uptake rates in most vitamin concentrations were obtained at 2 days with C. nana and M. lutheri and at 4 days with A. carterae after starved cultures were exposed to the vitamin. Radiocarbon uptake rates approximately reflect biomass increases. Cell numbers were proportional to vitamin concentrations when cells were incubated for 2 to 3 more days. Cell sizes varied depending on time of incubation. Chlorophyll a content did not always reflect vitamin concentrations. Maximum carbon assimilation rates (K_m) and saturation constants (K_s) determined from ¹⁴CO₂, uptake rates in different vitamin concentrations during early incubation were higher than when determined from cell number in log phase growth. Dissolved vitamin B₁₂, thiamine, and biotin in many samples of seawaters were in the ranges which influence the growth rate, cell size, and chlorophyll a content of C. nana, M. lutheri, and A. carterae, respectively, in laboratory studies. The effects of vitamins on these algae in situ may be similar.     

Analysis of pH-stat continuous cultivation and the stability of the mixed fermentation in continuous yogurt production

A pH-stat fermentor is a continuous cultivator in which the feed rate is controlled to maintain a constant pH, i.e., end-product acid concentration. This fermentor has application to the continuous cultivation of lactic acid-producing organisms in milk-based media. The equations describing the operation of this fermentor are developed. It is shown that, where the limiting substrate is the carbon and energy source, the operation of the fermentor is essentially equivalent to that of a turbidostat. In contrast to this, where the carbon and energy source is in excess and growth is limited by another substrate, pH-state fermentation is stable both in regions of substrate excess, where D = μ_max, comparable with turbidostat operation, and substrate limitation where D < μ_max, comparable with chemostat operation. These conditions are met in milk-based media. An analysis is presented, allowing the prediction of the degree of substrate limitation, cell density, and dilution rate in a pH-stat fermentor from batch-growth kinetics. This was confirmed using experimental data for the growth of Streptococcus thermophilus TS2 and Lactobacillus LB1 in skim milk. Stable simultaneous growth of two organisms in continuous culture occurs if their growth rates are determined by separate conditions, so that, at steady state, their growth rqtes are separately madeequal to the dilution rate. It is then predicted, and confirmed by experiment, that a mixed culture of S. thermophilus TS2 and L. bulgaricus LB1 in a pH-stat continuous fermentor in yogurt mix at pH 5.5 would be stable with the growth of L. bulgaricus being substrate unlimited and the fermentor operting with D = μ_max for L. bulgaricus LB1, and the growth of S. thermophilus TS2 being substrate limited so that its growth rate is equal to the existing dilution rate. Finally, it is predicted and confirmed by experiment that if the conditions are altered so that the growth of S. thermophilus TS2 is substrate unlimited the stable association is broken down, the fermentor operates with D approaching μ_max for S. thermophilus TS2, and L. bulgaricus LB1 is washed out to the level maintained by wall growth.     

COMPARATIVE KINETIC STUDIES OF NITRATE-LIMITED GROWTH AND NITRATE UPTAKE IN PHYTOPLANKTON IN CONTINUOUS CULTURE1

A comparative study of nitrate-limited growth and nitrate uptake was carried out in chemostat cultures of Ankistrodesmus falcatus (Corda) Ralfs., Asterionella formosa Hass., and Fragilaria crotonensis Kit. In each species growth rate (μ) was related to total cell nitrogen or cell quota (q) by the empirical Droop growth function. Nitrate uptake was a function of both external N concentration and q. The apparent maximum uptake rate (V_m') at a given μ was inversely related to q – q₀, where q₀ is the minimum quota. The apparent half-saturation constant for uptake, (K_m') appears to show a slight inverse trend with μ, although statistical analysis shows that this trend is inconclusive. When q approaches q₀, V_m' is several orders of magnitude greater than μq, the calculated steady-state uptake rate. As q increases, however, the difference between these two variables decreases sharply until q approaches q_m, the cell quota for nitrogen-rich cells. At this point the difference between μq and V_m' disappears. This behavior is explained by the feedback regulation of N uptake. The inverse relationship between V_m' and q – q₀ can be described by an empirical three-parameter equation.     

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.     

Growth and Antithraquinone Biosynthesis by Galium mollugo L. Cells in Batch and Chemostat Culture

The kinetics of growth, nutrient uptake, and anthraquinone biosynthesisby suspension cultures of Galium mollugo L. cells were examinedin batch and continuous (chemostat) culture. In batch culture,although the initial growth rate was constant (minimum doublingtime = 35 h) characteristic changes in cell composition wereobserved during the growth cycle particularly cell dry weight(between 3.9 and 9.2 g/10⁹ cells), cell anthraquinone (22–80mg/10⁹ cells), and cell protein (0.7–1.6 g/10⁹ cells).Using a chemostat steady state growth was established at twodifferent specific growth rates with mean doubling times of40 h and 25 h. Phosphate was established as the growth-limitingnutrient in chemostat culture at a concentration of 11 µgP ml^–1. In steady state growth at a doubling time of 40h the cell composition remained constant although this was differentfrom any cells grown in batch culture. The cell anthraquinonelevel in steady state growth was between 7 and 30 times lowerthan in batch culture. This result raises the question of therelative importance of growth rate and the growth-limiting nutrientin determining accumulation of secondary products by culturedplant cells.     

CONTINUOUS CULTURE OF MARINE DIATOMS UNDER SILICATE LIMITATION. II. EFFECT OF LIGHT INTENSITY ON GROWTH AND NUTRIENT UPTAKE OF SKELETONEMA COSTATUM1,2

Two replicate experiments were conducted to investigate the effect of light intensity on the growth and nutrient uptake of Skeletonema costatum (Grev.) Cleve in silicate-limited continuous culture. Each experiment began with 4 identical chemostat cultures of S. costatum growing at the normal laboratory light (0.14 ly · min^?1, continuous illumination) under strong silicate limitation. Screens were placed over 3 cultures reducing them to light intensities of 0.042, 0.021 and 0.0018 ly · min^?1. Based on growth rules, nutrient uptake rates, cell morphology and chemical composition, the cultures receiving 0.021, and 0.0018 ly · min^?1 appeared to he light-limited, whereas the culture receiving 0.14 ly.     

A Simple and Inexpensive Design of Chemostat Enabling Steady-state Growth of Acer pseudoplatanus L. Cells under Phosphate-limiting Conditions

Operational and constructional details are given of a relativelysimple and inexpensive chemostat designed for the continuousculture of plant cells in suspension. This apparatus permitscontrol of the growth rate of sycamore, Acer pseudoplatanusL. cells in steady-state conditions. By alteration of the rateof input of medium different steady-state growth rates wereobtained over a wide range (mean doubling times from 182 h to36 h). In order to establish a growth-limiting nutrient thetime course of nutrient uptake in batch culture was measured.In batch culture the maximum growth obtained was proportionalto the initial concentration of phosphate when this was belowa concentration of 17 µg P per ml (as phosphate). It isalso shown in chemostat culture that the steady-state cell densityis proportional to the phosphate concentration in the mediumwhen this is below 17 µg P per ml (as phosphate). Phosphatewas therefore established to be the growth rate-limiting nutrientin chemostat culture at a concentration of 8•5 µgP per ml (as phosphate).     

Growth of Suspension-cultured Acer pseudoplatanus L. Cells in Automatic Culture Units of Large Volume

A phytostat to mass culture higher plant cells in liquid medium is described. This apparatus allowed the culture in batch, turbidostat and chemostat of 20 liters of cells. Automatic control of cell suspension growth was based on culture turbidity. Changes with time of certain cell characteristics, particularly cell respiration and phospholipid content, indicated that the test time to harvest large amounts of sycamore cells (Acer pseudoplatanus L.) in good physiological state was about 2 days before the end of the exponential phase of growth, when the cell density reached one million cells per milliliter of culture.     

Influence of growth rate on pigment and lipid composition of the microalgaIsochrysis aff.galbana clone T.iso

A continuous culture ofIsochrysis aff.galbana clone T.iso, used to feedPecten maximus larvae at IFREMER (Brest, France), was carried out in a chemostat at its optimum temperature for growth (26 °C). Changes in pigments, lipid class (neutral, glyco- and phospholipids) and degree of fatty acid unsaturation were studied at three different growth rates (0.33, 0.5, 1 d^–1). As predicted by chemostat theory, a slow growth rate produced higher cell numbers and higher biomass per unit volume. These cells were low in chlorophylla and carotenoids, but rich in neutral lipids. In contrast, cultures with a fast growth rate yielded lower cell concentrations, buth higher chlorophylla, carotenoid and membrane lipid contents per cell. Changes in polyunsaturated fatty acid distribution were related to differences in algal growth rates. Neutral lipids contained mainly saturated and monounsaturated fatty acids (C18:19) at low growth rates whereas they were enriched in polyunsaturated fatty acids, especially C22:63, at high growth rates. Therefore, it is suggested that the growth rate in continuous cultures be controlled so as to adjust the relative proportions of polyunsaturated fatty acids in lipid classes of the diet meant for larval nutrition.Author for correspondence     

LIGHT/DARK-PHASED CELL DIVISION IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) IN PO4-LIMITED CONTINUOUS CULTURE1

Eugene gracilis Klebs (Z) was grown in a cyclostat (continuous culture on a light/dark cycle) at growth limiting levels of phosphate. Cell division was restricted to the dark period regardless of the proportion of the cells dividing during each 24 h period. Growth rate, as reflected by the amplitude of the cell density oscillation, was correlated with dilution rate. The width of the division gate was analyzed using a phasing index and found to be narrowest at dilution rates where the mean generation time of the cell population was an even multiple of 24 h. The effect was attributed to enhanced phasing of the cell division process by the biological clock of Euglena. Residual phosphate levels in the cyclostat were less than 0.3 μM PO₄ at all submaximal growth rates. Cellular phosphorus concentration increased with dilution rate as described by a hyperbola saturating at D_max= 0.74 day⁻¹ with 8 × 10⁻⁸μM P/cell as the minimum intracellular phosphorus concentration for growth. The results are discussed, in terms of the inherent similarities and differences between a cyclostat and a steady state chemostat, and the advantages of the cyclostat for studies in phytoplankton ecology.     

CIRCADIAN GROWTH RHYTHM IN JUVENILE SPOROPHYTES OF LAMINARIALES (PHAEOPHYTA)1

A circadian rhythm in growth was detected by computer-aided image analysis in 3–4-cm-long, juvenile sporophytes of the kelp species Pterygophora California Rupr. and in seven Laminaria spp. In P. californica, the free-running rhythm occurred in continuous white fluorescent light, had a period of 26 h at 10°or 15°C, and persisted for at least 2 weeks in white or blue light. The rhythm became insignificant in continuous green or red light after 3 cycles. Synchronization by white light-dark regimes, e.g. by 16 h light per day, resulted in an entrained period of 24 h and in a shift of the circadian growth minimum into the middle of the light phase. A morning growth peak represented the decreasing portion of the circadian growth curve, and an evening peak the increasing portion. The circadian growth peak was not visible during the dark phase, because growth rate decreased immediately after the onset of darkness. At night, some growth still occurred at 16 or 12 h light per day, whereas growth stopped completely at 8 h light per day, as in continuous darkness. During 11 days of darkness, the thallus area became reduced by 3.5%, but growth rate recovered in subsequent light–dark cycles, and the circadian growth rhythm reappeared in subsequent continuous light.     

Glucose uptake of Cytophaga johnsonae studied in batch and chemostat culture

The influence of different physiological states on the glucose uptake and mineralization by Cytophaga johnsonae, a freshwater isolate, was examined in batch and chemostat cultures. At different growth rates under glucose limitation in chemostat cultures, different uptake patterns for ¹⁴C labeled glucose were observed. In batch culture and at high growth rates the glucose uptake potential showed a higher maximum velocity and a much lower substrate affinity than at lower growth rates. These findings and the results of short-term labeling patterns could be explained by two different glucose uptake mechanisms which enable the strain to grow efficiently both at high and low substrate concentrations. Substrate specificity studies showed that a structural change of the C-2 atom of the glucose molecule was tolerated by both systems. The consequences of these results for the ecophysiological classification of the Cytophaga group and for the operation of continuous cultures are discussed.     

Dependence of cell yield and specific growth rate ofCandida boidinii on temperature during continuous cultivation

Investigation of the dependence of cell yield ofCandida boidinii in a chemostat and specific growth rate in a turbidostat on temperature was carried out using methanol as substrate. Each of the curves obtained had only one maximum, the optimum temperature for yield (Y _s )and for specific growth rate (μ) being somewhat different. With deviation of temperature from the corresponding optimum the μ value decreases more sharply than that ofY _s . The shape of two-factor dependencesY _s (μ, T) and μ(S, T) is analyzed on the basis of these data and the known dependences ofY _s on μ and μ on the limiting substrate concentrationS. It is shown that the decelerating action of methanol on the growth rate increases at elevated and lower temperatures.     

Photosynthetic carbon dioxide assimilation by Rhodospirillum rubrum

Summary Although Rhodospirillum rubrum, grown photoheterotrophically on malate, assimilates carbon dioxide less rapidly than it does when grown autotrophically, the difference is less marked than previously suggested.The rate of photoassimilation of carbon dioxide varies during batch culture on malate, reaching a maximum at about mid-exponential phase. It also varies with density and growth rate in a turbidostat continuous-flow culture on malate and increases with decreasing growth rate in a chemostat continuous-flow culture growing with limiting malate concentrations.The changing rates of carbon dioxide photoassimilation during photoheterotrophic growth under the various conditions are paralleled by changing activities of ribulose diphosphate carboxylase.Under conditions of maximum carbon dioxide fixation the rate by photoheterotrophic cultures approaches that shown by the bacterium growing autotrophically and is assimilated eight to ten times more slowly than is malate in chemostat cultures.The rate of carbon dioxide fixation also increases to that shown by autotrophic cells when photoheterotrophic cultures are deprived of malate, but without subjecting them to the conditions required for autotrophic growth.     

The use of the chemostat to study the relationship between cell growth rate, viability, and the effect of interferon on L 1210 cells

Mouse leukemia L 1210 cells were cultivated in the chemostat at growth rates ranging from 0.1 day⁻¹ (population doubling time (T_d) 166.3 h) to 2.0 day⁻¹ (T_d 8.3 h). At growth rates of 1.0 day⁻¹ and above, the viability of the steady-state culture was greater than 99%. However, below 1.0 day⁻¹ there was a progressive decrease in the viability of the culture with decreasing growth rate until a minimum growth rate (0.1 day⁻¹) was reached below which steady-state cultures of L 1210 cells could not be established. Interferon treatment had no effect on the viability (>99%) of L 1210 cells cultivated at fast growth rates in the chemostat, whereas at slow growth rates (0.35 day⁻¹) interferon treatment markedly reduced the viability of the culture, even though the percentage increase in the doubling time of interferon-treated cultures was the same for cells cultivated at both fast and slow growth rates. Thus, although interferon is not directly cytotoxic, it can cause cell death by reducing the rate of cell multiplication below the minimum value compatible with viability.     

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.