Division competence in Tetrahymena: determination of minimum cell volume and rate of nutrient uptake

Cell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased. When the cell volume increase per time per cell in a given medium is expressed as a function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 microns 3 for T. pyriformis at 28 degrees C. The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC. The increase in cell volume per unit of time for a given cell depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis-Menten-like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2.s. The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2.s) increases the ability of growth in more concentrated media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous ethanol production from pineapple cannery waste using immobilized yeast cells

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).     

Morphological properties ofSchizosaccharomyces pombe in a continuous culture

The dilution rate (specific growth rate) influences the mean volume ofS. pombe cells and their length—width ratio. The degree of asymmetry of the cell division is also a function of the dilution rare. The difference between the volume and length of both sibs of the dividing cell is significant under conditions of substrate limitation, whereas with excess substrate both parts of the cell are identical in volume.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Microbial sensor system which usesMethylomonas sp. for the determination of methane

Summary Pseudomonas putida (ATCC 111 72) was studied in a continuous culture at various dilution rates with asparagine as the sole carbon source and limiting factor. Under the experimental conditions applied, a considerable number of the cells became attached to the fermentor walls and equipment. The viable count of the attached cells was of the same magnitude as those in suspension. The following steady-state characteristics were obtained: The cell-mass (OD₆₂₀ and dry weight) versus dilution rate (D) had maxima at 0.63 and 1.1 h⁻¹. The corresponding plot of viable count had a minimum at 0.94 h⁻¹ whereafter it reached a maximum at 1.3 h⁻¹. Largest yield coefficient obtained was 0.44 g dry weight/g asparagine (D=1.1 h⁻¹). The productivity of the culture increased with D up to 1.1 h⁻¹, which is far above the D corresponding to the maximum specific growth rate (^μmax) of a batch culture (0.59 h⁻¹). The cell mass was not completly washed-out of the fermentor even at a D of 2.2 h⁻¹. The influence of attached growth for the steady-state characteristics, and the significance of the results in relation to chemostate as an instrument for testing environmental factors, are discussed. It is suggested that the attached cells had a significantly higher (^μmax) value than the suspended ones.     

Surface-limited growth: a model for the synchronization of a growing bacterial culture through periodic starvation

This article analyses the Surface-Limited Growth Model put forward to explain the very tight synchrony, over more than ten division cycles, obtained experimentally by subjecting a growing bacterial culture to alternating periods of starvation and dilution, using inorganic phosphate as the limiting substrate. The Model states that when an essential nutrient is in limited supply, the rate of growth of an individual cell will be proportional to its surface area (and the current concentration of the limiting substance) rather than to its volume. This decrease in dimensionality from volume to surface is expected to favor the smaller cells and so result ultimately in a narrower size distribution. The Surface-Limited Growth Model deals with cell growth under unusual nutritional conditions, and its predictions depend on how the cell replication cycle is assumed to behave under these same circumstances. Two alternatives are considered: the volume at which cells divide is the same during the starvation phase as during steady-state exponential growth, and the cells adjust immediately to the changing growth rate. In the latter case, we have tested both C + D constant with time and C + D variable (where C + D is the time between initiation of chromosome replication and the corresponding cell division), the incremental value at any instant being computed separately for each individual cell from its current effective growth rate. The simulation results are of two sorts depending on the auxiliary assumptions used. Either the dilution-starvation cycles have no effect whatsoever on the cell volume distribution, or the width of the distribution decreases gradually with time, approaching zero slowly and asymptotically, but the mean cell volume decreases as well--directly contradicting experimental observations. We conclude that the Surface-Limited Growth Model is incapable of explaining the synchronization of cells by periodic starvation of a growing bacterial culture.     

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

Physiological and Morphological Changes Induced by Nutrient Limitation of Pseudomonas fluorescens 378 in Continuous Culture

Pseudomonas fluorescens 378 was studied in continuous culture at a dilution rate of 0.05 or 0.15 h⁻¹ and under a limitation of carbon/energy, nitrogen, phosphorus, iron(III), or oxygen. Cultures were examined for nutritional consumption, production of biosurfactant AP-6 and lipase, and electron microscopy morphology. Morphological features were lysis and plasmolysis of the cells, vacuoles in the cells, granules in cell nuclei, and DNA coagulation during transmission electron microscopy preparation. Biosurfactant and lipase production were lost after 8 to 15 retention times, but under iron limitation and at low dilution rate they were maintained for more than 30 retention times. Consumption of nutrients varied between different cultures. Between 2.4 and 6.0 g of succinic acid per g (dry weight) was consumed; the highest value was obtained under phosphorus limitation. The uptake of nitrogen was mostly about 0.16 g/g (dry weight), and that of phosphorus varied between 13 and 58 mg/g (dry weight). Phosphorus-limited cells reduced their phosphorus consumption by at least 50% compared with other limitations. Cell morphology varied among different cultures. Up to 25% cell lysis occurred at the higher dilution rate. The frequencies of plasmolysis varied between 0 and 85%. Granules in nuclei were found in 65 to 100% of the cells. Vacuoles appeared mostly in low numbers, but at the lower dilution rate under phosphorus or iron limitation the frequencies increased to between 25 and 85%. At high dilution rate, the DNA coagulated in 30 to 70% of the cells. Multivariate data analysis demonstrated a general difference between the two tested dilution rates; i.e., both nutritional and morphological features differed more between the two tested dilution rates than between the different limitations. Cultures at the lower dilution rate changed more with time; this was especially pronounced for phosphorus or iron limitation. The data analysis also showed a correlation between plasmolysis or vacuoles in the cells and an increased carbon uptake under phosphorus limitation.     

Significance of the surface of immobilized Saccharomyces cerevisiae cells in ethanolic fermentation

S. cerevisiae cells immobilized in alginate beads show in many cases an increase of mean single cell volume during long-time fermentations (successive batch cycles). The biomass loading capacity of the gel beads is characterized by a maximum volume but not by a maximum number of cells occupying the gel volume. In our system this loading capacity, i.e. the maximum volume fraction of cells per volume of beads, amounted to about 0.54. As a more important result it must be stated that the specific product formation rate in the case of fermentations negligibly influenced by diffusion hindrance is related to the total surface of the viable cells but not to their total number, total volume or total dry weight.     

Continuous culture of RPMI 8226 human cells

Continuous culture of RPMI 8226 human hematopoietic cells was performed. The viable cell number and glucose, lactate and ammonium concentrations became constant within 3–4 days at a constant dilution rate. The viable cell number decreased at low and high dilution rates. The growth and product yields slightly depended on the dilution rate, except for product yield for lactate based on cell number. Growth characteristics of these cells at various dilution rates could be expressed by equations considering the maintenance energy in growth yield. Maximum specific growth rate could be evaluated from the wash-out profile and the known inhibition constants.     

High yeast concentration in continuous fermentation with cell recycle obtained by tangential microfiltration

Summary Continuous fermentation fed by 150 kg/m³ of glucose with total cell recycling by tangential microfiltration enabled yeasts concentration of 300 kg/m³ (dry weight) to be reached with a dilution rate of 0,5h^–1 and a cell viability greater than 75%. The stability of this system was tested for 50 residence times of the permeate. The method can be used both for the production of cell concentrates and for high rates of metabolite production.Nomenclature D. W. dry weight - X_T (kg/m³) total cell concentration D.W. - X_V (kg/m³) viable cell concentration D.W. - V viability of cell culture in per cent of total cell concentration - S (kg/m³) glucose concentration - P (kg/m³) ethanol concentration - D (h) dilution rate - R (kg/kg) fermentation yield - (h) specific growth rate - vp(kg/kg/h) specific alcohol production rate - (m) yeast size - (kg/kg) kg of intracellular water per kg of dry cells     

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates)

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.     

Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65 degrees C

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 degrees C. A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1). At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1). The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1). Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)). Additionally, yield coefficients for oxygen and ammonium are reported.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Influence of Ammonium on the Growth and Development of Suspension Cultures of Pauľs Scarlet Rose

In this work as in previous studies from this laboratory it was demonstrated that the presence of a trace amount of NH₄⁺ (72.8 μmol) stimulated the growth of Pau?s Scarlet Rose on a defined medium containing NO₃^? (1920 μmol) as the only other source of nitrogen. A kinetic analysis of several growth parameters showed that the rate of increase of dry weight, fresh weight, cell number, and cell volume were greater during early stages of growth (days 0–8) when NH₄⁺ was provided. During later stages (days 8–14) this relationship between the two cultures did not hold. The cells provided NH₄⁺ continued to increase in fresh weight and cell volume, but the cells which were not provided NH₄⁺ had a greater rate of dry weight and cell number increase. These differences led to 14-day-old cultures which were approximately equal in dry weight and cell number but differed by a factor of 2 in fresh weight. The presence of NH₄⁺ speeded up the development and growth of the cells.     

Air-bubbling, hollow-fiber reactor with cell bleeding and cross-flow filtration

Continuous asymmetric reduction of dyhydrooxoisophorone (DOIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) was achieved by a thermophilic bacterium Bacillus stearothermophilus NK86-0151. Three reactors were used: an air-bubbling hollow-fiber reactor with cell bleeding and cross-flow filtration, an air-lift reactor, and a CSTR with PAA immobilized cells. The maximum cell concentration of 11.1 g dry wt L(-1) was obtained in an air-bubbling hollow-fiber reactor, while in the other reactors the cell densities were between 3.5 and 4.1 g dry wt L(-1) The optimum bleed ratio was 0.1 at the dilution rate 0.3 h(-1) in the hollow-fiber reactor. The highest viable cell concentration was maintained in the dilution range of 0.4-0.7 h(-1) by a combination of proper cell bleeding and cross-flow filtration. The maximum volumetric productivity of 4-HTMCH reached 826 mg L(-1) h(-1) at the dilution rate 0.54 h(-1). This value was 4 and 2 times higher than those in the air-lift reactor and CSTR, respectively. The increasing viable cell concentration increased the volumetric productivity of 4-HTMCH. A cell free product solution was continuously obtained by cross-flow filtration.