Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady-state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady-state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. (c) 1992 John Wiley & Sons, Inc.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum free fed batch production of IgM

Summary Fed batch cultivation of a murine hybridoma secreting IgM in a serum free medium was successfully attempted. Cell growth and IgM productivity remained high for over a month, and compared well to a similar fed batch culture in medium supplemented with 10% fetal calf serum. The average specific secretion rates of antibody in both media were the same. Sufficient inoculum cell density was crucial to the establishment of a viable culture in serum free medium for the cell line, NS6.3, used.     

Investigation of metabolic variability observed in extended fed batch cell culture

A 13‐day fed‐batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell‐specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1519–1527, 2013     

Application of scale-down experiments in the study of kinetics of oxytetracycline biosynthesis

Scale-down experiments in antibiotic biosynthesis were performed by transferring the corresponding amounts of fermentation broth from industrial to laboratory and pilot-plant fermentors where the cultivation process was continued at different cultivation conditions. A previously proposed mathematical model was used to explain the experimental results. The effects of temperature, agitation-aeration intensity, and medium addition during the process were investigated. Computer simulation data were fitted to the experimental data, and good agreement was found. As a consequence of increasing temperature up to 37 degrees C, increases in the specific growth and autolysis rates as well as the specific rates of antibiotic synthesis and carbohydrate utilization were in evidence. Temperature increases of up to 40 degrees C caused a lower oxytetracycline yield. The effect of increased oxygen transfer rate on oxytetracycline biosynthesis was more pronounced at higher temperatures than at lower cultivation temperatures. Culture differentiation (strain segregation) was also studied; it was found that the increased cultivation temperature could be favorable for the growth of biomass active in oxytetracycline biosynthesis. Results of experiments at the pilot-plant scale showed that fed batch and repeated fed batch cultures could be successfully applied and the period of intensive antibiotic synthesis could be prolonged significantly.     

Effects of culture temperature shift and light-dark time cycle on the cellular sugar accumulation of Chlorella pyrenoidosa

We investigated the effect of culture temperature on the maximum specific growth rate and the cellular sugar accumulation, and the effect of a temperature shift on the sugar accumulation of Chlorella pyrenoidosa cells in a batch culture system. Increase in temperature below 30?°C appeared to correlate with increase in the maximum specific growth rate, on the contrary the cellular sugar content showed a reverse tendency against temperature. We attempted to utilize this tendency for improving sugar productivity in Chlorella. First, we cultured Chlorella at 28?°C during the logarithmic growth phase to obtain a high specific growth rate. The culture temperature was then shifted from 28?°C to 22?°C at the late logarithmic growth phase in order to reduce the specific growth rate and enhance the cellular sugar accumulation. As a result, we obtained a 15% increase in sugar production over that obtained by cultivation at 28?°C throughout the culture. We also investigated the effect of light-dark time cycle on the sugar productivity and found that this operating variable did not affect the cellular sugar content but influenced the final cell concentration. Among the examined light-dark time cycles, maximum sugar productivity was obtained in the case of 12?h light and 12?h dark period.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

Kinetic modeling of the autotrophic growth of Pavlova lutheri: study of the combined influence of light and temperature

The optimization and control of biochemical processes require the previous establishment of mathematical models that can describe the effect of process variables on their actual kinetics. Environmental temperature is a modulating factor to which the algal cells respond continuously by adjusting their rates of cellular reactions, their nutritional requirements, and, consequently, their biomass composition. Light intensity is an exhaustible resource, indispensable to autotrophic organisms. The effects of light intensity and temperature on growth of the microalga Pavlova lutheri, which have hardly been considered to date in a simultaneous fashion, were experimentally assessed using a factorial experimental design; in this way, the effects of each variable independently and their interactions could be quantified, using maximum biomass (X(max)) or maximum specific growth rate (mu(max)) as objective functions. The preliminary results produced indicated that light intensity plays a more important role on mu(max) than temperature; in the case of X(max), both temperature and, to a lesser extent, light intensity do apparently play a role. The highest values of X(max) were associated with low temperatures and high light intensities; a similar behavior could be observed for mu(max) concerning light intensity, although the dependency on temperature did not seem to be as important. A more complex mechanistic model was then postulated, incorporating light and temperature as input variables, which was successfully fitted to the experimental data generated during batch cultivation of P. lutheri.     

Reexamination of the Acid growth theory of auxin action

Some crucial arguments against the acid growth theory of auxin action (U Kutschera, P Schopfer [1985] Planta 163: 483-493) have been reinvestigated by simultaneous measurements of proton fluxes and growth of maize (Zea mays L.) coleoptiles. Special care was taken to obtain a mild, effective, and reproducible abrasion of the cuticle. Proton secretion rates were determined in a computer-controlled pH-stat. In some experiments, equilibrium pH was measured. Growth rates were determined simultaneously in the same vessel using a transducer-type auxanometer. It was found that (a) the timing of auxin and fusicoccin-induced (FC) proton secretion and growth matches well, (b) the equilibrum external pHs in the presence of IAA and FC are lower than previously recorded and below the so-called `threshold-pH,' (c) neutral or alkaline unbuffered solutions partially inhibit FC and IAA-induced growth in a similar manner, (d) the action of pH, FC, and IAA on growth are not additive. It is concluded that the acid-growth-theory correctly describes incidents taking place in the early phases of auxin-induced growth.     

The effect of temperature and growth rate on the susceptibility of Listeria monocytogenes to environmental stress conditions

R.A. PATCHETT, N. WATSON, P.S. FERNANDEZ AND R.G. KROLL. 1996. The effect of growth temperature and growth rate on the susceptibility to heat and pH stress were investigated in Listeria monocytogenes grown in continuous culture where these two growth variables could be varied independently of each other, and in batch culture. After growth at 30°C or 10°C at constant growth rate, or at 30°C at different growth rates, cells did not differ in their resistance to heat at 55°C. Cells grown at 30°C were more resistant to acid stress at pH 2.5 than cells grown at the same growth rates at 10°C. Cells grown at low growth rate at 30°C gave greater resistance to acid stress than those grown at high growth rate. Growth temperature and growth rate had independent effects on the susceptibility of L. monocytogenes to acid stress conditions. This may have implications for the survival of L. monocytogenes in acidic foods.     

Element content of Ochromonas danica: a replicated chemostat study controlling the growth rate and temperature

Ecological stoichiometry focuses on the balance between multiple nutrient elements in resources and in consumers of those resources. The major consumers of bacteria in aquatic food webs are heterotrophic and mixotrophic nanoflagellates. Despite the importance of this consumer-resource interaction to understanding nutrient dynamics in the aquatic food web, few data are available addressing the element stoichiometry of flagellate consumers. Ochromonas danica, a mixotrophic bacterivore, was used as a model organism to study the relationships among temperature, growth rate and element stoichiometry. Ochromonas danica was grown in chemostats at dilution rates ranging between 0.03 and 0.10 h(-1) and temperatures ranging between 15 and 28 °C. Cells accumulated elements as interactive functions of temperature and growth rate, with the highest element concentrations corresponding to cells grown at a low temperature and high growth rates. The highest concentrations of elements were associated with small cells. Temperature and growth rate affected the element stoichiometry (as C:N, C:P and N:P) of O. danica in a complex manner, but the growth rate had a greater effect on ratios than did temperature.     

Continuous cultivation of recombinant Escherichia coli: Existence of an optimum dilution rate for maximum plasmid and gene product concentration

Growth yield factors, plasmid stability, cellular plasmid content, and cloned gene product activity for Escherichia coli HB101 containing plasmid pDM246 were measured at several dilution rates in continuous culture. Cell mass yield per mass of glucose consumed declined with increasing dilution rate. There was no evidence of plasmid segregational instability in any experiments, none of which employed selective medium. Plasmid content per cell varied with population-specific growth rate as observed in earlier batch experiments with the same strain. Plasmid content declined with increasing specific growth rate following indication of a maximum number of plasmids per cell at specific growth rates of ca. 0.3 h(-1). Cloned gene product (beta-lactamase) activity exhibited a sharp maximum with respect to dilution rate in continuous culture. Qualitatively different results were observed in previous experiments in batch cultivation in which specific growth rate changes were effected by altering medium composition.     

Coupling of transcription termination to RNAi

Microbes require multiple essential elements that they acquire from the environment independently. Here we investigate how microbial stoichiometry and uptake rates depend on the conditions in which they grow. We modify a recent model of growth based on a multinutrient extension of the Droop model to allow a trade-off between ability to acquire two essential resources. In a static analysis, we show that the optimal allocation strategy is the one that results in co-limitation by both nutrients. We then add a dynamic equation to model the physiological acclimation uptake rates in changing conditions. This dynamic model predicts that the response of organismal stoichiometry to nutrient supply ratio can vary over time. The response of organismal stoichiometry and growth rate to a nutrient pulse depends on the speed at which cells adapt their uptake rates. In a variable environment, very fast or very slow acclimation may be better strategies than intermediate speed acclimation. We suggest experimental tests of the model and avenues for future model development.     

Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature

Microalgal lipid is a promising feedstock for biodiesel production. Effect of cultivation temperature on the growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. LX1 was studied. Scenedesmus sp. LX1 could grow in a wide range of temperature (10～30°C), and the growth activation energy was 49.3 kJ·mol(-1). The optimal temperature to produce microalgal biomass and lipid was 20°C, and after 15 days of batch cultivation the productivities of 313.3 g biomass·(g P)(-1), 112 g lipid (g P)(-1) and 14.7 g TAGs·(g P)(-1) were obtained. The content of polyunsaturated fatty acids decreased with the increase of cultivation temperature. The reactive oxygen species (ROS) levels at 10°C and 20°C were higher than that under higher temperature. For the first time the cultivation temperature, ROS level, specific growth rate and lipid content per microalgal biomass were correlated together.     

Determination of diauxic lag in continuous culture

A procedure was developed to characterize diauxic lag of bacteria switching between electron acceptors in continuous culture. In this procedure, a virtual batch growth curve is developed by integrating the time-dependent net specific growth rates of bacteria observed under continuous flow conditions. The length of diauxic lag and the highest net specific growth rate following lag are conveniently estimated from the virtual batch curve. The procedure was found to give reproducible diauxic lag lengths and highest net specific growth rates when applied to experimental data from replicate continuous culture trials.     

Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.     

Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth-rate-limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate-starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.