Rapid media transition: An experimental approach for steady state analysis of metabolic pathways

Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated in fed‐batch mode under non‐stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup—rapid media transition—enables steady state perturbation of metabolism on a time scale of several minutes in parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab‐scale stirred‐tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed‐batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the short‐term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Metabolic flux analysis of hybridoma continuous culture steady state multiplicity.

Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL-1606 cultivated in glutamine-limited steady state chemostats. At the same dilution rate (0.04 h-1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 x 10(5) and 1.36 x 10(6) cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low-efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h-1. The high-efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h-1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h-1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns.     

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.     

Kinetic analysis of batch and continuous culture of Streptococcus cremoris HP1.

The growth of Streptococcus cremoris on a semidefined medium was studied at initial lactose concentrations of 0.2-5.0% in batch culture, and in lactose-limited chemostat cultures at 0.5% lactose. Kinetic analysis of the batch data, using statisitcal techniques, indicated the importance of lactose limitation and lactic acid inhibition of the growth of S. cremoris. A model for the biomass production, lactose utilization, and lactic acid production in batch culture was proposed. In continuous culture, it was found that steady state populations were maintained at higher dilution rates (D = 0.6-0.7 h-1) than the maximum predicted by batch culture (0.56h-1). No evidence for a selection of fast growing mutants was obtained. Copious growth adhering to the walls of the fermentor (i.e. wall growth) occurred very rapidly at higher dilution rates and this undoubtedly affected steady-state growth and wash-out and, as a consequence, the apparent maximum dilution rate.     

Differences in membrane proteins,cyclic AMP levels,and glucose transport between batch and chemostat cultures ofEscherichia coli

Escherichia coli bacteria were grown in minimal-salt medium with glucose as carbon source under either batch or chemostat culture conditions. The physiological state of chemostatgrown bacteria is significantly different from those grown in batch cultures. Differences were measured in outer membrane protein composition,K _m for glucose uptake, and intracellular cyclic AMP levels. Since chemostat growth conditions more closely resemble conditions bacteria encounter in nature, these physiological differences may be significant for bacterial adaptation and survival.     

Comparative study of the growth of Listeria monocytogenes in defined media and demonstration of growth in continuous culture

C.E. JONES, G. SHAMA, P.W. ANDREW, I.S. ROBERTS AND D.JONES. 1995 A basic requirement for physiological studies with Listeria monocytogenes is a chemically defined medium that supports growth of the bacterium in batch and continuous culture. A number of such media have been devised but comparative studies of their efficiency are few and none has been used in continuous culture. Six of the media were compared for their ability to sustain sequential growth of L. monocytogenes in static and aerated batch culture with glucose as sole carbon source. The most suitable, judged on the basis of ease of preparation, growth rate and yield, was that of Trivett and Meyer (1971). This medium was shown to support growth of L. monocytogenes NCTC 7973 in continuous culture in a chemostat. A lytic phenomenon, noted with the same strain under anaerobic conditions and in batch culture in the chemostat, is discussed.     

Studies on the Growth in Culture of Plant Cells: XII. A VERSATILE SYSTEM FOR THE LARGE SCALE BATCH OR CONTINUOUS CULTURE OF PLANT CELL SUSPENSIONS

A versatile large-scale batch culture unit has been developedfor the growth of plant cell suspension cultures. This unithas been modified to permit of intermittent or continuous renewalof culture medium and, in a modified form, incorporated intoopen continuous culture systems of the chemostat and turbidostattype. A fully automatic culture sampler has been incorporatedinto the basic culture unit. The culture systems described havebeen successfully operated using a cell suspension derived fromAcer pseudoplatanus and results are presented demonstratingsynchronous growth in batch culture, prolonged logarithmic increassein cell number under conditions of high aeration and culturemedium renewal, and steady states of growth resulting from automaticregulation of the optical density of the cell suspension andfrom fixed rates of displacement of cell suspension by new medium.The potentialities of the culture systems are discussed in thelight of the experimental results presented.     

Steady state growth space study of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in D-stat cultures

Growth space of Lactococcus lactis subsp. lactis IL1403 was studied at constant growth rate using D-stat cultivation technique. Starting from steady state conditions in a chemostat culture (μ = 0.2 h⁻¹), the pH and/or temperature were continuously changed in the range of 5.4–6.4 and 26–34°C, respectively, followed by the return to the initial environmental conditions. Based on substrate consumption and product formation yields and expression changes of 1,920 genes, it was shown that changes of physiological state were not dependent on the direction of movement (from pH 6.3 to 5.4 or from 5.4 to 6.3), showing that quasi steady state values in D-stat corresponded to the steady state values in chemostats. Relative standard deviation of growth characteristics in triplicate D-stat experiments was below 10%. Continuing the experiment and reestablishing initial growth conditions revealed in average 7% difference (hysteresis) in growth characteristics when comparing chemostat steady state cultures prior and after the change of environmental conditions. Similarly, shifts were also seen at gene expression levels. The large amount of quantitatively reliable data obtained in this study provided a new insight into dynamic properties of bacterial physiology, and can be used for describing the growth space of microorganisms by modeling cell metabolism.     

Inhibition kinetics of phenol degradation from unstable steady-state data

Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady-state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation).The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well-known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady-state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567-576, 1997.     

In vitro gene expression dissected: chemostat surgery for mycobacterium tuberculosis

A unique approach, combining defined and reproducible in vitro models with DNA microarrays, has been developed to study environmental modulation of mycobacterial gene expression. The gene expression profiles of samples of Mycobacterium tuberculosis, from independent chemostat cultures grown under defined and reproducible conditions, were found to be highly correlated. This approach is now being used to study the effect of relevant stimuli, such as limited oxygen availability, on mycobacterial gene expression. A modification of the chemostat culture system, enabling largevolume controlled batch culture, has been developed to study starvation survival. Cultures of M. tuberculosis have been maintained under nutrient-starved conditions for extended periods, with 10(6) - 10(7) bacilli surviving in a culturable state after 100 days. The design of the culture system has made it possible to control the environment and collect multiple time-course samples to study patterns of gene expression. These studies demonstrate that it is possible to perform long-term studies and obtain reproducible expression data using controlled and defined in vitro models.     

Variable-volume continuous cultivation

Equations are developed which describe variable-volume cultivations, including fed-batch systems. An analogy is drawn between the quasi-steady state in variable-volume cultivation and a dynamic steady state in variable-flow, constant-volume chemostat bioreactors. Switching procedures are developed to give a steady-state transition from batch to fed-batch and to continuous operation. In this respect, considerations in the literature have been extended. Computer solutions of the governing differential equations verify the theory and provide insight into the behavior of variable-volume stirred tank reactors. Application of variable-volume cultivation as a tool in investigating growth rates at low substrate levels is suggested. Variable-volume bioreactor systems could be also to obtain controlled dynamic conditions for research or production purposes.     

Studies on the stability of solvent production by Clostridium acetobutylicum in continuous culture

Various methods of continuous flow culture of Clostridium acetobutylicum NCIB 8052 were investigated, with the aim of obtaining prolonged production of acetone and butanol. In ammonia-limited chemostat culture, maximal concentrations of solvents were obtained at pH 5–5 at a relatively high biomass concentration of 1.3–2.0 g/1 dry weight maintained at a dilution rate of 0.06/h. Similar dependence of solvent production on the sustenance of a relatively high cell density was observed in magnesium- or phosphate-limited chemostat cultures. Solvent production was always transient, however, with a shift to production of only acetic and butyric acids being observed after 4–16 volume changes. Longer term solvent production was obtainable under conditions of glucose limitation but the solvent yield was low. Cultivation in a pH-auxostat permitted solvent production in reasonably high yield over at least 70 volume changes with no signs of culture degeneration. Although none of the continuous flow cultures achieved a true steady state, we conclude that turbidostat or pH-auxostat culture are the methods of choice for continuous solvent production by Cl. acetobutylicum NCIB 8052.     

Mathematical investigations of growth of microorganisms in the gradostat

Microbial ecosystems with spatial distribution of substrate (nutrient) supply in the form of gradients have been studied in a laboratory system called the gradostat, which is a series of coupled chemostats. We investigate analytically and numerically a mathematical model, similar to the one for a single chemostat based on Michaelis-Menten kinetics, of the growth of one species of microorganisms in the gradostat in the presence of one limiting substrate and two limiting complementary substrates. Our analysis predicts various patterns of spatial distribution of microorganisms at steady state and suggests further experiments to be performed with the gradostat.This work was supported by the Deutsche Forschungsgemeinschaft     

Yield Constant for Acanthamoeba polyphaga in Batch and Continuous Culture1

ABSTRACT. A two-stage chemostat modified to accommodate the growth of adhesive organisms was used to determine the yield constant, Y, of a representative soil amoeba, Acanthamoeba polyphaga, utilizing as its prey Pseudomonas paucimobilis. The first stage consisted of a glucose-limited bacterial culture in steady state. The second stage consisted of a simplified predator-prey system, nongrowing bacteria serving as the limiting substrate for amoebae. A refined methodology to more accurately determine Y was developed, and Y for Acanthamoeba polyphaga in batch and continuous culture was determined to be 19.1%.     

Kinetic studies of the lactic acid fermentation in batch and continuous cultures

The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose-yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady-state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.     

Microbial film formation: dental plaque deposition on acrylic tiles using continuous culture techniques

A chemostat system has been developed to model the attachment of oral bacteria, and the subsequent development of plaque film, to acrylic surfaces immersed in steady state cultures. Plaque was removed from the teeth and gingival margin of volunteers who refrained from oral hygiene for at least 72 h. Samples were pooled and inoculated into a complex growth medium maintained at 37 degrees C. Glucose-limited continuous culture was established at a dilution rate of 0.05/h and at pH 7.0. Microbiological analysis of the culture indicated that a complex community of oral bacteria was established, typical of that found in dental plaque. Acrylic tiles were immersed in the fermenter through a modified fermenter head and incubated therein for up to 21 d. Scanning electron microscopy showed that either side of the tiles contained a rough and a smooth surface and these initially favoured the attachment of fusiform bacteria, particularly on the rough surface. Cocci attached to those surfaces which were not heavily colonized by the fusiforms and eventually grew into and on the colonial sheets of the fusiforms.     

Control of nitrogenase in chemostat cultures of Rhodobacter capsulatus grown on ammonium at different illuminations

Rhodobacter capsulatus strain 37b4 was grown phototrophically in chemostat cultures with 2 mM of ammonium chloride and 30 mM of malate at a constant dilution rate of 0.075 h^-1. When illumination was raised from 3000 to 30000 lx, steady state biomass levels as well as malate uptake increased linearly with increasing illumination. Yet, in no case external ammonium could be detected in the culture fluid. Specific nitrogenase activity increased by a factor of ten between 3000 and 15000 lx and approached constancy above 15 000 lx. When samples were anaerobically withdrawn from the chemostat and subsequently grown in batch cultures under saturating light conditions, biomass increased to a constant level, independently of the illumination used in the previous chemostat culture. In fact, the specific nitrogen contents of cells were 0.195 and 0.154 (g of N per g of protein) with chemostat cultures adapted to 3000 and 30000 lx, respectively. With the former cultures, specific nitrogen contents decreased to 0.142 g of nitrogen per g of cell protein upon incubation in a batch system. This suggests the existence of free nitrogen compounds in cells of chemostat cultures, the concentrations of which decrease while protein levels increase with increasing energy supply. Intracellular amino acid pools revealed slightly elevated levels of major amino acids in low-light cultures as compared to high-light cultures. On the basis of intracellular levels of ammonium, however, no significant differences could be detected. Since, in addition, malate consumption increased linearly with increasing illumination, it is proposed that light controls nitrogenase in Rhodobacter capsulatus via the C/N ratio, as represented by malate and ammonium consumption, rather than directly.     

Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any give dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.     

Cell weight kinetics simulation in chemostat and batch culture of the rhodophyte Porphyridium cruentum

Experimental observations of cell size variations in the proliferating rhodophyte Porphyridium cruentum cultured under fully controlled conditions showed significant decreases from inoculation to a steady state in the chemostat with 0.23 d(-1) dilution rate and to a minimum in batch, dropping in size by ratios of over 10. To numerically simulate these variations, we assumed that the cell is made up of two categories of components that behave differently during the interphase and mitosis. These have been called essential (EC) and accessory (AC) components. It is assumed that the cell divides once the EC have doubled in size, regardless of the AC's state. The experimental cell weight time courses were correctly simulated by a model of synchronous cell kinetics based on these assumptions. The EC's specific growth rate was 1.5 times that of the whole cell, when no limitation occurred. The increase in cell weight observed during batch cultures after nutrient exhaustion was suitably simulated by assuming that EC growth stops when a limiting nutrient is exhausted. Several parameters characterizing the cell kinetics were defined, particularly the minimum minimorum EC or cell weight (26 and 15 pg for chemostat and batch, respectively), which was influenced by the cultivation method, and the maximum whole cell weight (224 to 244 pg), which depended on the inoculum's age. The influence of culture conditions on the amount of essential and accessory components contained in a cell was examined. A new approach was developed with respect to these compartments to determine the most suitable strategy and conduct a predictive approach for valuable molecule production.