High cell density continuous culture ofEscherichia coli producing penicillin acylase

Summary We studied high cell density continuous culture (HDCC) of a recombinant (E. coliHB101 (pPAKS2)) and a mutant (E. coli ATCC 11105) strains ofE. coli producing penicillin acylase(PA). Using pure oxygen, high cell density up to 95 g/l was obtained without significant inhibition by a main byproduct, acetic acid. The operation was simple and productivity was several times higher than those of conventional batch and continuous culture. Dissolved oxygen level and CO₂ concentration were important variables, and glucose concentration was naturally regulated in HDCC.     

Oscillatory behaviour control in a continuous culture under double-substrate limitation

The paper discusses the possibility of using a mixture of two growth limiting substrates to induce or eliminate self-sustained oscillations in a continuous culture process. The proportion of both substrates in the mixture is treated as a new control variable. The presented approach is based on the assumption that the oscillatory behaviour occurs for selected substrates in some range of dilution rates. Because a double-substrate limitation may occur, the analysis is performed for two fundamental substrate utilization patterns: simultaneous consumption and diauxic growth. By using model simulations and bifurcation analysis, we show that an appropriate proportion of two substrates in the mixture allows for the control of the oscillatory behaviour.     

Physiological constraints in increasing biomass concentration ofEscherichia coli B in fed-batch culture

Summary Fed-batch culture ofE.coli B was carried out to obtain high concentration of biomass. After requirement of oxygen was met by sparging the pure oxygen, physiological constraints were delineated. High partial pressure of CO₂ caused the decrease of the maximum specific growth rate, whereas fermentative byproducts caused the decrease of biomass yield as well as the maximum specific growth rate.     

DNA synthesis in a synchronized culture ofEscherichia coli 15 TAUbar after continuous and interrupted thymine starvation

After transfer to a thymine-containing medium the DNA synthesis did not increase with increasing intervals of thymine starvation. On the other hand, the starvation interrupted at regular intervals by 5 min thymine pulses resulted in an increased DNA synthesis. Induction of a bacteriophage which is prevented by the pulses is discussed as a possible reason for the observed difference in kinetics of the DNA synthesis following continuous and interrupted thymine starvation. Turbidity of the culture increased roughly three times, both during the continuous and the interrupted thymine starvation. The increase of the turbidity after prolonged interrupted starvation was lower than that which would correspond to the observed increase of the DNA synthesis according to the hypothesis of a critical mass of the cell resulting in the initiation (Donachie, 1968).     

Adaptation ofEscherichia coli to chlortetracycline during continuous cultivation

При трехступенчатой проточной культивации штаммаЕscherichia coli SZÚ В питательной среде с повышениением концентраци хлортетрациклина быстро наступает адаптаця к этому антибитику. Процесс адаптации протекает интенсивнее у ?изиологически более молодых клеток в первых двух ступенях культивации, но достигает максимума в конечной стадии развития клеточой поиуляции. Полученные результаты говораят микроорганизмов к антибиотикам, а, быть может, и к другим ингибиторам.     

Growth limitation of submerged aquatic macrophytes by inorganic carbon

1. This study determined the effects of CO₂ and HCO₃- enrichment on in situ growth of two submerged macrophytes, Elodea canadensis and Callitriche cophocarpa, in two Danish lakes: Lake Hampen and Lake Væng. Lake Hampen is an oligotrophic low-alkaline lake (0.4 meq ^?1) and Lake Væng is mesotrophic with an alkalinity of 1.1 meq 1-^?1. In Lake Hampen experiments were carried out throughout the growth season, whereas experiments in Lake Væng were restricted to late summer. The CO₂ and HCO₃-enrichment procedures used increased the concentration of free-CO₂ by 500–1000 μM and the concentration of HCO₃- by about 80 μM. 2. The concentration of free-CO₂ in Lake Hampen was about five times atmospheric equilibrium concentration (55 μM) in early summer declining to virtually zero at the end of summer. 3. Under ambient conditions Callitriche, which is restricted to CO₂ use, was unable to grow and survive in both lakes. In contrast, Elodea, which has the potential to use HCO₃- in photosynthesis, grew at rates varying from 0.046 to 0.080 day^?1 over the season. 4. Under CO₂ enrichment the growth rate of Callitriche varied from 0.089 to 0.124 day^?1 and for Elodea from 0.076 to 0.117 day^?1 over the season. Enrichment with HCO₃-affected Elodea only and only to a limited extent. This may be a result of insufficient increase in [HCO₃-] upon enrichment or to a limited capacity of the plants to take up HCO₃-. 5. The substantial stimulation of in situ growth of Elodea and Callitriche by enhanced concentrations of free-CO₂ shows that inorganic carbon is an important determinant of growth of submerged macrophytes and that inorganic carbon limitation of in situ growth may be a common phenomenon in nature, even in lakes with an alkalinity as high a 1 meq 1-^?1. Inorganic carbon, however, is only one of many parameters important for growth, and the growth rates of Elodea at both ambient and high free-CO₂ were closely coupled to day length and photon irradiance, indicating that light had an ultimate control on growth.     

Fed-batch culture ofEscherichia coli W by exponential feeding of sucrose as a carbon source

Fed-batch cultures ofEscherichia coli W were carried out in a chemically defined medium containing sucrose as a sole carbon source. The substrate feed rate was changed every 30 minutes based on the immediate off-line analysis of medium. When the target specific growth rate was set at 0.15 and 0.2 h⁻¹, the final cell concentrations of 105 and 110 g dry cell weight/L were obtained in 25 and 20 h, respectively.     

Growth and phosphate uptake of a high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6 in continuous culture

A high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6, was grown in a chemostat under glucose-limitation. Two different growth patterns at steady state with various dilution rates were obtained. In one case, cells having a coccus shape tended to washout at a low dilution rate, 0.2 (h(-1)). In another, cells with a rod shape grew faster and gave a good steady-state growth at a dilution rate of 0.4. Such a close relationship between growth rate and cell morphology was found both in continuous and batch cultures. The amount of phosphate uptake per cell mass was almost constant irrespective of the dilution rate, but the rate of the uptake was maximum at about the dilution rate of 0.4. A clone of PAB-6 was isolated from the continuous culture with high dilution rate and had maximum specific growth rate of 0.7 in a simple glucosesalt medium.     

Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture ofEscherichia coli in a non-selective medium

Summary Experimental results were obtained withEscherichia coli C600 galK (GAPDH), a genetically engineered strain that synthetizes a large quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (80 % of the soluble proteins). Data concerning the stability of plasmid-containing cells and gene expression as a function of dilution rate have been obtained in continuous cultures. Contrary to other studies, our results show a clear indication that the rate of the recombinant activity was dependent on dilution rate. The results support the finding that the apparent stability of the plasmid decreases with dilution rate.     

Effect of cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation

Summary An overflow filtration unit for cell recycle with Clostridium acetobutylicum was developed. A cellulose-triacetate ultrafiltration membrane with a cut-off volume of 20 000 MW was found to work best. C. acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH value of 4.4 with cell recycle, the cell dry weight in the culture vessel reached 13.1 g/l at a dilution rate of D=0.10 h^-1 and 37°C. 377 mM of glucose were fermented to 190 mM butanol, 116.2 mM acetone and 25.8 mM ethanol. Total acids were 47.6 mM. The butanol productivity was 1.41 g/l/h. At a dilution rate of 0.40 h^-1 the butanol productivity was increased to 4.1 g/l/h but glucose consumption was decreased to 285 mM and butanol, acetone and ethanol production to 138.2, 97.5, 16.5 mM, respectively.     

Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture

A system for growing Geobacter sulfurreducens under anaerobic conditions in chemostats was developed in order to study the physiology of this organism under conditions that might more closely approximate those found in the subsurface than batch cultures. Geobacter sulfurreducens could be cultured under acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was threefold higher with fumarate as the electron acceptor than with Fe(III), despite the lower mid-point potential of the fumarate/succinate redox couple. When growth was limited by availability of fumarate, high steady-state concentrations were detected, suggesting that fumarate is unlikely to be an important electron acceptor in sedimentary environments. The half-saturation constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that the growth of Geobacter species is likely to be acetate limited in most subsurface sediments, but that when millimolar quantities of acetate are added to the subsurface in order to promote the growth of Geobacter for bioremediation applications, this should be enough to overcome any acetate limitations. When the availability of electron acceptors, rather than acetate, limited growth, G. sulfurreducens was less efficient in incorporating acetate into biomass but had higher respiration rates, a desirable physiological characteristic when adding acetate to stimulate the activity of Geobacter species during in situ uranium bioremediation. These results demonstrate that the ability to study the growth of G. sulfurreducens under steady-state conditions can provide insights into its physiological characteristics that have relevance for its activity in a diversity of sedimentary environments.     

Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Macromolecular composition of a Cellulomonas sp. cultivated in continuous culture under glucose and zinc limitation.

The mutant strain of Cellulomonas sp. (ATCC 21399) was cultivated under glucose and zinc limitation at a variety of growth rates in continuous culture. The growth characteristics and macromolecular composition of the population varied with the limitation imposed and the growth rate. Glucose- and zinc-limited cultures maintained a constant relative protein content. The relative ribonucleic acid content increased, whereas the carbohydrate and deoxyribonucleic acid contents decreased with an increase in the population growth rate in glucose-limited cultures. Free unbound lipid remained constant. The maximum population growth rate in zinc-limited cultures was directly proportional to the zinc concentration and demonstrated a traditional steady-state function. The nucleic acid content increased with increased growth rate; however, the relative nucleic acid content was significantly depressed when compared to glucose limited cells. This manner of cultivation may prove to be a useful tool for the production of single cell protein with lowered nucleic acid content and the elucidation of micronutrient involvement in growth-related processes.     

Macromolecular composition of a Cellulomonas sp. cultivated in continuous culture under glucose and zinc limitation.

The mutant strain of Cellulomonas sp. (ATCC 21399) was cultivated under glucose and zinc limitation at a variety of growth rates in continuous culture. The growth characteristics and macromolecular composition of the population varied with the limitation imposed and the growth rate. Glucose- and zinc-limited cultures maintained a constant relative protein content. The relative ribonucleic acid content increased, whereas the carbohydrate and deoxyribonucleic acid contents decreased with an increase in the population growth rate in glucose-limited cultures. Free unbound lipid remained constant. The maximum population growth rate in zinc-limited cultures was directly proportional to the zinc concentration and demonstrated a traditional steady-state function. The nucleic acid content increased with increased growth rate; however, the relative nucleic acid content was significantly depressed when compared to glucose limited cells. This manner of cultivation may prove to be a useful tool for the production of single cell protein with lowered nucleic acid content and the elucidation of micronutrient involvement in growth-related processes.     

Continuous culture ofEscherichia coli for extracellular production of recombinant amylase

Summary A recombinantEscherichia coli, grown in continuous culture, expressed aBacillus stearothermophilus -amylase at 100-fold higher activities than theB. stearothermophilus itself. Excretion of the -amylase to the supernatant was shown and found to be independent of the growth rate of the organism. Eleven to eighteen percent of the -amylase was found in the supernatant. Dilution rates, or cell growth rates, ranging from 0.1 to 1.0 hours^–1 were shown not to affect the compartmentation of the amylase and -galactosidase.     

Growth kinetics of a bacteriophage in continuous culture

Lytic coliphage Qbeta was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qbeta infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. (c) 1996 John Wiley & Sons, Inc.