Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Stable production of hyaluronic acid in Streptococcus zooepidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h-1) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci.     

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.     

Growth and enzymological characteristics of a pink-pigmented facultative methylotrophMethylobacterium sp. MB1

Growth characteristics of batch and continuous cultures of the pink facultative methylotrophMethylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C₁-metabolism were measured in a chemostat culture at different dilution rates.     

Characteristics of the constituent substrains of Bacillus popilliae growing in batch and continuous cultures.

Continuous culture of Bacillus popilliae was achieved for the first time in a small chemostat. Initially, variable cell yields during steady-state chemostat growth led to a re-examination of growth rates in batch cultures. B. popilliae NRRL B-2309 and a wild strain were both found to be natural mixtures of three substrains characterized by different growth rates and colony morphologies and varying stability. Selected subcultures grown continuously provided data for three different cell production curves. Cell yields were two to three times greater per unit of medium in continuous than in batch culture, and about 1% of slow-growing chemostat cells formed typical spores.     

Characteristics of the constituent substrains of Bacillus popilliae growing in batch and continuous cultures.

Continuous culture of Bacillus popilliae was achieved for the first time in a small chemostat. Initially, variable cell yields during steady-state chemostat growth led to a re-examination of growth rates in batch cultures. B. popilliae NRRL B-2309 and a wild strain were both found to be natural mixtures of three substrains characterized by different growth rates and colony morphologies and varying stability. Selected subcultures grown continuously provided data for three different cell production curves. Cell yields were two to three times greater per unit of medium in continuous than in batch culture, and about 1% of slow-growing chemostat cells formed typical spores.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

Comparative stability of ethanol production by Escherichia coliKO11 in batch and chemostat culture

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded. Received 13 January 1999/ Accepted in revised form 23 April 1999     

Mechanism of growth limitations in a recombinant strain of Escherichia coli

A recombinant Escherichia coli strain with the cloned gene of restriction endonuclease EcoR-V was studied. The diauxic growth of this strain under batch conditions, the composition of excreted products and the hyperbolic shape of the chemostat growth curve has made it possible to formulate the following hypothesis. At a high flow rate, one of the first reactions in the tricarboxylic acid cycle or a reaction directly preceding the cycle is a limiting step in the metabolism of E. coli cells. The saturation of the limiting reaction with a substrate at a flow rate higher than the critical one may be responsible for the hyperbolic profile of chemostat curves. The above hypothesis was confirmed by experiments conducted under extreme conditions, in which the composition of the growth medium was changed and the temperature was raised so that the cloned protein synthesis was depressed. Under such conditions, the enzyme composition of the cell was fixed and determined by the physiological state of the culture at a time when the temperature was elevated. The hypothesis was also supported by the results obtained in growing the strain with the induced protein synthesis in media to which various compounds were added.     

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.     

Continuous cultivation in a chemostat of the phototrophic procaryote,anacystis nidulans,under nitrogen-limiting conditions

Anacystis nidulans was grown photoautotrophically in a chemostat in the presence of light, air and CO2 as the sole carbon source. Either the amount of the nitrogen source in the medium or light intensity were used as growth-limiting parameters. 1. Cells of high glycogen content obtained by pre-incubation under nitrogen starvation conditions maintained their glycogen content during continuous cultivation. Both growth rate and the amount of cell-mass and of glycogen depended on the nitrate content of the medium and the light intensity. The values for the growth rate, the maximal rates of glycogen synthesis and of cell mass formation were 0.1 h-1, 6 mg/l.h and 17 mg/l.h, respectively. 2. Cells without glycogen which had been transferred from an exponentially growing batch culture to chemostat conditions showed increasing rates of growth and of cell mass formation when the light intensity was increased. A determination of specific values resulted in 0.15 h-1 for growth rate and 23 mg/1.h for cell mass formation. 3. The chemostat apparatus is described in detail.     

Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus sciuri biofilms

The adhesion and subsequent development of Listeria monocytogenes on stainless steel was studied in the absence and in the presence of a Staphylococcus sciuri biofilm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day biofilms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day biofilms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3.5 to 35% of the stainless steel depending on the growth media. This suggests that extracellular substances rather than cell-to-cell interactions were involved in the decreased adhesion. After 3 days of culture, Staphylococcus biofilms prevented the adherent L. monocytogenes population from increasing within the biofilm, leading to an average logarithmic cfu difference of 0.9-2.7 between the pure and mixed culture. A competition for nutrients by Staph. sciuri was observed in one of the three media. A role for extracellular polysaccharides produced by the Staphylococcus biofilm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and biofilm phase is hypothesized. A higher proportion of L. monocytogenes cells was observed in the planktonic phase in mixed cultures, suggesting that the extracellular substances produced by Staph sciuri biofilms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and biofilm populations. In addition, co-cultures of L. monocytogenes and Staph. sciuri in broth showed competition for nutrients for Staph. sciuri in one of the three media.     

Kinetics of Streptomyces baarnensis growth and antibiotic synthesis in batch and dialysis cultures

The kinetics of biomass and antibiotic formation in batch and dialysis culture of Streptomyces baarnensis at various initial concentrations limiting the substrate growth (glucose) has been studied. The antibiotic substances were synthesized by actively growing culture, its concentration in the cultural media was maximum in the log-phase. In continuous dialysis culture on the background of biomass lianer growth in the course of time the constant antibiotic concentration in the media proportional to the glucose input concentration has been established. The inactivation (decomposition) of antibiotic was immediately initiated after discontinuation of substrate supply and followed first kinetics order. Observed features were used for construction of kinetical model of antibiotic biosynthesis. A conclusion has been made that the dialysis culture gives opportunity for more effective antibiotic synthesis as compared with the batch one.     

Influence of growth conditions on diverse polysaccharide production by Campylobacter jejuni

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide. The present study was undertaken to determine the forms of polysaccharide-related compounds (PRCs) produced by C. jejuni and the culture conditions influencing their production. Expression of polysaccharides by C. jejuni was influenced by culture medium composition and growth phase. In addition to the production of lipooligosaccharide and capsular polysaccharide, a previously undescribed polysaccharide, not related to capsular polysaccharide, was shown to occur in C. jejuni in batch liquid and chemostat cultures. Thus, a variety of PRCs are produced by C. jejuni, and this should be considered when growing the bacterium in vitro for pathogenesis studies.     

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.     

Physiological and biochemical studies on psychrotolerance in Listeria monocytogenes

Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat. It was found that when grown in continuous culture at a dilution rate of 0·02 h⁻¹ L. monocytogenes had a lower proportion of anteiso -17:0, and a higher proportion of anteiso -15:0, and smaller chain fatty acids when grown at 10°C compared to 30°C. A previously unreported glycolipid was only seen after growth at low temperature. Growth temperature had no effect on the rate of glucose uptake.     

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.     

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Summary The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

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).