Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline,glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg^–1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na⁺ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h^–1 and 0.4 h^–1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.     

Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

Growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by Corynebacterium glutamicum

The organophosphorous pesticide, demeton-S-methyl was transformed byCorynebacterium glutamicum in co-metabolism with more readilydegradable substrates. Glucose, acetate and fructose were tested as growth substrates, and the highest demeton-S-methyl biotransformation average rate (0.78 mg l^-1 h^-1) and maximum instantaneous rate (1.4 mg l^-1 h^-1) were achieved on fructose. This higher efficiency seems to be linked to the atypical behavior of C. glutamicum grown on fructose, characterized by a prolonged period of accelerating growth instead of a constant growth rate observed on glucose or acetate. More precisely, for growth rates in the 0.1–0.4 h^-1 range, a direct coupling between the specific demeton-S-methyl consumption rate and the growth rate was demonstrated on fructose during batch –, steady state continuous – or continuous cultures with a controlled transient growth rate (accelerostat technology). The demeton-S-methyl biotransformation was more favoured during an acceleration phase of the growth rate.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Phenotypic characterization of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under osmotic stress conditions using elementary mode analysis

Corynebacterium glutamicum, a soil bacterium, is used to produce amino acids such as lysine and glutamate. C. glutamicum is often exposed to osmolality changes in its medium, and the bacterium has therefore evolved several adaptive response mechanisms to overcome them. In this study we quantify the metabolic response of C. glutamicum under osmotic stress using elementary mode analysis (EMA). Further, we obtain the optimal phenotypic space for the synthesis of lysine and formation of biomass. The analysis demonstrated that with increasing osmotic stress, the flux towards trehalose formation and energy-generating pathways increased, while the flux of anabolic reactions diminished. Nodal analysis indicated that glucose-6-phosphate, phosphoenol pyruvate, and pyruvate nodes were capable of adapting to osmotic stress, whereas the oxaloacetic acid node was relatively unresponsive. Fewer elementary modes were active under stress indicating the rigid behavior of the metabolism in response to high osmolality. Optimal phenotypic space analysis revealed that under normal conditions the organism optimized growth during the initial log phase and lysine and trehalose formation during the stationary phase. However, under osmotic stress, the analysis demonstrated that the organism operates under suboptimal conditions for growth, and lysine and trehalose formation.     

The concentration of cadmium,lead, copper and zinc inChironomus gr.thummi larvae (Diptera,Chironomidae) with deformedversus normal menta

The concentrations of Cd, Pb, Cu and Zn inChironomus gr.thummi were determined for 4th instar larvae from the polluted Dyle River, tributary of the Scheldt River (Belgium). Comparison was made between larvae with deformed and normal menta. Deformed larvae showed higher overall metal concentrations than normal larvae. Especially Pb and Cu had higher concentrations in deformed larvae (16.22 mg kg^–1 dry weight and 39.66 respectively) than in normal larvae (12.80 mg kg^–1 dry weight and 35.70 respectively). No significant differences were found in the concentrations of Cd and Zn (mean [Cd] = 0.81 mg kg^–1 dry weight and mean [Zn] = 313.12 mg kg^–1 dry weight). There was no difference between the two larval groups as far as total length, dry weight and developmental stage of the imaginal discs are concerned.     

Influence of L-tryptophan and auxins applied to the rhizosphere on the vegetative growth of Zea mays L.

Glasshouse experiments were conducted to evaluate the influence of L-TRP in comparison with indole-3-acetamide (IAM), tryptophol (TOL) and indole-3-acetic acid (IAA) on the growth of Zea mays L. var. Early Sunglow. L-TRP (25 to 2.5×10^–5 mg kg^–1 soil), IAM (22 to 2.2×10^–5 mg kg^–1 soil), TOL (20 to 2.0×10^–5 mg kg^–1 soil), and IAA (22 to 2.2×10^–5 mg kg^–1 soil) were applied as a soil drench to established uniform seedlings. All treatments were applied in a completely randomized design with 10 replicates. IAM had no significant effect on the plant growth parameters. Shoot height, uppermost leaf collar base distance, internodal distance, and shoot dry and fresh weights were significantly improved upon the addition of TOL (2.0×10^–2 mg kg^–1 soil), however, the highest concentration (20 mg kg^–1 soil) caused a 14.6% reduction in leaf width. L-TRP (2.5×10^–3 mg kg^-1 soil) also had a significant influence on shoot height, uppermost leaf collar base distance, internodal distance and fresh weight of shoot compared with the control. The highest concentration of L-TRP (25=mg kg^–1 soil) had a negative effect on leaf width and dry weight of the shoot. The most pronounced response on the corn growth parameters was observed with the application of IAA at lower concentrations (2.2×10^–5 to 2.2×10^–2 mg kg^–1 soil) specifically improving root growth. The highest concentration (22 mg kg^–1 soil) of IAA had a significant negative effect on plant height, leaf width, stem diameter, shoot fresh and dry weight. These findings indicate that L-TRP applied at the appropriate concentrations can have positive effects on corn growth comparable to pure auxins (TOL and IAA).     

Glutamate excretion by Corynebacterium glutamicum: a study of glutamate accumulation during a fermentation course

Summary Corynebacterium glutamicum was used in fed-batch fermentation for glutamate production. Both intracellular and extracellular concentrations were determined which allowed us to study the repartition of the amino acid according to the culture conditions and in the presence or absence of surfactants. A decrease in cell volume was observed after addition of surfactants during the exponential phase of growth; glutamate accumulates in the cell, whereas in standard industrial conditions the glutamate concentration in the medium during the production phase can be 30-fold higher than that found inside the cell. The level of excretion is compatible with industrial production.     

<Emphasis Type="Italic">Narcissus</Emphasis> bulblet formation <Emphasis Type="Italic">in vitro</Emphasis>: effects of carbohydrate type and osmolarity of the culture medium

Shoot clump cultures of Narcissus cultivars St. Keverne and Hawera were used to investigate the effects of culture medium carbon supply, type of carbohydrate and osmolarity on in vitro bulblet development. Increasing the medium osmolarity using mannitol or sorbitol, which did not act as substrates for growth, failed to stimulate bulblet formation with either cultivar. An exception to this was a relatively small increase in total bulblet dry weight per culture, in the cultivar Hawera only, caused by adding 30 g l ^–1 sorbitol in combination with 30 g l^–1 sucrose. Simultaneously increasing the medium osmolarity and carbon supply using the metabolisable carbohydrate sources, sucrose, glucose, fructose or an equimolar mixture of glucose and fructose stimulated bulblet production, total dry matter accumulation and partitioning into bulblets. At comparable levels of carbon supply up to 19.0 g l^–1, bulblet development of both cultivars was similar with monosaccharide and sucrose media. This indicates that substrate supply is more important for bulblet development than osmolarity of the culture medium. The cultivar Hawera also showed similar responses to monosaccharide and sucrose media supplying 37.9 g C l^–1, despite the high osmolarity of monosaccharide media (c. 650 m Osm kg^–1, equivalent to –1.6 MPa, compared to 380 m Osm kg^–1 for sucrose medium). However in St. Keverne total dry matter accumulation and dry weight per bulblet were further stimulated only by increasing the sucrose supply from 19.0 to 37.9 g C l^–1, not by increasing the monosaccharide supply. Implications of the findings for Narcissus micropropagation are discussed.     

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.     

Requirement of chelating compounds for the growth of Corynebacterium glutamicum in synthetic media

Summary The effects of various iron-complexing substances on the growth of Corynebacterium glutamicum in synthetic medium were investigated. The data obtained indicate that C. glutamicum has an absolute requirement for the presence of an iron-complexing compound as a growth factor for rapid and abundabnt growth in synthetic media. This requirement can be met by adding low concentrations (10^–5 M) of certain dihydroxyphenols (catechol, protocatechuate) or relatively high concentratuions (0.1%) of citrate to the medium or by autoclaving a small amount of glucose together with other media components. The addition of catechol or protocatechuate to synthetic media has advantages over media preparation with citrate or autoclaved glucose. In the described synthetic broth supplemented with catechol or protocatechuate growth is largely independent of the iron content of the medium in a range between 0.037 and 0.37 mM.Offprint requests to: W. Liebl     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Heavy metal and PCB contamination of Bryozoan colonies in the River Meuse (Belgium)

In the Meuse River (Liège area, Belgium), large amounts of three species of Bryozoans, Fredericella sultana (BLUM.), Plumatella emarginata ALL. and Plumatella fungosa (PALL.) occur. They cover 3 to 40% of the bank walls and their biomass ranges from 12 to 293 g m^–2 (dry weight). In the heated waters of a nuclear power plant (Tihange) and of industries lined up along the river, colony development occurs 3 to 4 weeks earlier than at an upstream station. The heavy metal content of living colonies ranges from 4 to 21 mg kg^–1 Cd, 45 to 182 mg kg^–1 Cu, 803 to 2232 mg kg^–1 Zn, 150 to 483 mg kg^–1 Pb and 21 to 138 mg kg^–1 Cr (DW). The mean concentration of PCBs was 925 µg kg^–1 (DW). Heavy metal and PCB concentrations in the sediments were close to those of colonies, suggesting that most of the pollutants found in the Bryozoans is in fact in the sediment trapped by the colonies.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5–7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg^-1 dry weight) and P (400 mmol kg^-1 dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

The effect of specific growth rates on the recovery of amino acids

Three specific growth rates, 0.23, 0.45 and 0.51 h^–1, were used to cultivate Corynebacterium glutamicum in a pH-auxostat. The specific formation rates of most amino acids increased by raising the specific growth rates. The highest specific growth rate, 0.51 h^–1, favors the production of LEU; whereas the highest production yield for ALA and GLU were at = 0.23 h^–1. A correlation among specific growth rates, glucose consumption rate, and production yields of amino acids was obtained.     

Characterisation of kinetic parameters and metabolic transition of Corynebacterium glutamicum on l-lysine production in continuous culture

Kinetic parameters and physiological states of Corynebacterium glutamicum at the growing and l-lysine-overproducing phase were characterised in continuous culture on threonine-limited complex and minimal media. High l-lysine productivity occurred at dilution rates ranging from 0.1 h^–1 to 0.3 h^–1 on threonine-limited complex medium, and at dilution rates ranging from 0.1 h^–1 to 0.15 h^–1 in minimal medium. l-Lysine yields of 0.25 g/g (0.31 g/g as l-lysine hydrochloride) in complex medium, and of 0.17 g/g (0.21 g/g as l-lysine hydrochloride) in minimal medium, corresponding respectively to intrinsic yields of 0.533 g/g and 0.572 g/g were obtained. These intrinsic yield factors are closed to the theoretical ones (0.608 g/g, 0.75 mol/mol). Intrinsic biomass yields were calculated as 0.658 g/g in complex medium and 0.283 g/g in minimal medium. CO₂ production has been clearly related to l-lysine production. According to our results on specific uptake rates and specific productivities in complex medium, metabolic rearrangement should occur during the transition from the growing phase to the l-lysine-overproducing phase. This phenomenon was further investigated.     

Effects of Benzyladenine and Naphthalene Acetic Acid on Growth and Camptothecin Accumulation in Camptotheca acuminata Seedlings

The effects of the cytokinin benzyladenine (BA) and the auxin naphthalene acetic acid (NAA) on Camptotheca acuminata Decaisne growth and camptothecin (CPT) accumulation (leaf CPT concentration and total leaf CPT yield) were studied in a hydroponic culture system for three weeks. Increasing BA concentrations from 0 to 3 mg l^–1 in growth medium decreased plant height, stem weight, and leaf weight but increased root weight. High BA levels (1 and 3 mg l^–1) increased leaf CPT concentration (% of dry weight), whereas BA applications had no effect on total leaf CPT yield, the product of leaf CPT concentration and total leaf dry weight per seedling. There was a positive correlation between root weight and leaf CPT concentration under BA treatments. NAA supplementations (from 0.5 to 4 mg l^–1) to growth medium reduced plant height, leaf number, leaf length, specific leaf weight, plant weight, stem weight, and leaf weight compared with the NAA control. Meanwhile, there were no differences in plant height, leaf length, and specific leaf weight among the NAA supplementations. NAA applications had no effect on leaf CPT concentration and NAA applications decreased total leaf CPT yield. There were negative correlations between leaf number and leaf CPT concentration, leaf length and leaf CPT concentration under NAA treatments. Our results suggest that BA applications from 0.3 to 3 mg l^–1 are not helpful for achieving high total leaf CPT yield and NAA applications from 0.5 to 4 mg l^–1 decrease total leaf CPT yield.     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.