Changes of Exponential Growth Rates in Relation to Differentiation of Geotrichum candidum in Submerged Culture

Growth and differentiation of the imperfect fungus Geotrichum candidum were followed in submerged cultures containing a simple synthetic glucose salt medium. Uptake of glucose, ammonium and oxygen from the medium were measured during the entire growth perod. In 0.1% glucose the fungus grows with one exponential growth phase until all the glucose has been consumed. The arthrospores are formed in the stationary phase. In 0.5% glucose the growth curve has two exponential growth phases, one with a doubling time of 1.8 h and a second one with a doubling time of 4.9 h. The second exponential growth phase, which starts when less than 15% of the glucose and less than 30% of the ammonium have been consumed, is shown to be the sporulation phase. During this growth phase the oxygen saturation in the culture remained constant at about 50%.     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Conditioning of the culture medium by suspension cells and formation of somatic proembryo in Araucaria angustifolia (coniferae)

Summary Cell masses of Araucaria angustifolia cultured in LP liquid medium showed different uptake and utilization patterns for different sugars. Fructose was slowly utilized by cell growth during the lag phase. After this lag period, fructose concentration decreased quickly, corresponding to the start of linear growth. Glucose rapidly decreased following fructose depletion. The level of extracellular proteins in the culture medium increased for the first 5 d after cell inoculation, during the transition from the lag phase to the high-growth phase. The pH of the medium decreased through the first half of the culture period (4.94) and increased (5.6) thereafter. Proembryos originated from a single cell, without a cleavage process. Suspensor cells and proembryonal groups developed independently. Only embryogenic cells divided and formed a suspensor or a proembryo group, which differentiated further along the longitudinal axis. The pathway observed for proembryo formation in Araucaria differs from the model proposed for other conifers, which show cleavage polyembryony.     

Differential sugar uptake by cell suspension cultures of <Emphasis Type="Italic">Rudgea jasminoides</Emphasis>, a tropical woody Rubiaceae

The primary utilization of carbohydrates by cell suspension cultures of Rudgea jasminoides, a native woody Rubiaceae from tropical forests, was investigated. Sucrose, glucose + fructose, glucose, or fructose were supplied as carbon sources. The growth curves of R. jasminoides cultured in glucose + fructose, glucose, or fructose showed similar patterns to that observed when sucrose was supplied to the cells, except that an increase in dry mass was observed at the beginning of the stationary growth phase in the media containing only one monosaccharide. The increase in hexose levels in the media during the early stages of the cultures indicated extracellular hydrolysis of sucrose, which was further supported by the increase in the activity of acid invertase bound to the cell wall. Glucose was preferentially taken up, whereas uptake of fructose was delayed until glucose was nearly depleted from the medium. Measurements of intracellular sucrose content and cytoplasmatic and vacuolar invertases indicate that the enzymatic activity seems to be correlated with a decrease in the hexose flux into the cells of R. jasminoides. Our results indicate that the behavior of cell suspension cultures of R. jasminoides regarding sugar utilization seems to be similar to other dicotyledonous undifferentiated cell suspension cultures.     

Extracellular invertase from Aspergillus flavus

An extracellular invertase was induced in cultures of Aspergillus flavus Link during growth in liquid medium that contained sucrose as the sole carbon source. Synthesis of this enzyme was repressed by the addition of glucose or fructose to sucrose-metabolizing cells, and was induced in a glucose or fructose-metabolizing culture by the addition of sucrose. A. flavus invertase had a pH optimum of 6.0 and an apparent Km of approximately 133 mM for sucrose. The enzyme required potassium phosphate for maximum activity, optimum concentration being 250 mM. The enzyme was partially purified by ammonium sulphate precipitation followed by dialysis and separated by molecular exclusion into three components with molecular weights ranging from approximately 40,000 to 55,000.     

Growth of Cephalotaxus harringtonia plant-cell cultures

Summary Cell cultures of Cephalotaxus harringtonia were examined to characterize growth kinetics. The requirement for an undefined medium supplement (coconut water) was eliminated by maintaining high cell concentrations in semicontinuous and batch growth. Sucrose fed to batch-cultured cells was completely hydrolyzed and a diauxic growth pattern was observed corresponding to first glucose and then fructose uptake. Examination of increases in cell concentrations on the basis of fresh and dry weight showed that a substantial lag period existed between the initiation of substrate uptake and increases in cell volume. Specific growth rates were highest during periods of glucose uptake, but cell yields were comparable for the two sugars. In contrast, studies with glucose or fructose as the sole carbon source indicated that cell yields were significantly lower with fructose but specific growth rates were comparable for the two sugars.Offprint requests to: P. J. Westgate     

Effects of initial medium pH on growth and metabolism ofCatharanthus roseus hairy root cultures —A study with31P and13C NMR spectroscopy

Summary Hairy root cultures ofCatharanthus roseus were grown for 26 days in half-strength Gamborg's B5 liquid medium at different initial pH values of 4.2, 5.7, 6.5, and 7.3. Maximum growth was obtained for cultures with an initial medium pH 6.5. The lowest growth rate was found in cultures at initial pH values of 4.2 and 7.3. Roots in cultures at initial pH of 4.2 had a thickened and stunted morphology in contrast to the other cultures. Also, cultures at initial medium pH of 4.2 exhibited an increase in medium pH in the first few days instead of the characteristic acidification. All cultures maintained a cytoplasmic pH of 7.4 throughout the growth cycle. However, vacuolar pH was 5.1–5.2 in cultures of initial pH 4.2, as opposed to 5.4–5.5 for other cultures. Sucrose was hydrolyzed completely to glucose and fructose by day 26 except for cultures at initial pH of 7.3. Glucose was the preferred substrate throughout the growth cycle for cultures with initial pH values of 7.3 and 6.5, after day 20 for an initial pH of 5.7, and after day 26 for pH 4.2.     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Glucose and fructose consumption in a phosphoglucoseisomeraseless mutant in Saccharomyces cerevisiae

Saccharomyces cerevisiae with a practically complete absence of phosphoglucoseisomerase activity when grown in fructose or glucose minimal medium showed different consumption of fructose and glucose during different periods of the culture. At the beginning of growth, cells had a great quantity of glucose available relative to their requirements and a large quantity of trehalose accumulated from ¹⁴C-glucose in comparison with the wild type strain. A second phase arises when the concentration of glucose in the medium was practically absent and the cells obtain glucose by mobilisation of stored glucose containing compounds. It is very likely that at this moment a balance rate between glucose 6-phosphate formation and consumption occurs. Finally cells reach conditions of glucose starvation and fructose consumption increases in this last stage. The different consumption of fructose throughout different periods of cell growth most probably indicates a strict regulation at the level of sugar uptake.Non Standard Abbreviation pgi phosphoglucoseisomerase     

Diauxic growth of Penicillium camembertii on glucose and arginine

The growth of Penicillium camembertii during batch culture in a synthetic medium containing glucose and arginine was examined. The diauxic growth observed can be well characterized. Indeed, in a first phase, glucose and arginine were, respectively, assimilated as carbon and nitrogen sources, with an acidification of the medium (until 3.5), since arginine was taken up in exchange for protons. During this phase of growth, arginine, in addition to glucose, was also assimilated as an energy source, resulting in the release of the arginine carbon content as CO₂. Then, in a second phase, characterized by reduced growth rates after glucose depletion, arginine was assimilated as a carbon and nitrogen source, as well as an energy source, resulting in ammonium release which raised the pH (final pH 6.3), despite the amino acid/H⁺ exchange, since amino acids contain excess nitrogen in relation to their carbon content for fungi.     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

Retinoic Acid Improves a Hybridoma Culture in a Fructose-Based Medium by Up-Regulation of Fructose Incorporation via Retinoid Nuclear Receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

Improvement of the catharanthine productivity in hairy root cultures ofCatharanthus roseus by using monosaccharides as a carbon source

Summary Sucrose, glucose, and fructose as carbon sources in culture medium were assessed in hairy root cultures ofCatharanthus roseus. The cultures preferentially consumed sucrose, resulting in about 40% (dry wt) higher growth rate. However, fructose enhanced the cathranthine yield about two-fold. The elevated yield was not seemingly ascribed to the higher osmolarity per unit weight of fructose than sucrose. A two stage culture using sucrose (1st) and fructose (2nd) improved volumetric yields of catharanthine about two-fold, i.e. 41 mg/l.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Fructose production from sucrose and high fructose syrup: A mycelial fungal system

Summary The use of Mucor sp. M105 and Fusarium sp. F5 in the production of fructose from sugarcane sucrose and high fructose syrup (HFS) was investigated. Although Mucor sp. could not utilize sucrose as the sole carbon and energy source for cell growth, Mucor sp. preferentially utilized glucose in a glucose:fructose (1:1) mixture during fermentation to ethanol. In contrast, Fusarium sp. utilized sucrose as sole carbon source by secretion of extracellular hydrolytic enzymes that degraded the disaccharide. In Fusarium sp., glucose formation in the medium was faster than fructose. Due to the low consumption rate of fructose, this substrate remained in the fermentation broth. The application of these biological systems for the production of fructose from either sucrose or HFS is discussed.     

Growth pattern and ginsenoside production of Agrobacterium-transformed Panax ginseng roots

Panax ginseng roots transformed by Agrobacterium rhizogenes grew rapidly in a hormone-free medium. The transformed roots showed biphasic growth: rapid during the first two weeks and slower thereafter. Sucrose in the medium was almost all converted to glucose and fructose during the first two weeks, and the root growth slowed down after the depletion of sucrose in the medium. Periodic changes of the medium maintained the high growth rate, and the dry weight increased by 31 times in 32 days, which is the highest growth rate so far reported for cultured tissues of ginseng. The medium exchange also increased the ginsenoside content in the roots. Effective scale-up of the root culture was achieved in a turbine-blade type bioreactor.     

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30° C but no detectable diauxic lag at 5° C.At 30° C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed unitl onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with respression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase.At 5° C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase.Thus, at low temperatures, psychrotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.     

Metabolic regulation in Pseudomonas oxalaticus OX1. Diauxic growth on mixtures of oxalate and formate or acetate

Diauxic growth was observed in batch cultures of Pseudomonas oxalaticus when cells were pregrown on acetate and then transferred to mixtures of acetate and oxalate. In the first phase of growth only acetate was utilized. After the exhaustion of acetate from the medium enzymes involved in the metabolism of oxalate were synthesized during a lag phase of 2 h, followed by a second growth phase on oxalate. When the organism was pregrown on oxalate, oxalate utilization from the mixture with acetate completely ceased after a few hours during which acetate became the preferred substrate. Similar observations were made with formate/oxalate mixtures in which formate was the preferred substrate. Until formate was exhausted, it completely suppressed oxalate metabolism, again resulting in diauxic growth. However, when the organism was pregrown on oxalate and then transferred to mixtures of oxalate and formate, both substrates were utilized simultaneously although the initial rate of oxalate utilization from the mixture was strongly reduced as compared to growth on oxalate alone.Since both preferred substrates cross the cytoplasmic membrane by diffusion, whereas oxalate is accumulated by an inducible, active transport system, the effect of acetate and formate on oxalate transport was studied at different external pH values. At pH 5.5 both substrates completely inhibited oxalate transport. However, at pH 7.5, the pH at which the diauxic growth experiments were performed, formate and acetate did not affect oxalate transport. Growth patterns and enzymes profiles suggest that, at higher pH values, formate and acetate possibly affect oxalate utilization via an effect on the internal pool of oxalyl-CoA, the first product of oxalate metabolism.Abbreviations PMS phenazine methosulphate - RuBPCase ribulosebisphosphate carboxylase - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - p.m.f. protonmotive force