Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content

Summary The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.Abbreviations LS Linsmaier and Skoog - S sucrose - N mineral nitrogen - K K₂SO₄ - F fructose     

Genetic algorithm-based medium optimization for a toxic dinoflagellate microalga

A genetic algorithm stochastic search strategy was used to optimize the culture medium for producing the toxic marine dinoflagellate microalga Protoceratium reticulatum. The optimized medium contained 26 different components (macronutrients, trace elements, vitamins). The use of this medium allowed a 60% enhancement in the final cell concentration relative to the control culture based on L1 medium. The final titer of yessotoxins was improved by 40% relative to the control medium.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Strain development and medium optimization for fumaric acid production

Rhizopus oryzae RUR709 mutant was isolated based on halo size from selection medium via mutagenesis with UV and γ-rays, and the production of fumaric acid in the submerged fermentation was assessed. The maximum concentration of fumaric acid was obtained using 0.5% corn steep liquor (CSL) as the nitrogen source. Organic nitrogen sources were shown to be more effective in fumaric acid production than inorganic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentration of fumaric acid achieved in flask culture was 26.2 g/L, which is fairly close to the 27.4 g/L predicted by the model. The highest concentration of fumaric acid in the stirred-tank reactor generated by the R. oryzae RUR709 mutant was 32.1 g/L and yield (0.45 g/g) and productivity (0.32 g/L/h) were highest at 4 days.     

Cholinesterase,acid phosphatase,and phospholipase C ofPseudomonas aeruginosa under hyperosmotic conditions in a high-phosphate medium

The presence of low choline or betaine concentrations in a culture medium containing succinate, NH₄Cl, and inorganic phosphate (Pi) as the carbon, nitrogen, and phosphate sources, respectively, permits the growth ofPseudomonas aeruginosa in a hyperosmolar medium. Dimethylglycine, acetylcholine, and phosphorylcholine were less effective as osmoprotectants than choline or betaine. Other alkylammonium compounds tested were virtually ineffective in this capacity. Bacterial growth was also observed in a hyperosmolar medium when choline was the sole carbon and nitrogen source. Choline could act as an osmoprotectant under all the conditions tested. However, the production of cholinesterase (ChE), acid phosphatase (Ac. Pase) and phospholipase C (PLC) took place only when choline was the carbon and nitrogen source. This fact confirms that the synthesis of PLC may occur even in the presence of a high Pi concentration in the medium. Inasmuch as in a high-P_i medium the synthesis of PLC and Ac. Pase (phosphorylcholine phosphatase) is dependent only on choline metabolism, it is postulated that both enzymes are involved in a set of reactions coordinated to produce the breakdown of the membrane phospholipids of the host cell in a hyperosmotic medium.     

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.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Induction of flocculation of brewer's yeast strains of Saccharomyces cerevisiae by changing the calcium concentration and pH of culture medium

Strains of Saccharomyces cerevisiae (NCYC 1190, 1214 and 1364) behaved as nonflocculent in defined culture medium, as well as the strain NCYC 1214 in rich medium. Flocculation was induced by Ca²⁺ addition and/or by correcting the pH to a suitable value. Since the free Ca²⁺ concentration is pH-dependent, these two factors (total Ca²⁺ concentration and pH) cannot be dissociated and are the critical parameters governing flocculation, in culture medium, of the brewing strains studied.     

A comparison of amide and ureide nitrogen sources in tissue culture of tree legume Prosopis alba clone B2 V50

The effects of medium nitrogen sources on the recalcitrant nature of Prosopis alba clone B2V50 in tissue culture were compared involving shoot development using axillary bud explants from 2 to 4-year-old greenhouse-grown trees. A significant difference (P<0.05) was found between the amino acids aspartic acid and glutamic acid and their corresponding amide-containing compounds asparagine and glutamine. A comparison between amide and ureide nitrogen sources showed that allantoin, a ureide, was an acceptable replacement for asparagine or glutamine. Allantoin, asparagine, and glutamine could be used as the sole nitrogen sources. Allantoin at a concentration of 20 mM was adopted for use in future research. Although shoots were consistently induced, all explants showed complete shoot-tip necrosis after 12 weeks of in vitro culture.     

Lipid accumulation in <Emphasis Type="Italic">Schizochytrium</Emphasis> G13/2S produced in continuous culture

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h⁻¹, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h⁻¹, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.     

Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans

Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4 g l^–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5 g l^–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16 g l^–1 h^–1 were obtained when the C/N ratios in the feed were 10, 20 and 100 g octanoic acid g^–1 ammonium nitrate, respectively.     

Simultaneous production of hydrogen and bioflocculant by Enterobacter sp. BY-29

Hydrogen and a bioflocculant could be produced simultaneously by anaerobic culture of Enterobacter sp. BY-29. For production of hydrogen and the bioflocculant by cell culture of the bacterium in batch cultures, cultivation at 37 °C in a medium containing glucose as a carbon source and Polypepton as a nitrogen source was found to be suitable. In continuous production of hydrogen and the bioflocculant by cell culture or immobilized cells of the bacterium, the hydrogen production rate and hydrogen yield by the immobilized cells on porous glass beads in stirred and column reactors were higher than those by the cell culture in a stirred reactor. However, production of the bioflocculant by the cell culture was superior to that by the immobilized cells in continuous production.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Effect of phosphate concentration on the production of dextransucrase by<Emphasis Type="Italic"> Leuconostoc mesenteroides</Emphasis> NRRL B512F

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K₂HPO₄. In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.     

Serum-free liquid medium forNeisseria gonorrhoeae

A liquid culture medium (GB) forNeisseria gonorrhoeae is described. Except hemin, the GB medium does not contain blood products. Liver digest, proteose peptone and Yeastolate were used as the main carbon/nitrogen sources. The medium permitted growth, with a satisfactory cell yield, of all but one of the 68 gonococcal strains tested. Cysteine-hydrochloride was used to achieve an adequate redox-potential of the medium. The optimal pH of the ready-to-use medium was found to be 7.4. Comparative culture studies showed that the GB medium has advantages compared to earlier recommended broth media for the growth of gonococci in the percentage of consecutive gonococcal strains isolated from clinical specimens that grew in the medium. The addition of 1.4% purified agar permitted the use of the medium as a transparent solid medium for the culture of gonococci. The possibility to make the GB medium selective for gonococci by addition of trimethoprim, polymyxin B and E, and natamycin was also evaluated. In this respect, vancomycin-colistin-nystatin and mycostatin were also tested. The GB medium is inexpensive and easy to prepare.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

A semi-defined medium for culturing Nomuraea rileyi

A semi-defined medium was successfully used to culture several natural and mutant isolates of the entomopathogenic fungus, Nomuraea rileyi. The medium contains only two complex ingredients at very low levels, yet permits germination and mycelial growth of N. rileyi. The medium also facilitates recovery and detection of hydrolytic enzymes. Use of the formulation could simplify nutritional, biochemical and physiological studies with N. rileyi and also might be used to culture other entomopathogenic fungi.This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by USDA.     

Modification of an algal culture medium for sustained growth of a saxitoxin-producing isolate of Alexandrium minutum

In order to study saxitoxin (STX) production bymicro-algae in the laboratory, a defined algal culture medium which supports optimum growth over a longtime-period is a requirement. In the development of such a medium, a number of modifications were made to a standard algal culture medium (GP) and growth of a STX-producing isolate of Alexandrium minutum in the different formulations was assessed by measuring maximum cell densities and mean generation times (MGT). All experiments were carried out under controlled conditions in an aerobic atmosphere with increased CO₂. Whilst maximum cell densities in the different modifications were similar, the MGT was significantly shortened by the addition of Tris buffer and the trace metals strontium, selenium and molybdenum. Replacement of natural with artificial seawater and removal of soil extract did not adversely affect algal growth. Five of the six media formulations supported the growth of A. minutumover a 9-month period. This revised version was published online in August 2006 with corrections to the Cover Date.