Production of HBsAg by growth rate control with recombinantSaccharomyces cerevisiae in fed-batch

Summary To maintain a constant specific growth rate for a recombinantS.cerevisiae in fed-batch, the medium feeding rate has been controlled with respect to the hourly calculated glucose uptake rate. The recombinant yeast producing HBsAg showed the exponential production trend in proportion to the exponential cell growth. Total cell yield in fed-batch was about 0.402 g cells/g glucose, and HBsAg was produced about ten times more than in batch. Decrease of growth rate by HBsAg produced was not shown.     

Effect of low-dose ultrasonic treatment on phystological variables inAnabaena flos-aquae andSelenastrum capricornutum

Summary Cell protein content in two species of cultured algaeAnabaena flos-aquae andSelenastrum capricornutum, was markedly enhanced by low-dose, short-duration ultrasonic treatment. Chlorophylla levels and¹⁴C-bicarbonate uptake rates were not affected by ultrasonic treatment in either species. Ultrasonically-activatedAnabaena cultures placed in media deficient in nitrogen and phosphorus produced more biomass per unit time, exhibited less cell-surface alkaline phosphatase activity per cell, and had a higher heterocyst frequency than non-sonicated, nutrient-deficient cultures. In contrast, sonicated, nutrient-deficientSelenastrum cultures grew more slowly and had higher alkaline phosphatase activity than non-sonicated variants. Collectively, the data suggest that key metabolic variables may be altered by ultrasonic treatment in algal cultures and that the magnitude and direction of change may be species-specific.     

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.     

TEMPERATURE DEPENDENCE OF GROWTH AND PHOSPHORUS UPTAKE IN TWO SPECIES OF VOLVOX (VOLVOCALES,CHLOROPHYTA)1

The growth of Volvox globator L. and Volvox aureus Ehr. was measured at five temperatures and nine phosphorus concentrations. Growth rates were hyperbolically related to phosphorus concentrations for all temperatures using a Monod growth model. Optimal growth rates of 1.17 and 1.00 doublings d^?1 were obtained at 20°C for V. globator and V. aureus, respectively. Neither species grew at 5°C. The half-saturation constants for growth, K_s, were lower for V. aureus. Phosphorus uptake by both species was also dependent upon external phosphorus concentrations and temperature. At all temperatures, maximum phosphorus uptake (μmol P colony^?1 min^?1) was similar for both species; however, the half-saturation constants for uptake showed significant differences between the species. Comparisons of the kinetic constants for growth and phosphorus uptake suggest that V. aureus will outcompete V. globator under phosphorus limited, conditions.     

Kinetics of alcohol production by zymomonas mobilis at high sugar concentrations

Summary Studies on the growth ofZ.mobilis revealed that high concentrations of glucose (10-25%) can be efficiently and rapidly converted to ethanol in batch culture. By comparison withS. carlsbergensis,Z.mobilis had specific glucose uptake rates and specific ethanol productivies several times greater than the yeast.Z.mobilis also had ethanol yields of up to 97% of a theoretical value.     

Studies on Ailanthus altissima cell suspension cultures

The uptake of L-[methylene ¹⁴C]-tryptophan from culture medium and the subsequent incorporation of the radiolabel into canthin-6-one, 1-hydroxycanthin-6-one and 1-methoxycanthin-6-one has been demonstrated in cell suspension cultures of Ailanthus altissima. Efficient incorporation has been shown to depend significantly on the time of feeding. Furthermore, feeding of L-tryptophan, at levels of 500 mg/l resulted in improved alkaloid yields, particularly when fed during the lag phase of the growth cycle.     

Studies on Ailanthus altissima cell suspension cultures. Uptake of L-[methyl-14C]methionine and incorporation of label into 1-methoxycanthin-6-one

Time-course studies of the uptake of L-[methyl ¹⁴C]-methionine have shown rapid uptake by Ailanthus altissima cells when fed at weekly stages throughout the growth cycle. The radio-label from [¹⁴C]-methionine was shown to be incorporated into 1-methoxycanthin-6-one with the highest level of incorporation being achieved with cells fed late in the growth phase.     

SIZE-DEPENDENT PHOSPHORUS UPTAKE KINETICS AND CELL QUOTA IN PHYTOPLANKTON1

The allometric equation, y = aX^b, described the interspecific variation of phosphate uptake kinetics and cell quota with phytoplankton cell size and showed that smaller cells are superior in uptake rate to large. Species-specific measurements, made by track autoradiography in phosphorus deficient cultures of communities from a phosphorus-limited lake, revealed that eight different species did not differ significantly in the Michaelis-Menten half-saturation constant, K_m. However, both saturated uptake rates (V_max) and the initial slope of the uptake curve (V_max:K_m) decreased per unit biomass with increasing cell size. Biomass-specific cell phosphorus quotas also decreased with increasing cell volume, but less rapidly than did V_max or V_max: K_m. Comparable data from the literature showed that marine species were superior in phosphorus uptake to freshwater species of similar size, but allometric variation of kinetics appeared to exist within both groups. Together with a variable internal stores model of phosphorus-limited growth, the allometric relationships of uptake kinetics and quotas predicted competition to favor smaller cells, with a differential in growth rate diminishing as competitive intensity increased.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

PheMaDB: A solution for storage,retrieval, and analysis of high throughput phenotype data

Background  

OmniLog™ phenotype microarrays (PMs) have the capability to measure and compare the growth responses of biological samples upon exposure to hundreds of growth conditions such as different metabolites and antibiotics over a time course of hours to days. In order to manage the large amount of data produced from the OmniLog™ instrument, PheMaDB (Phenotype Microarray DataBase), a web-based relational database, was designed. PheMaDB enables efficient storage, retrieval and rapid analysis of the OmniLog™ PM data.     

NON-STEADY STATE DYNAMICS OF ALGAL POPULATION GROWTH: EXPERIMENTS WITH TWO CHLOROPHYTES1

The relations among dissolved phosphorus, cell quota of phosphorus, and population growth rate were determined for two Chlorophytes, Chlorella sp. and Scenedesmus quadricauda var. longispina (Chod.) G. M. Smith, in two types of non-steady state continuous culture. One of these types had relatively smooth transitions between growth under different degrees of phosphorus limitation. Under these conditions, two equations often applied to growth kinetics in steady state cultures were found to apply to non-steady state growth. Monad's equation described the relation between dissolved phosphorus concentration and population growth rate, and Droop's equation described the relation between cell quota and population growth rate. The second type of culture received phosphorus only as periodic pulses, leading to sharp changes in dissolved phosphorus, cell quota, and growth rate. A simulation model based on Droop's equation described much of the observed dynamics of cell numbers and quotas in these cultures. Droop's equation could not be convincingly fitted directly to the data, however, due to its incorrect prediction of an immediate growth response to phosphorus pulses. A third relation, predicting that saturated rates of phosphorus uptake would depend on the recent nutrient history of the cells as reflected by the cell quota, was not supported.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Growth and mineral nutrition in Stylosanthes fruticosa (Retz) Alston

Interacting effects of phosphorus, potassium and sodium on the growth and nutrient uptake in Stylosanthes fruticosa were studied in its native sandy soil and solution culture. High phosphorus in the medium had adverse effects on growth. High potassium level completely masked or lessened adverse effects of high phosphorus. High sodium level adversely affected growth only in combination with high phosphorus and low potassium. Shoot nutrient content data indicated that phosphorus per se may not inhibit growth. Selective uptake of potassium against sodium was eliminated by high phosphorus. It is suggested that high concentration of phosphorus in the medium effects membrane permeability and the effect of phosphorus on plant growth is primarily through its influence on uptake of other ions.     

Temperature effects on steady-state growth,phosphorus uptake,and the chemical composition of a marine phytoplankter

The marine chrysophyteMonochrysis lutheri was grown in phosphorus-limited continuous cultures at temperatures of 15°, 18.8° and 23°C. The effect of temperature on the maximum growth rate was well-defined by the Arrhenius equation, but the Q₁₀ for this alga (1.7) was somewhat lower than has been determined previously for many other phytoplankton species (2.0–2.2). The minimum phosphorus cell quota was relatively unaffected by temperature at 18.8°C and 23°C, but doubled in magnitude at 15°C. As a result, the internal nutrient equation of Droop described the relationship between specific growth rate and phosphorus cell quota well at 18.8° and 23°C, but was less successful at 15°C. The major limitation in using the Droop equation is that the ratio between the minimum and maximum cell quotas must be known, thus necessitating the need to establish the true maximum growth rate by the cell washout technique. In addition, the phosphorus uptake rate on a cell basis at a given steady state growth rate (=specific uptake rate) increased dramatically at 15°C, whereas the turnover rate of total available phosphorus was unaffected by temperature. Both the nitrogen and carbon cell quotas were relatively unaffected by growth rate at a given temperature, but the average values increased slightly with decreasing temperature. The overall conclusion is that phytoplankton growth and limiting-nutrient uptake rates are only synchronous at or near the optimum temperature. Because these types of responses are species specific, much additional data on temperature effects will be required before the importance of including such effects in phytoplankton-nutrient models can be determined.     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Dominant expression of a gene amplification-related herbicide resistance in medicago cell hybrids

Protoplasts from phosphinotricin resistant M. sativa and M. varia cell lines carrying an amplified glutamine synthethase gene were fused with leaf protoplasts of kanamycin resistant M. varia transformants. The dominant nature of both PPT and kanamycin resistant traits was shown by the double resistant phenotype of the intra- and interspecific cell hybrids obtained. The presence of amplified GS gene in the hybrid genomes and the expression of chimeric neomycin phosphotransferase II gene was detected. The highly embryogenic character of the M. varia parent was not expressed after cell fusion. All hybrid cell lines with the double resistant phenotype showed non-morphogenic growth similarly to the PPT resistant parent. The possible role of GS gene amplification and other factors in the dominant behaviour of unorganized cell growth in alfalfa somatic hybrids is discussed.     

Increased accumulation of indole alkaloids by some cell lines of Catharanthus roseus in response to addition of vanadyl sulphate

Vanadyl sulphate (10–500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.Abbreviations Ajm ajmalicine - Cath catharanthine - CAS ceric ammonium sulphate - VOSO₄ vanadyl sulphate - FW fresh weight - n.d. not detected     

Regeneration of plantlets from mesophyll protoplasts of Brassica juncea (L.) Czern

Isolated mesophyll protoplasts of Brassica juncea (L.) Czern., cv. RLM 514 upon culture in suitable growth medium, regenerated cell wall, underwent cell division and formed cellular colonies. Subsequent induction of embryoid (embryogenesis) and shoot bud (organogenesis) formations in such cell masses resulted in regeneration of 186 and 42 plantlets respectively.Abbreviations NT Nagata and Takebe, 1971 - B5 Gamborg et al. 1968 - KM Kao and Michayluk, 1975 - GK₂ Schenck and Hoffmann, 1978 - MS Murashige and Skoog, 1962 - BAP 6-benzyladenine - 2, 4-D 2, 4-dichlorophenoxyacetic acid - NAA napthaleneacetic acid - GA₃ gibberellic acid     

Production of recombinant human interleukin-z by Escherichia coli with computer controlled temperature induction

Summary The amount of alkali added to the fermentation broth of a recombinant Escherichia coli strain for pH control was monitored on-line by an electronic balance interfaced to a computer. It was successfully correlated with the cell mass, and consequently the cell growth could be well estimated. Using this cell growth estimation technique, an automatic temperature induction was successfully carried out to produce human interleukin-2 in a high yield from E. coli harbouring a temperature controlled expression vector system.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.