Response of the adenosine phosphate pool level to changes in the catabolic pattern of Saccharomyces cerevisiae

Changes in the catabolic pattern of Saccharomyces cerevisiae, growing in continuous culture, were effected by altering the glucose feed rate or the dissolved oxygen concentration. The cytochrome concentrations and the adenosine phosphate pool level of the yeast in a series of steady states and during three transitions were measured and compared with the glucose uptake rate (Q_G), the respiration rate (QO₂), and the rate of ethanolic fermentation (Q_E). Respiration was decreased at high glucose feed rates only if oxygen was low but cytochromes were glucose repressible at both high and low oxygen concentrations. In the main, Q_E and the levels of ATP, ADP, and AMP were decreased and cytochrome concentration were elevated at low Q_G values. No consistent relationship between any of the adenosine phosphate parameters and QO₂ was discernible. Evidence is presented for the concept that the Q_G directly controls the adenosine phosphate pool level and that a relationship between the concentration of adenosine phosphate anhydride bonds and the adenosine phosphate level is constantly maintained.     

Metabolic cost of nitrogen incorporation by N2-fixingAzotobacter vinelandii is affected by the culture pH

Summary N₂-fixing continuous cultures ofAzotobacter vinellandii ATCC 9046 were carried out under various dissolved oxygen tensions (2, 25 and 50% air saturation) and, for each of these oxygen concentrations, the culture pH was controlled at 6.2 and 7.4. The culture pH exerted a profound influence on the specific consumption rates of glucose and oxygen and on the growth yields. Parallely, the total metabolic cost for N-incorporation was affected: for incorporating a given amount of N an extra glucose consumption of more than 70% took place when the culture pH was changed from 7.4 to 6.2. This effect was observed when the dissolved oxygen tension in the cultures was 25 or 50% but was less pronounced when it was 2% air saturation.     

Some factors affecting the respiration of some aquatic plants

Summary The oxygen consumption of four aquatic plants has been determined at various concentrations of dissolved oxygen and at three different temperatures.Oxygen consumption rates (mg oxygen/g dry weight per hr) at 20°C in air-saturated water were Berula erecta, 1.25; Callitriche obtusangula, 2.8; Hippuris vulgaris, 1.96; and Ranunculus pseudofluitans, 1.90.Oxygen consumption rates increased with increase in dissolved-oxygen concentration within the experimental limits of 1.2–17 p.p.m. dissolved oxygen. The relation of oxygen consumption to this range of oxygen concentrations can be described by the empirical equation R = aC ^b. Increase of temperature has been shown to increase the rates of oxygen consumption. Q₁₀ values ranging from 1.32 to 3.48 have been obtained.     

Occurrence of cytochrome P450 in continuous cultures of Saccharomyces cerevisiae

Summary Cytochrome P450 of Saccharomyces cerevisiae is an inducible enzyme system. Hitherto, its induction was related to semi-anaerobic culture conditions and high glucose concentrations in the growth medium respectively. Since glucose and oxygen are main regulatory effectors in this yeast, the relationship between the occurrence of cytochrome P450 and these two effectors was established in continuous culture. At glucose-derepressed conditions it was not possible to induce the formation of cytochrome P450 by oxygen limitation alone. The oxygen supply had to be decreased to a level where glucose repression also became active. At glucose-repressed conditions cytochrome P450 was obtained in good yield (3 to 5 pmol per mg dry cell weight) below a dissolved oxygen tension of appproximately 15%. There was a correlation between the content of mitochondrial cytochromes and that of cytochrome P450. The presence of mitochondrial cytochromes was reciprocal with cytochrome P450 when its content was increased by lowering the dissolved oxygen tension.     

The response by microorganisms to steady-state growth in controlled concentrations of oxygen and glucose. II. Saccharomyces carlsbergensis

The growth of Saccharomyces carlsbergensis in continuous culture has been studied when dissolved oxygen and glucose concentrations were held constant at a series of steady-state levels. Both oxygen and glucose controlled the degree of aerobic metabolism and of ethanolic fermentation. When the glucose uptake rate was low (between 1.2 and 2.8 mmoles per hour per gram of yeast) the relative distribution of glucose between ethanolic and aerobic fermentation was sensitive to oxygen: when dissolved oxygen was near to saturation, glucose metabolism was 0.98 aerobic; when dissolved oxygen was 0.01 saturated, 0.8 of intake glucose metabolism was by ethanolic fermentation. On the other hand when glucose intake was high (between 7.6 and 18.2 mmoles) metabolism was predominately by ethanolic fermentation even when dissolved oxygen concentration was at saturation. The extent, to which catabolism proceeded by an anaerobic or aerobic pathway, as judged by ethanol production, was controlled more by the uptake of glucose than of oxygen.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. II. Candida utilis

Candida utilis was grown in batch culture with and without oxygen control. The concentrations of A-, B-, and C-type cytochromes were found to vary with the initial glucose concentration, with the dissolved oxygen concentration, and with time. A-type was the most sensitive. After glucose was essentially exhausted, the yeast catabolized ethanol, if it had been growing in a relatively low initial glucose concentration, or non-glucose carbohydrate, including some of that previously accumulated within the cell, if it had been growing in a high initial glucose concentration. This difference in metabolic pattern could explain why cytochrome derepression was initiated soon after glucose uptake ceased only if initial glucose had been relatively low. The effects of glucose and dissolved oxygen concentrations on yeast cytochromes and respiratory activity are discussed.     

Use of continuous-culture techniques for determining the growth kinetics of a celluloytic Thermoactinomyces sp.

In order to better understand the kinetics of cellulose degradation by Thermoactinomyces sp., continuous-culture experiments were performed utilizing the various intermediates of cellulose degradation as the feed substrates. Steady-state data from the glucose runs suggest that this organism has a growth yield of 0.42 g cell/g glucose, and a specific maintenance of 0.24 g glucose/g cell/hr. The Monod equation did not seen to model the growth well, since a plot of 1/D vs. 1/S gave a maximum specific growth rate that was even lower than one of the steady-state dilution rates. A dynamic washout experiment suggested a maximum specific specific growth rate of 0.36 hr^?1 and indicated that glucose is only slightly growth inhibitory as the inhibition constant, K_i, is 19 g glucose/liter. An equation for substrate concentration for washout conditions was derived. This equation predicted the transient glucose concentration relatively well. A fill-and-draw technique was investigated for determination of the growth parameters. It was not successful because of difficulties in contamination and accurately monitoring the dissolved oxygen in the small highly agitated vessel. However, the technique could be useful in studying the growth characteristics of sludge in a waste treatment system where contamination is not a worry. One could cover the medium surface and use a nonsterilizable dissolved oxygen probe of high sensitivity membrane to overcome these difficulties.     

Influence of dissolved oxygen concentration on growth,mitochondrial function and antibody production of hybridoma cells in batch culture

The effects of dissolved oxygen concentration on hybridoma cell growth, metabolism, and antibody production were studied. A mouse hybridoma cell line producing an IgG1 directed at a consensus -interferon was grown in batch cultures in a 5 dm³ stirred bioreactor at dissolved oxygen (DO) concentrations of 5, 30, 90 and 95% or air saturation. High oxygen tension (95% of air saturation) reduced specific growth rate without affecting cell viability. At lower dissolved oxygen levels, specific growth rates were approximately independent of DO, although changes in mitochondrial function and antibody production were observed. Flow cytometry assays of a fluorescent mitochondria-specific marker (Rhodamine 123) show significant single-cell heterogeneities during late exponential growth and greater average fluorescence in cultures grown at 95% DO. The quantity of cell-surface immunoglobulin, measured by an immunofluorescent flow cytometric technique, was the same at high (95%) and low (5%) dissolved oxygen concentrations. Myeloma cells of the type used in constructing the above hybridoma line were much less sensitive to dissolved oxygen level. Specific respiration rate, pyruvate utilization rate, cytochrome oxidase activity, and succinate-cytochrome c oxidoreductase activity were significantly greater (62–116%) for the hybridoma cells than for the myeloma cells in T-flask cultures.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Kinetics of biosynthesis of polyene macrolide antibiotics in batch cultures: Cell maturation time

The changes in several parameters of the candicidin fermentation (total, mycelium-associated, and extracellular product formation; growth rate; DNA content; glucose utilization; dissolved oxygen in the broth; and oxygen uptake rate) during the trophophase-idiophase transition are compared with previously reported data for the polyene macrolides, candidin and candihexin. The maturation time, t_m, and the productformation rate constant, k_p, have been calculated for each of the three polyene macrolides using a simple mathematical model. Slow-feeding of glucose, which resulted in candidin and candihexin overproduction has been shown to increase the polyene formation rate constant and toretard trophophase to idiophase transition (longer maturation time). The opposite effect is achieved by repeated feeding of soybean meal. The values of the maturation times and polyene formation rates obtained were used to predict the production of polyene macrolide antibiotics in batch cultures.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

Influence of hyperbaric oxygen tension on growth parameters of Candida tropicalis and Rhodococcus erythropolis

Summary Oxygen-limited growth was avoided by means of oxygen-enriched aeration in aerobic fermentation processes. Studies were carried out with Candida tropicalis (Cast.) Berkhout and Rhodococcus erythropolis (DSM 43215). The effect of hyperbaric dissolved oxygen tension on growth parameters was examined by varying the dissolved oxygen concentration and the carbon source (glucose, ethanol, and n-alkanes). Up to an oxygen concentration of 40 mg/l in the culture suspension no impairment of the economic coefficients and no promotion of cell lysis was found. It was observed that raised oxygen concentrations in the aeration gas led to enhanced specific growth rates. At cell concentrations above 20 g/l dry weight an uncoupling of carbon source dissimilation and biomass production was observed even at non-limiting oxygen concentrations.     

Embryonic nutrition,growth and energetics inZoarces viviparus L. as indication of a maternal-fetal trophic relationship

Summary The embryos ofZoarces viviparus (L.) show linear growth during their intraovarian development. In early gestation before hatching, the embryos take up very small amounts of low molecular tracer compounds such as glucose, glycine or taurine. Later in gestation (two months after hatching), the embryos accumulate substantial amounts of the tracer compounds. The uptake rates of the tracer compounds in vitro are correlated with ambient concentrations of unlabelled compounds within the natural concentration range of the ovarian fluid. The highest uptake rates are found for glucose and the lowest for taurine. Release of¹⁴CO₂ and dissolved organic carbon (DO¹⁴C) from assimilated tracers in the embryos is low. Oxygen uptake and body weight of the embryos appear to be linearly correlated, and the average oxygen uptake is 4.20 (SD 0.73) mol O₂ g^–1 h^–1 WW at 11°C. The contribution of glucose respiration to total aerobic respiration is 13.9%. A growth to respiration ratio of 0.91 indicates a relatively high efficiency for converting food to growth.Symbols and abbreviations DW dry weight - WW wet weight - DO ¹⁴ C dissolved organic carbon (¹⁴C-labelled) - t _1/2 half life time - turnover time (replacement time)     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

Relationship of respiratory pathways in soybean cell suspensions to growth of the cells at various glucose concentrations

Green soybean cells (Glycine max L.) were grown in suspension culture on B₅-medium containing various glucose concentrations (2%, 4% or 8% glucose and 2% glucose + 2% or 6% mannitol). Respiratory parameters have been determined in these cells in relation to growth.The length of the growth phase and the amount of dry weight produced increased with higher glucose concentrations. In cells cultured in the presence of 2% glucose, 4% glucose or 2% glucose + 2% mannitol, the activity of the cytochrome pathway (V _cyt )and the capacity of the alternative pathway (V _alt )were not significantly different. Also the contribution of the alternative pathway to total respiration was essentially the same.The glucose content of cells grown in 2% or 4% glucose was roughly equal, which might explain the absence of any effect of the increased glucose concentration on the respiratory parameters.In cells grown in 8% glucose or on 2% glucose + 6% mannitol, both the activity of the cytochrome pathway and the capacity of the alternative pathway were lower. High values of (approaching 1) were found in these cells, meaning that the alternative pathway is nearly fully operative. This is probably caused by the impairment of the cytochrome pathway due to the high osmotic value of the medium.ATP-production per batch per day has been calculated from oxygen uptake data, integrated and compared with cellular dry weight production to give values of 13-15.3 g dry weight produced mole ATP^-1.The described experiments produce no direct evidence for a function of the alternative pathway as energy overflow in soybean cells. The higher alternative pathway activity in the lag phase might be attributed to the great demand for intermediates during this phase.Abbreviations FW Fresh Weight - BHAM Benzhydroxamate     

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.     

The influence of dissolved oxygen partial pressure on the level of various enzymes in mouse LS cells

The intracellular levels of seven enzymes in mouse LS cells growing in suspension culture at controlled dissolved oxygen partial pressures (pO₂) have been measured. During the growth of each culture large fluctuations were observed in the levels of some enzymes, particularly aldolase and cytochrome oxidase. Mean values for the concentration of each enzyme during the growth phase have been calculated. These results are discussed in relation to previous observations made on the growth of mouse LS cells.     

Studies of fungal growth and intermediary carbon metabolism under steady and non-steady state conditions

The physiology of Aspergillus nidulans strain 224 has been studied under conditions of batch- and glucose-limited chemostat-culture and the effect of different steady state growth rates and dissolved oxygen tensions (DOT) examined. Measurements of the specific activities of selected glucose enzymes, the extent of oxygen uptake inhibition by glycolytic inhibitors, and radiorespirometric analyses were made in order to follow the variations in glucose catabolism, which occurred under these conditions. Greatly increased activity of the hexosemonophosphate (HMP) pathway was found during: (i) exponential growth of batch cultures; (ii) at near maximum specific growth rates (μ = 0.072 hr^?1) (DOT = 156 mm Hg); and (iii) at low DOT levels (<30 mm Hg) (μ = 0.050 hr^?1) in chemostat cultures. These changes in glucose eatabolism have been discussed in terms of the biosynthetic demands of the fungus under the influence of changing growth pressures. Preliminary studies also have been made of transition state behavior following stepwise alteration of the DOT. A new steady state was established after 4–5 culture doublings during which period an “overshoot” in HMP pathway activity occurred; these kinetics are indicative of a derepression of certain glucose enzymes. Low molecular weight phenols are synthesized during the exponential phase in batch cultures and these are further metabliized to a major secondary metabolite, melanin, at the onset of stationary phase conditions. The kinetics of tyrosinase production in steady state chemostats differs from those that might be predicted for an enzyme associated solely with secondary metabolism. A primary physiological role for this oxidase in Aspergillus nidulans has been postulated.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.