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1.
The yeast S. cerevisiae was grown on dilute, chemically defined media in continuous culture with either glucose or ammonium sulfate as the growth-limiting ingredient. Changes in dilution rate or glucose concentration induced decaying oscillations in the numbers of yeast growing on ammonium sulfate-limited media. Spot checks indicated that Cell dry weight and Kjeldahl nitrogen followed the cell numbers during these oscillations. With glucose-limited media, there was no response to step changes in ammonium sulfate concentration, and dilution rate step changes gave non-oscillatory transient responses.  相似文献   

2.
The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h-1 (H. polymorpha) and 0.26h-1, respectively (Kloeckera sp. 2201) which significantly exceeded max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C1 Methanol - C6 glucose; D dilution rate (h-1) - D c critical dilution rate (h-1) - q s specific, rate of substrate consumption (g substrate [g cell dry weight]-1 h-1) - q CO2 and q O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]-1 h-1) - RQ respiration quotient (q CO2 q O2 1 ) - s 0(C1) and s 0(C6) are the concentrations of methanol and glucose in the inflowing medium (g l-1) - s residual substrate concentration in the culture liquid (g l-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl-1) - Y X/C6 growth yield on glucose (g cell dry weight [g substrate]-1  相似文献   

3.
The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h−1. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997  相似文献   

4.
Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h–1 with a synthetic glucose medium of which the molecular ratio of KH2PO4 to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h–1) - Ei invertase concentration in culture (enzyme unit l–1) - Ep acid phosphatase concentration in culture (enzyme unit l–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l–1) Greek Letter specific rate of growth (h–1) Suffix f feed - 0 initial value  相似文献   

5.
Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.  相似文献   

6.
Citrobacter freundii was grown aerobically in a chemostat on a mineral medium witn galactose or glucose as carbon and energy sources under limitation by carbon or nitrogen source respectively. At various specific growth rates ranging from 7 to 95% μmax the culture in steady state was analysed and growth yield, specific metabolic rate of substrate utilization, intracellular concentration of pyruvate, ATP, ADP, AMP and energy charge were determined and plotted as functions of dilution rate. In all four types of experiments the physiological state of cells remained practically independent of dilution rate up toD = 0.6 μmax, and at a given specific growth rate nearly independent on μmax and type of limitation. At approximatelyD = 0.6 μmax, which is close to the maximum output dilution rateD m, the physiological state of the cells changed: growth yields decreased and intr cellular pyruvate and adenylates concentrations increased. Consequently, in a given medium two dilution rates exist at which growth rate dx/dt is the same but the physiology of the population is quite different.  相似文献   

7.
Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h–1. For such a range, the glucose and lactate metabolic quotients and the steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h–1, where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates below 0.02 h–1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h–1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.Abbreviations EFBC exponentially-fed batch culture - CSC continuous suspended culture - MAb monoclonal antibody - D dilution rate - q i metabolic quotient or specific rate of consumption or production of i  相似文献   

8.
Summary Conidiation of Aspergillus niger was studied in carbon-limited and nitrogen-limited chemostat culture. Under citrate-limitation conidiation intensity varied inversely with dilution rate. Conidiophores were less complex than in aerial conidiation and at high dilution rates conidia occasionally developed from modified hyphal tips. Conidiation was difficult to achieve under glucose-limitation. At the low dilution rates that allowed limited conidiation steady state could not be maintained due to onset of autolysis. At higher dilution rates when steady state was readily obtained conidiation did not occur. The maximum yield constants under citrate-limitation and glucose-limitation were respectively 0.145 and 0.4 mg dry weight/mg substrate, while the relative specific maintenance values were 0.045 and 0.018 mg substrate/mg dry weight/h. Under ammonium-limitation with citrate as the carbon source there was no conidiation. When nitrate became the limiting nitrogen source conidiophore initiation occurred but biomass production was low and wash-out occurred at D=0.034 h-1.  相似文献   

9.
The kinetic behavior of a nonproducing hybridoma clone AFP-27-NP was investigated in continuous culture under glucose-limited conditions. A total of more than 21, 000 h of cultures were operated at dilution rates ranging from 0.01 to 0.06 h(-1). The viable cell concentrations, dead cell concentrations, and cell volumes all varied with the dilution rate. A steady-state model was developed based on the biomass concentration and the glucose concentration. The specific growth rate as a function of glucose concentration is described by a model similar to the Monod model with a threshold glucose concentration and a minimum specific growth rate incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. A death rate is included in the model which is described by an inverted Monod-type function of glucose concentration. The yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper region of specific growth rates. No maintenance term for glucose consumption was needed; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepted the specific growth rate axis at a value close to the minimum growth rate. The values for the model parameters were determined from regression analysis of the steady-state data. The model predictions and experimental results fit very well.  相似文献   

10.
Batch growth characteristics of various organisms were determined on a number of pairs of sugars to find a stable system showing clear-cut classical diauxie. The system selected for further study was a strain of Klebsiella (Acrobacter) aerogenes, NCIB 8021 growing on a mixture of glucose and maltose in minimal salts medium at 30°C. This showed a specific growth rate (μ) of 1.19 ± 0.03 hr.?1 on 0.01% (w/v) glucose, followed by a diauxie lag of 0.73 ± 0.04 hr and then further growth on 0.01% (w/v) maltose at μ = 0.60 ± 0.03 hr?1. This system was applied to a two-stage continuous, stirred, aerated fermentor system, with working volumes of 1.85 and 2.77 liters, respectively, and growth was followed (mainly by optical density, referred to dry weights and viable counts) and also the concentrations of the sugars were measured. Except at the very highest flow rates, glucose was immediately and virtually completely consumed, but the utilization of maltose showed interesting variations: (a) At low feed rates between 0.09 and 0.4 vol./hr. exactly the same response was found with mixed sugars as with double concentration glucose, showing that the organism was able to metabolize maltose as well and as quickly as glucose. (b) At medium feed rates of 0.46 to 1.03 vol./hr. two deviations were observed, both of which increased as the dilution rate increased: the system showed a time lag on maltose before the cell population began to rise and the volume of medium used before the steady state was established was greater than predicted, (c) At fast feed rates, approaching “washout” condition of 1.055 to 1.135 vol./hr. the first culture vessel showed no reaction to a step change which included maltose, although, of course, with doubled glucose it responded immediately. The second vessel, however, quickly metabolized the overflow maltose, and showed a steady increase of cell population to the theoretical steady state. These results may have significance for industrial systems using complex commercial substrates.  相似文献   

11.
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.  相似文献   

12.
The fructose supply rate to continuous cultures of Ocimum basilicumL. cells was changed in two ways. The concentration of fructosein the medium feed was diminished or the dilution rate was increasedin fructose-limiting conditions. When fructose in the feed wasdecreased, whilst maintaining constant dilution rate (0.87 µmax),the specific rate of chlorophyll production increased—thebiomass became greener. Actual photosynthesis rate (dry biomass–1)measured in the steady state conditions also increased. When fructose supply rate was increased by increasing the dilutionrate, the specific rate of chlorophyll production and actualphotosynthesis rate (dry biomass–1) increased until acritical dilution rate (0.64 µmax) was reached, thereafterdecreasing. The potential photosynthesis rate was measured on samples ofcells supplied with additional photons. This was inversely relatedto dilution rate. It seemed possible that the concentration of residual fructoseoutside the cells was related to specific growth rate but probablynot according to the Monod model. The specific production ofchlorophyll may be regulated by the intracellular concentrationof a catabolite of fructose, possibly glucose. Results suggestthat the specific production rate of chlorophyll was inhibitedwhen glucose concentration in the cells was above a thresholdof about 1.2% dry biomass. The degree of inhibition was a functionof glucose concentration above this threshold. Key words: Continuous culture, Photosynthesis, Greening, Ocimum basilicum  相似文献   

13.
14.
Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.  相似文献   

15.
Vancomycin production in batch and continuous culture   总被引:5,自引:0,他引:5  
Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.  相似文献   

16.
Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.  相似文献   

17.
Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h-1, it accounted for only 1.3% at D=0.26 h-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h-1 and 0.26 h-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.  相似文献   

18.
Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.  相似文献   

19.
Summary Continuous fermentation fed by 150 kg/m3 of glucose with total cell recycling by tangential microfiltration enabled yeasts concentration of 300 kg/m3 (dry weight) to be reached with a dilution rate of 0,5h–1 and a cell viability greater than 75%. The stability of this system was tested for 50 residence times of the permeate. The method can be used both for the production of cell concentrates and for high rates of metabolite production.Nomenclature D. W. dry weight - XT (kg/m3) total cell concentration D.W. - XV (kg/m3) viable cell concentration D.W. - V viability of cell culture in per cent of total cell concentration - S (kg/m3) glucose concentration - P (kg/m3) ethanol concentration - D (h) dilution rate - R (kg/kg) fermentation yield - (h) specific growth rate - vp(kg/kg/h) specific alcohol production rate - (m) yeast size - (kg/kg) kg of intracellular water per kg of dry cells  相似文献   

20.
Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.  相似文献   

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